Dispersion of Brazilian green propolis extract in poloxamer 188 improves depression-like behavior and neuroinflammation in mice subjected to transient global cerebral ischemia

Dispersion of Brazilian green propolis extract in poloxamer 188 improves depression-like behavior and neuroinflammation in mice subjected to transient global cerebral ischemia | 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 Dispersion of Brazilian green propolis extract in poloxamer 188 improves depression-like behavior and neuroinflammation in mice subjected to transient global cerebral ischemia Gabriella Candido, Nathalia Akemi Kohara, Pablo Pompeu Quini, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6513240/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Sep, 2025 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted 6 You are reading this latest preprint version Abstract Brazilian green propolis (BGP) has notable anti-inflammatory and neuroprotective properties, making it a potential candidate for treating neurological conditions. However, its impact on the sequelae of cerebral ischemia remains insufficiently explored. This study aimed to evaluate the effects of the hydroalcoholic extract (HE) of BGP dispersed (HE-D) in Poloxamer 188 (P188) on adult C57BL/6 mice subjected to bilateral common carotid artery occlusion (BCCAO), a model of transient global cerebral ischemia. P188 was selected as the surfactant due to the low solubility of HE, which could compromise its pharmacokinetic profile, reducing bioavailability and potentially limiting its neuroprotective effects. Male C57BL/6J mice were subjected to BCCAO for 20 minutes. P188 or the HE-D at doses of 50, 100, and 150 mg/kg was administered orally once daily for seven consecutive days. The animals were assessed for locomotor activity, anxiety-related and antidepressant-like behaviors of the treatments. Following the behavioral evaluations, the brains were collected to investigate the impact of HE-D on neuronal death and neuroinflammation induced by BCCAO. HE-D (150 mg/Kg) decreased anxiety-like behavior induced by BCCAO in mice. Also, HE-D induced antidepressant-like effects in mice with BCCAO. HE-D mitigated hippocampal neuroinflammation induced by BCCAO, as seen by a decreased immunoreactivity to both inflammatory markers, Iba-1 and GFAP. These results suggest that HE-D may present beneficial effects on the consequences of BCCAO by decreasing hippocampal neuroinflammation. Bilateral common carotid arteries occlusion behavior P188 Brazilian Green Propolis neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Ischemic brain disease is a leading cause of disability and death worldwide. Global cerebral ischemia (GCI), most often due to cardiac arrest, can also result from dysrhythmias, hypotensive shock, or medical procedures. Long-term survivors may suffer cognitive, emotional, and motor impairments linked to neuronal loss in the hippocampus, thalamus, and cortex (Anderson and Arciniegas 2010 ; Horstmann et al. 2010 ; Sawyer et al. 2020 ; Feigin et al. 2022 ). These effects can be modeled in animals, enabling the study of neuroprotective agents in mice (Soares et al. 2013 ; 2016 ; Mori et al. 2017 ; 2021 ; Aguiar et al. 2020 ) and rats (Bacarin et al. 2016 ; Godinho et al. 2018 ; Bonato et al. 2021 ). Despite advances, effective treatments remain unavailable, reinforcing the need for new neuroprotective strategies. Propolis, a complex resin produced by Apis mellifera from plant sources, is used by bees to seal and reinforce hives (Burdock 1998 ; Marcucci 1996 ). Its composition varies by region and flora, contributing to diverse biological properties (Kumazawa et al. 2004 ). Brazilian Green Propolis (BGP) exhibits antibacterial (Meccatti et al. 2023 ), antifungal (Kischkel et al. 2020 ), antiviral (Fiorini et al. 2021 ), immunomodulatory (Rebouças-Silva et al. 2023 ), anti-inflammatory (Xu et al. 2020 ), antioxidant (Costa et al. 2020 ), and antitumor effects (Sepúlveda et al. 2020 ). BGP also prevents cognitive impairments in Alzheimer’s disease models by reducing neuroinflammation and modulating hippocampal calcium signaling (Ito et al. 2023 ; Inagaki et al. 2024 ), and supports cognitive function in elderly populations at high altitudes (Zhu et al. 2018 ). Under ischemic conditions, BGP shows neuroprotective effects. It inhibits oxidative stress in retinal ganglion cells exposed to oxygen-glucose deprivation/reoxygenation and reduces retinal damage induced by N-methyl-D-aspartate in mice (Nakajima et al. 2009 ). In vitro , BGP prevents lipid peroxidation in mouse forebrain homogenates and scavenges DPPH-induced free radicals. BGP (30 or 100 mg/kg) reduced brain infarction 24 h after transient middle cerebral artery occlusion (tMCAO) in mice (Shimazawa et al. 2005 ). Similarly, commercial propolis showed protective effects after tMCAO in rats, linked to increased TGF-β1 and reduced MMP9 mRNA expression (Abdel-Rahman et al. 2020 ), suggesting anti-inflammatory action. However, BGP's impact on ischemia-induced functional deficits remains underexplored. Despite its therapeutic potential, BGP has a complex, plant-dependent chemical composition and poor aqueous solubility, limiting its bioavailability (Burdock 1998 ). The most common pharmaceutical formulation of BGP is the hydroethanolic extract (HE) which can be used as final or intermediary dosage form (Burdock 1998 ; Bruschi et al. 2003 ; 2007 ). However, the HE has some disadvantages like aromatic odor, strong and unpleasant taste, high ethanol concentration, and poor aqueous solubility as well (Bruschi et al. 2003 ; Pereira et al. 2013 ). Therefore, poloxamer 188 (P188), a non-ionic, nontoxic biocompatible copolymer, has been found promising for its surfactant action (Alexandridis and Hatton 1995 ), it is commonly utilized for its adsorption properties in applications like cosmetics and emulsion formulations (Chen et al. 2022 ). Therefore, this study examined the effects of the HE of BGP dispersed (HE-D) in P188 on adult C57BL/6 mice subjected to BCCAO. Functional impairments were assessed using a comprehensive battery of behavioral tests, including the open field test (OFT), elevated zero maze (EZM), and tail suspension test (TST). Hippocampal neurodegeneration was evaluated using NeuN as a neuronal marker, while the expression of neuroinflammatory markers, including Iba-1 (microglial marker) and GFAP (astrocytic marker), were also analyzed. 2. Materials and methods 2.1 Animals Male C57BL/6J mice (2–3 months old, 25–30 g) were housed in polyethylene cages under controlled temperature (22 ± 1°C) and a 12/12 h light/dark cycle, with free access to food (Nutrilab-CR1; Nuvital Nutrients) and water. All procedures were approved by the Ethics Committee of the State University of Maringá (CEUA 3943160921). 2.2 Preparation and evaluation of HE BGP sample was obtained from Apis mellifera L . bee hives at an apiary of the Iguatemi Experimental Farm, the State University of Maringá, located in the city of Maringá (PR, Brazil) (latitude: 23° 25' 38" South, longitude: 51° 56' 15" West). The extraction of HE was obtained by turbo extraction, using BGP/ethanol ratio 30/70 (w/w) (Bruschi et al. 2003 ). HE was filtered though filter paper and the initial weight was made using ethanol. The quality control of 30% (w/w) HE of the BGP was evaluated as previously described by Pereira et al. ( 2013 ), where all results were expressed as percentages (%, w/w), representing the average of at least three assays (Table 1 ). The work was registered in Brazil with authorization from the National System for the Management of Genetic Heritage (SISGEN No. AC7A2F5). Table 1 Physicochemical analysis for HE of BGP Physicochemical analysis Result (mean ± SD) pH 4.74 ± 0.04 Relative density (g/mL) 0.8743 ± 0.0006 Dryness residue (%, w/w) 18.04 ± 0.99 Ethanol content (%, w/w) 67.47 ± 1.25 Total polyphenol content (%, w/w) 1.67 ± 0.08 2.3 Surgery Cerebral ischemia was induced by BCCAO as described previously (Mori et al. 2021 ). The animals were placed under anesthesia with a mixture of isoflurane/ oxygen (1.3–1.5% isoflurane in 100% oxygen, Isoforine®, Cristália) delivered through a universal vaporizer (Oxigel). An incision was made in the ventral neck to expose the common carotid arteries. Next, the arteries were occluded for 20 minutes using aneurysm clips (ADCA, Belo Horizonte, Brazil). After the 20 minutes occlusion time, the aneurism clips were carefully removed, and the arteries were visually inspected for reperfusion. The incision was then closed with sutures. In the sham-operated animals, the carotid arteries were exposed but not occluded and the animals were designated as controls. 2.4 Experimental design HE was dispersed in 5% P188 (veh; Sigma-Aldrich, USA) to create the HE-D formulation, administered by gavage. C57BL/6J mice were divided into five groups: Sham + veh (n = 19), BCCAO + veh (n = 15), and BCCAO + HE-D at 50 (n = 20), 100 (n = 17), or 150 mg/kg (n = 19). Treatments began 1 h after BCCAO and continued once daily for 7 days. Mice underwent behavioral tests as following Open field test (OF) and elevated zero maze (EZM) on day 7, and tail suspension test (TST) on day 15 post-surgery. Apparatuses were cleaned with 70% ethanol between tests. After the final test, mice were euthanized with sodium thiopental (Thiopentax, Cristalia, Brazil), and brains were collected for histological and molecular analysis. 2.5 Behavioral tests 2.5.1 The open field test The open field (OF) test is an experimental model used to evaluate the locomotor activity in mice and rats (Prut and Belzung 2003 ). It consists in a circular arena that was made of transparent polyvinyl chloride (43 cm in diameter and 40 cm high wall). The animal was put into the OF apparatus and its exploratory activity was recorded for 5 min, by measuring the distance traveled (in meters, ANY-maze software). 2.5.2. The elevated zero maze The EZM has been used to assess anxious-like behavior in rodents (Carola et al. 2002 ). The apparatus consists of a circular arena (46 cm in diameter, 5.5 cm wide) made of gray plastic, elevated 20 cm above the ground. The arena is divided into 4 quadrants: two open quadrants with small side walls (3 mm high), and two closed quadrants with higher side walls (11 cm high). Each animal was placed individually in one of the open quadrants and allowed to explore the arena for 6 min under low light conditions. The time spent in the open quadrants and the number of entries into the open quadrants of the maze were recorded. 2.5.3. The tail suspension test This test is based on the premise that in situations of inescapable stress, animals adopt a posture of immobility, which is reduced by drugs with antidepressant activity (Pitzer et al. 2022 ), where longer immobility time is considered depressive-like behavior. Mice were suspended 50 cm above the ground using adhesive tape, and the test lasted for 6 min. The parameters analyzed were latency to the first immobility episode and immobility time during the test period. 2.6 Immunohistochemistry The animals were anesthetized with an intraperitoneal injection of sodium thiopental (Thiopentax, Cristália, SP, Brazil) and were transcardially perfused with 0.1 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.2 M phosphate buffer. Brains were removed and subsequently fixed in 4% PFA for 24 hours. The brains were cryoprotected by immersion in 30% sucrose in 0.1 M sodium phosphate buffer pH 7.4. Then, the brains were frozen and then sectioned using a cryostat (Criocut 1800, Reichert-Jung, Heidelberg, Germany), into serial coronal slices of 40 µm, spanning from bregma coordinates − 3.14 mm to -4.30 mm (Franklin and Paxinos 2019). Free floating sections were washed with 0.01 M PBS and the endogenous peroxidase activity blocked in 0.3% H 2 O 2 in PBS. The sections were incubated with 2% bovine serum albumin (BSA) and subsequently incubated with the following polyclonal antibodies: rabbit anti-NeuN (neuronal specific nuclear, 1:500; Abcam, Cambridge, USA), rabbit anti-Iba-1 (ionized calcium-binding adapter molecule 1, 1:1500; Wako Chemicals, Cambridge, USA), rabbit anti-GFAP (glial fibrillary acidic protein, 1:2000; Abcam, Santa Cruz, CA, USA). Then, the sections were incubated with respective biotinylated secondary antibodies (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hours. Next, the incubation in ABC solution (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA) for 2 hours at room temperature. The peroxidase colorimetric reaction was performed using 3,3'-diaminobenzidine (DAB; Sigma) and H 2 O 2 . Nickel sulfate II was added to the DAB solution to enhance color contrast. 2.6.1 Immunohistochemistry analysis Immunohistochemical analyses were conducted using an Olympus BX41 microscope (Tokyo, Japan) coupled with a high-performance color camera (QColor3, Ontario, Canada). Camera settings and microscope parameters were meticulously maintained constant. ImageJ software (NIH, Bethesda, MD, USA) was used to calculate the number of cells and integrated optical density (IOD) when indicated. The IOD measurements (Iba-1, GFAP and NeuN) were determined in the prefixed areas of interest in the hippocampus (CA1 = 0.12 mm 2 ; CA3 = 0.15 mm 2 0.04). For IOD measurements, selected images were converted to 32-bit grayscale, and the background was subtracted. The threshold for a positive signal was predefined, and IOD was calculated. 2.6 Statistical analysis GraphPad Prism-9 software was used. One-way ANOVA followed by Dunnett’s multiple comparison test was used to evaluate the effects of HE-D or vehicle on the behavioral and immunohistochemical changes caused by BCCAO. Shapiro-Wilk’s and Brown-Forsythe’s or Bartlett’s tests were used to test the assumption of normality and homoscedasticity, respectively. In the case where the homoscedasticity assumptions was not found, Welch’s ANOVA was used, as recommended by the statistic package. Data are presented as means ± SEM, and differences were considered significant at p < 0.05. 3. Results and Discussion 3.1. Open field test The distance traveled (Fig. 2 A), the number of entries (Fig. 2 B), and the time spent in the center (Fig. 2 C) of the OF were not changed by BCCAO (F 4.83 = 0.67, p > 0.5, BCCAO + veh vs. sham + veh). Compared to the sham group, the BCCAO groups that received the HE-D formulation at the doses of 100 mg/kg or 150 mg/kg exhibited a greater number of entries (F 4.80 = 3.07, p = 0.01–0.04 and spent more time in the center (C, W = 5.76, p = 0.008–0.01). 3.2. Elevated zero maze test ANOVA detected the main effect of groups for both the parameter number of entries (Fig. 3 A) and the time spent (Fig. 3 B) in the open arm (F 4,79−81 = 3.66–5.35, p < 0.001–0.01). The sham-operated mice preferred the closed quadrant over the open quadrant of the EZM, indicating their anxiety-like behavior toward open places. This anxiety-like behavior was not changed by BCCAO (p > 0.05 vs. sham). However, the number of entries and the time spent in the open arm increased significantly in the BCCAO group that received HE-D 150 mg/kg compared to the sham-operated group (p < 0.001–0.01). Further, compared to the BCCAO + veh group, the time spent in the open arm also increased significantly in the BCCAO + HE-D 150 mg/kg group (p < 0.05). These data indicate that HE-D can improve emotional performance in the EZM test. 3.3. Tail-suspension test A highly significant main effect of groups appeared for both the parameters of latency (Fig. 4 A, F 4,76 = 9.36, p < 0.0001) and immobility time (Fig. 4 B, W 4,37.31 = 6.23; p < 0.001). Compared to the sham group, BCCAO increased the immobility time (p 0.05), indicating that cerebral ischemia caused depressive-like behavior. Compared to the BCCAO + veh group, the latency to immobility increased after treatment with HE-D at doses of 100 or 150 mg/kg (Fig. 4 panel A, p < 0.001–0.01); in contrast, the immobility time was significantly reduced after HE-D 150 mg/kg (Fig. 4 panel B, p < 0.001). These data indicate that HE-D can mitigate depressive-like behavior that was induced by BCCAO in the TST. 3.4. Immunohistochemistry Figure 5 shows that Ischemia-induced neuronal loss in the hippocampus was significantly expressed in the CA3 (F 4.20 = 7.064, p 0.05). Figure 6 and 7 show the impact of BCCAO on the immunoreactivity (IR) of the Iba-1 (microglia) and GFAP (astrocyte) markers in the CA1 and CA3 subfields of the hippocampus, and the effect of the HE-D formulation thereon. Either in the CA1 and CA3, a main effect of groups was revealed by ANOVA for both Iba-1 and GFAP (F 4.23−25 = 6.46–10.19, p < 0.001–0.01). Compared to sham operation, BCCAO increased the IOD of the Iba-1 and GFAP in both hippocampal regions examined (p < 0.001–0.05), indicating an inflammatory effect of BCCAO. Compared to the vehicle-treated group, treatments with the HE-D formulation reduced the IOD for both Iba-1 (p < 0.05) and GFAP (p < 0.001–0.05), either in the CA1 or CA3. Although this effect varied with the doses used, these results indicate the HE-D formulation exerted an anti-inflammatory effect after BCCAO. 4. Discussion This study explored the effects of the HE-D formulation administered post-ischemia in C57BL/6J mice. In those animals, HE-D reduced both anxiety and depressant-like behaviors. Although HE-D did not prevent ischemia-induced neurodegeneration in the hippocampus, it effectively mitigated hippocampal neuroinflammation, as evidenced by a reduction in the expression of both Iba-1 and GFAP-IR. BCCAO leads to significant emotional and cognitive impairments. Twenty minutes of BCCAO has been shown to increase anxiety- and despair-like behaviors in mice (Soares et al. 2013 ; 2016 ; Mori et al. 2017 ; Aguiar et al. 2020 ). In this study, unexpectedly, we did not observe significant behavioral differences when comparing ischemic animals to their sham-operated controls. However, the control animals in this study received P188 as a vehicle. Upon investigating the pharmacological effects of P188, we observed that P188 may help to alleviate anxiety-like behaviors even in healthy animals. In pilot studies, our group observed the anxiolytic-like effects of P188 in healthy animals compared to those that received saline (data not shown). Consistent with these findings, the inclusion of P188 with curcumin reduced anxiety-like behavior in mice tested in the elevated plus maze test, as well as despair-like behaviors measured in the TST and forced swim test (Rubab et al. 2021 ). The authors of this related study proposed that some of the observed effects may be attributable to the surfactant action of P188 (Rubab et al. 2021 ). Beyond ischemia/reperfusion, the protective effects of P188 have recently been reviewed for models of traumatic brain injury or mechanical injury in cells, neurotoxicity, Parkinson’s disease, and amyotrophic lateral sclerosis (Chen et al. 2022 ). Therefore, it can be hypothesized that P188 may have exerted neuroprotective effects per se , preventing the differences between sham and ischemic animals from manifesting in the behavioral tests employed. The OFT assessed locomotor activity and anxiety-related behaviors in mice. While ischemic animals did not exhibit significant changes in anxiety-related behaviors compared to sham animals, BCCAO mice treated with HE-D (100 and 150 mg/kg) demonstrated increased exploration in the center of the open field, suggesting anxiolytic-like effects. This finding was consistent with the results obtained from the EZM test. HE-D (150 mg/Kg) resulted in an anxiolytic-like effect in ischemic animals compared to their controls. In agreement, Reis et al. ( 2014 ) reported that BGP obtained using edible vegetable oil increased open-arm entries in both the OFT and the elevated plus maze tests. Similarly, Li et al. ( 2012 ) showed that propolis essential oil effectively alleviated anxiety-like behaviors and inhibited the hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis in rats. Ischemic mice presented an increase in the immobility time in the TST, reflecting despair-like behaviors. HE-D (150 mg/kg) produced an antidepressant-like effect in BCCAO mice. Similar effects were observed by others (Reis et al. 2014 ; Li et al. 2012 ). In a recent study, it has been demonstrated that Turkish propolis decreased depression-like behaviors linked to chronic unpredictable stress in rats through its anti-inflammatory and antioxidant effects (Celebi et al. 2023 ). The present study confirmed significant cell loss in the CA3 hippocampal region of BCCAO mice, as indicated by reduced NeuN expression (Soares et al. 2013 ; Mori et al. 2017 ). However, treatment with HE-D did not protect hippocampal neurons from ischemic damage. This contrasts with the findings of Abdel-Rahman et al. ( 2020 ), who reported neuroprotective effects of commercial propolis extract in the hippocampus of mice subjected to transient, focal cerebral ischemia (tMCAO model). Using hematoxylin and eosin staining, the authors demonstrated that propolis exerted neuroprotective effects, potentially through increased TGF-β1 expression and decreased MMP9 expression (Abdel-Rahman et al. 2020 ). It is important to note, however, that functional recovery does not always align with cellular protection, including neuronal preservation. Subcellular ultrastructural changes, such as dendritic remodeling, reactive synaptogenesis, and growth-promoting processes, may contribute significantly to functional recovery after cerebral ischemia (García-Chávez et al. 2008 ; Bacarin et al. 2016 ; Godinho et al. 2018 ). The hallmark of neuroinflammation is the activation of glial cells, accompanied by the infiltration and recruitment of peripheral inflammatory cells (Zhang et al. 2010 ). We observed an increase in brain inflammation, evidenced by elevated Iba-1-IR and GFAP-IR in the hippocampus. Treatment with HE-D (100 mg/kg) alleviated neuroinflammation in the CA1 and CA3 subfields. These findings align with those of Ito et al. ( 2023 ). Using an Alzheimer's disease model, the authors demonstrated that BGP prevented changes in the gene expression of microglial and astrocytic markers, suggesting that prophylactic administration of propolis may effectively suppress inflammation and modulate immune responses in glial cells (Ito et al. 2023 ). Moreover, the administration of commercial propolis attenuated astrogliosis, and microgliosis in the brains of mice subjected to tMCAO (Abdel-Rahman et al. 2020 ). Although evidence regarding the mechanisms underlying the improvement of neuroinflammation remains limited, anti-inflammatory effects of propolis may be mediated by the inhibition of nuclear factor-κB (NF-κB) activation in microglia (Wu et al. 2013 ). In conclusion, post-treatment with HE-D exerts both functional and structural positive effects in mice following BCCAO. HE-D induced anxiolytic-like and antidepressant-like actions, while also mitigating neuroinflammation associated with BCCAO. These findings suggest that the observed neuroprotective effects of HE-D may be attributed to its anti-inflammatory properties. Declarations Ethics approval The local Ethics Committee on Animal Experimentation of the State University of Maringá approved the experimental procedures in accordance with the guidelines of the U.S. National Institutes of Health and Brazilian College for Animal Experimentation (CEUA no. 3943160921). All efforts were made to minimize the number of animals used and reduce their suffering. Competing interests There is no competing or conflict of interests. All authors contribute for this study. G Candido and NA Kohara performed the animals’ surgeries. GC and RS dos Santos performed the extract dilution tests. GC performed behavioral testing and histological experiments. CA Terdardi and PP Quini analyzed the histological tests. ML Bruschi provided the propolis hydroalcoholic extract. GC wrote the first draft of the manuscript. H Milani analyzed the data and helped with statistical analysis. RMW Oliveira conceived the experimental design, planned the experiments, and performed the data workup. All authors discussed the results and commented on the manuscript. Funding This study received finantial support from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and State University of Maringá. Availability of data and material All the experimental data will be made available upon request. References Abdel-Rahman RF et al. (2020). Propolis ameliorates cerebral injury in focal cerebral ischemia/reperfusion (I/R) rat model via upregulation of TGF-β1. Saudi Pharm J 28:116–126. https://doi.org/10.1016/j.jsps.2019.11.013 Aguiar RP et al. (2020). Activation of 5-HT1A postsynaptic receptors by NLX-101 results in functional recovery and an increase in neuroplasticity in mice with brain ischemia. Prog Neuro-Psychopharmacol Biol Psychiatry 99:109832. https://doi.org/10.1016/j.pnpbp.2019.109832 Alexandridis P and Hatton TA (1995). Poly(ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces: thermodynamics, structure, dynamics, and modeling. Colloids Surf A: Physicochem Eng Asp 96(1-2):1-46. https://doi.org/10.1016/0927-7757(94)03028-X Anderson CA and Arciniegas DB (2010). Cognitive sequelae of hypoxic-ischemic brain injury: a review. NeuroRehabilitation 26(1):47–63. https://doi.org/10.3233/NRE-2010-0535 Bacarin CC et al. (2016). Postischemic fish oil treatment restores long-term retrograde memory and dendritic density: An analysis of the time window of efficacy. Behav Brain Res 311: 425–439. https://doi.org/10.1016/j.bbr.2016.05.047. Bonato JM et al. (2021). Roflumilast protects against spatial memory impairments and exerts anti-inflammatory effects after transient global cerebral ischemia. Eur J Neurosci 53(4):1171–1188. https://doi.org/10.1111/ejn.15089 Bruschi ML et al. (2003). Gelatin microparticles containing propolis obtained by spray-drying technique: preparation and characterization. Int J Pharmac 264(1-2): 45–55. https://doi.org/10.1016/s0378-5173(03)00386-7 Bruschi ML et al. (2007). Semisolid systems containing propolis for the treatment of periodontal disease: in vitro release kinetics, syringeability, rheological, textural, and mucoadhesive properties. J Pharmac Sci 96(8): 2074–2089. https://doi.org/10.1002/jps.20843 Burdock GA (1998). Review of the biological properties and toxicity of bee propolis (propolis). Food Chem Toxicol 36:347–363. https://doi.org/10.1016/S0278-6915(97)00145-2 Carola V et al. (2002). Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behavior in inbred mice. Behav Brain Res 134: 49–57. https://doi.org/10.1016/S0166-4328(01)00452-1 Celebi G et al. (2023). Propolis prevents vascular endothelial dysfunction by attenuating inflammation and oxidative damage in the chronic unpredictable stress model of depression in rats. J Pharm Pharmacol 75:1418–1429. https://doi.org/10.1093/jpp/rgad071 Chen WN et al. (2022). Poloxamer 188 (P188), a potential polymeric protective agent for central nervous system disorders: A systematic Review. Curr Neuropharmacol 20(4):799–808. https://doi.org/10.2174/1570159X19666210528155801 Costa P et al. (2020). Role of the antioxidant properties in the gastroprotective and gastric healing activity promoted by Brazilian green propolis and the healing efficacy of Artepillin C. Inflammopharmacology 28:1009–1025. https://doi.org/10.1007/s10787-019-00649-7 Feigin VL et al. (2022). World Stroke Organization (WSO): global stroke fact sheet 2022. Int J Stroke 17:18–29. https://doi.org/10.1177/17474930211065917 Fiorini AC et al. (2021). Antiviral activity of Brazilian green propolis extract against SARS-CoV-2 (Severe acute respiratory syndrome-coronavirus 2) infection: Case report and review. Clinics 76:e2357. https://doi.org/10.6061/clinics/2021/e2357 García-Chávez D et al. (2008). Long-term evaluation of cytoarchitectonic characteristics of prefrontal cortex pyramidal neurons, following global cerebral ischemia and neuroprotective melatonin treatment, in rats. Neurosci letters 448(1):148–152. https://doi.org/10.1016/j.neulet.2008.10.043 Godinho J et al. (2018). Ethyl-acetate fraction of Trichilia catigua restores long-term retrograde memory and reduces oxidative stress and inflammation after global cerebral ischemia in rats. Behav Brain Res 337:173–182. https://doi.org/10.1016/j.bbr.2017.08.050 Horstmann A et al (2010). Resuscitating the heart but losing the brain: brain atrophy in the aftermath of cardiac arrest. Neurology 74(4):306–312. https://doi.org/10.1212/WNL.0b013e3181cbcd6f Inagaki R et al. (2024). Preventive effect of propolis on cognitive decline in Alzheimer's disease model mice. Neurobiol Aging 139:20–29. https://doi.org/10.1016/j.neurobiolaging.2024.03.002 Ito T et al. (2023). Brazilian green propolis prevents Alzheimer’s disease-like cognitive impairment induced by amyloid beta in mice. BMC Complement Med Ther 23:416. https://doi.org/10.1186/s12906-023-04247-7 Kischkel B et al. (2020). Silver nanoparticles stabilized with propolis show reduced toxicity and potential activity against fungal infections. Futur Microbiol 15:521–539. https://doi.org/10.2217/fmb-2019-0173 Kumazawa S, Hamasaka T, Nakayama T (2004). Antioxidant activity of propolis of various geographic origins. Food Chem 84:329–339. https://doi.org/10.1016/S0308-8146(03)00216-4 Li YJ et al. (2012). Therapeutic effects of propolis essential oil on anxiety of restraint-stressed mice. Hum Exp Toxicol 31:157–165. https://doi.org/10.1177/0960327111412805 Marcucci MC (1996). Biological and therapeutic properties of chemical propolis constituents. Quim Nova 19:529–536. https://doi.org/10.20396/chemkeys.v0i10.9640 Meccatti VM et al. (2023). Synergistic antibiofilm action of Cinnamomum verum and Brazilian green propolis hydroethanolic extracts against multidrug-resistant strains of Acinetobacter baumannii and Pseudomonas aeruginosa and their biocompatibility on human keratinocytes. Molecules 28:6904. https://doi.org/10.3390/molecules2819690 Mori MA et al. (2017). Cannabidiol reduces neuroinflammation and promotes neuroplasticity and functional recovery after brain ischemia. Prog Neuro-Psychopharmacol Biol Psychiatry 75:94–105. https://doi.org/10.1016/j.pnpbp.2016.11.005 Mori MA et al (2021). Differential contribution of CB1, CB2, 5‐HT1A, and PPAR‐γ receptors to cannabidiol effects on ischemia‐induced emotional and cognitive impairments. Eur J Neurosci 53:1738–1751. https://doi.org/10.1111/ejn.15134 Nakajima Y et al. (2009). Neuroprotective effects of Brazilian green propolis and its main constituents against oxygen‐glucose deprivation stress, with a gene-expression analysis. Phytother Res 23:1431–1438. https://doi.org/10.1002/ptr.2797 Pereira RRA et al. (2013). Preparation and characterization of mucoadhesive thermoresponsive systems containing propolis for the treatment of vulvovaginal candidiasis. J Pharm Sci 102:1222–1234. https://doi.org/10.1002/jps.23451 Pitzer C, Kurpiers B, Eltokhi A (2022). Sex differences in depression-like behaviors in adult mice depend on endophenotype and strain. Front Behav Neurosci 16:1–12. https://doi.org/10.3389/fnbeh.2022.838122 Prut L and Belzung C (2003). The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 463:3–33. https://doi.org/10.1016/S0014-2999(03)01272-X Rebouças-Silva J et al. (2023). Leishmanicidal and immunomodulatory properties of Brazilian green propolis extract (EPP-AF®) and a gel formulation in a pre-clinical model. Front Pharmacol 14:1013376. https://doi.org/10.3389/fphar.2023.1013376 Reis JSS et al. (2014). Antidepressant-and anxiolytic-like activities of an oil extract of propolis in rats. Phytomedicine 21:1466–1472. https://doi.org/10.1016/j.phymed.2014.06.001 Rubab S et al. (2021). Enhanced neuroprotective and antidepressant activity of curcumin-loaded nanostructured lipid carriers in lipopolysaccharide-induced depression and anxiety rat model. Int J Pharm 603:120670. https://doi.org/10.1016/j.ijpharm.2021.120670 Sawyer KN et al. (2020). Relationship between duration of targeted temperature management, ischemic interval, and good functional outcome from out-of-hospital cardiac arrest. Crit Care Med 48(3):370–377. https://doi.org/10.1097/CCM.0000000000004160 Sepúlveda C et al. (2020). Antitumor activity of propolis: Recent advances in cellular perspectives, animal models and possible applications. Food Rev Int 36:429–455. https://doi.org/10.1080/87559129.2019.1649692 Shimazawa M et al. (2005). Neuroprotection by Brazilian green propolis against in vitro and in vivo ischemic neuronal damage. Evid Based Complement Altern Med 2:201–207. https://doi.org/10.1093/ecam/neh078 Soares LM et al. (2016). Rolipram improves cognition, reduces anxiety- and despair-like behaviors and impacts hippocampal neuroplasticity after transient global cerebral ischemia. Neuroscience 326:69–83. https://doi.org/10.1016/j.neuroscience.2016.03.062 Soares LM et al. (2013). Cognitive impairment and persistent anxiety-related responses following bilateral common carotid artery occlusion in mice. Behav Brain Res 249:28–37. https://doi.org/10.1016/j.bbr.2013.04.010 Wu Z et al. (2013). Brazilian green propolis suppresses the hypoxia‐induced neuroinflammatory responses by inhibiting NF‐κB activation in microglia. Oxid Med Cell Longev 2013:906726. https://doi.org/10.1155/2013/906726 Xu X et al. (2020). The chemical composition of Brazilian green propolis and its protective effects on mouse aortic endothelial cells against inflammatory injury. Molecules 25:4612. https://doi.org/10.3390/molecules25204612 Zhang L et al. (2010). Escin attenuates cognitive deficits and hippocampal injury after transient global cerebral ischemia in mice via regulating certain inflammatory genes. Neurochem Int 57:119–127. https://doi.org/10.1016/j.neuint.2010.05.001 Zhu A et al. (2018). Brazilian green propolis prevents cognitive decline into mild cognitive impairment in elderly people living at high altitude. J Alzheimers Dis 63:551–560. https://doi.org/10.3233/JAD-170630 Supplementary Files GraphAbstract.jpg Cite Share Download PDF Status: Published Journal Publication published 06 Sep, 2025 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted Editorial decision: Major revisions 23 Jun, 2025 Reviewers agreed at journal 02 Jun, 2025 Reviewers invited by journal 09 May, 2025 Editor invited by journal 29 Apr, 2025 Editor assigned by journal 24 Apr, 2025 First submitted to journal 24 Apr, 2025 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-6513240","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454177636,"identity":"0e2e1dbb-ff91-47c2-855b-8aa6ab7f8476","order_by":0,"name":"Gabriella Candido","email":"","orcid":"","institution":"UEM: Universidade Estadual de Maringa","correspondingAuthor":false,"prefix":"","firstName":"Gabriella","middleName":"","lastName":"Candido","suffix":""},{"id":454177637,"identity":"1ad16607-3872-4f82-8c92-a2f3fb15aa6b","order_by":1,"name":"Nathalia Akemi Kohara","email":"","orcid":"","institution":"UEM: Universidade Estadual de Maringa","correspondingAuthor":false,"prefix":"","firstName":"Nathalia","middleName":"Akemi","lastName":"Kohara","suffix":""},{"id":454177638,"identity":"8a3a72d4-a6ea-4108-a05d-7fbda1f5692b","order_by":2,"name":"Pablo Pompeu Quini","email":"","orcid":"","institution":"UEM: Universidade Estadual de Maringa","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"Pompeu","lastName":"Quini","suffix":""},{"id":454177639,"identity":"9d45893b-5679-419f-943d-5d4125eec00d","order_by":3,"name":"Carolina de Alencar Tedardi","email":"","orcid":"","institution":"UEM: Universidade Estadual de Maringa","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"de Alencar","lastName":"Tedardi","suffix":""},{"id":454177640,"identity":"96bd6b4f-bd6a-47a6-b2b0-287f752eacbe","order_by":4,"name":"Rafaella Said dos Santos","email":"","orcid":"","institution":"UEM: Universidade Estadual de Maringa","correspondingAuthor":false,"prefix":"","firstName":"Rafaella","middleName":"Said dos","lastName":"Santos","suffix":""},{"id":454177641,"identity":"df8605c1-8398-4025-af5c-acbe76bd7457","order_by":5,"name":"Humberto Milani","email":"","orcid":"","institution":"State University of Maringá: Universidade Estadual de Maringa","correspondingAuthor":false,"prefix":"","firstName":"Humberto","middleName":"","lastName":"Milani","suffix":""},{"id":454177642,"identity":"ef6db3ba-0e4f-43a5-a75e-b1d8f5d62f5e","order_by":6,"name":"Marcos Luciano Bruschi","email":"","orcid":"","institution":"State University of Maringá: Universidade Estadual de Maringa","correspondingAuthor":false,"prefix":"","firstName":"Marcos","middleName":"Luciano","lastName":"Bruschi","suffix":""},{"id":454177643,"identity":"6db912fe-6517-4c76-adb3-e2d1da0d3d58","order_by":7,"name":"Rubia Maria Weffort de Oliveira","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYNCCAgkGBmYg/QHEkSBKiwFEC+MMErRAKGYeYrSYTzudJvHBwCKxn5334WfbNptoBuneB3i1yNzO3SY5w0AicWYzu7F0bltaboPMcQO8WiSkc7fd5jGQMDY4zMYA1HI4t0EiDb/D4FrsD7Mx/7Zs+0+8FjkDZjY2aca2A0Rp2f4T6Bc5icNsbJY955Jz22SOEdSy2eBDRR0Pf/8x5hs/yuxy+6Xb8GvBBGykahgFo2AUjIJRgAkA7B8501YM4wYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6181-1881","institution":"UEM: Universidade Estadual de Maringa","correspondingAuthor":true,"prefix":"","firstName":"Rubia","middleName":"Maria Weffort","lastName":"de Oliveira","suffix":""}],"badges":[],"createdAt":"2025-04-23 13:44:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6513240/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6513240/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43450-025-00698-w","type":"published","date":"2025-09-06T15:57:04+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82647071,"identity":"4c5e62b1-eefd-4588-abdb-3532ee9156ee","added_by":"auto","created_at":"2025-05-13 16:18:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58166,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design. HE-D (50, 100, 150 mg/kg) or vehicle were administered by gavage 1h and during 7 days after BCCAO surgery. Behavioral tests were performed on the 7\u003csup\u003eth\u003c/sup\u003e and 15\u003csup\u003eth\u003c/sup\u003e days after BCCAO. Brains were removed after the last behavioral test for further processing and immunohistochemistry. BCCAO, bilateral occlusion of the common carotid arteries; HE-D, dispersion of hydroalcoholic extract of Brazilian green propolis in poloxamer 188; OFT, open field test; EZM, elevated zero maze; TST, tail suspension test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6513240/v1/106b47f050bb8d5270a39c23.png"},{"id":82647072,"identity":"49d91bfd-a281-4a25-a8a9-bfd5e7304884","added_by":"auto","created_at":"2025-05-13 16:18:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":695297,"visible":true,"origin":"","legend":"\u003cp\u003eMice’s performance in the open field test (OFT) after sham-operation, BCCAO + veh or BCCAO + HE-D at doses of 50, 100 or 150 mg/kg. \u003cstrong\u003e(A)\u003c/strong\u003e Distance traveled (m), \u003cstrong\u003e(B)\u003c/strong\u003e Number of entries into the OFT center, and \u003cstrong\u003e(C)\u003c/strong\u003e Time spent in the OFT center. BCCAO, bilateral common carotid artery occlusion; Veh, vehicle. The data are expressed as the means ± SEM, n = 10-20. *p \u0026lt; 0.05 and **p \u0026lt; 0.001 compared to sham + veh (one-way ANOVA or Welch’s ANOVA).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6513240/v1/11b1084d0171b36a7aca61cc.png"},{"id":82647824,"identity":"a1a3d347-eb97-485d-bdd7-80015cbc5a92","added_by":"auto","created_at":"2025-05-13 16:26:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":788498,"visible":true,"origin":"","legend":"\u003cp\u003eMice’s performance in the elevated zero-maze (EZM) test after sham-operation, BCCAO + veh, or BCCAO + HE-D at doses of 50, 100 or 150 mg/kg. \u003cstrong\u003e(A)\u003c/strong\u003e Entries and \u003cstrong\u003e(B)\u003c/strong\u003e time spent in the open arms of the EZM. BCCAO, bilateral common carotid artery occlusion; Veh, vehicle. Data are expressed as means ± SEM, n = 14-19/group. **p \u0026lt; 0.001 and ***p \u0026lt; 0.0001 compared to sham + veh, and # p \u0026lt;0.05 compared to BCCAO + veh group (one-way ANOVA).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6513240/v1/e928fa05fb3b1f25e97f0ac5.png"},{"id":82647076,"identity":"dcb6f89c-4844-45e9-aa60-d63fb375f383","added_by":"auto","created_at":"2025-05-13 16:18:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":832354,"visible":true,"origin":"","legend":"\u003cp\u003eMice’s performance in the tail suspension test (TST) after sham-operation, BCCAO + veh, or BCCAO + HE-D at doses of 50, 100 or 150 mg/kg. \u003cstrong\u003e(A)\u003c/strong\u003e Latency to the first immobility, and\u003cstrong\u003e (B)\u003c/strong\u003e immobility time. BCCAO, bilateral common carotid artery occlusion; Veh, vehicle. Data are expressed as means ± SEM, n = 14-19/group. *p \u0026lt; 0.05 and **p \u0026lt; 0.001 compared to sham + veh; ##p \u0026lt; 0.001 and ###p \u0026lt; 0.0001 compared to BCCAO + veh group (Welch’s ANOVA).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6513240/v1/7139a824d75458b8cc4c2168.png"},{"id":82647079,"identity":"6b62bc41-0d41-4c4e-8aa4-5bc533198ecc","added_by":"auto","created_at":"2025-05-13 16:18:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":15543290,"visible":true,"origin":"","legend":"\u003cp\u003eIntegrated optical density (IOD) of Neu-N positive cells in the hippocampus of mice subjected to BCCAO and treat with HE-D formulation. \u003cstrong\u003eA)\u003c/strong\u003e Diagrams of hippocampal coronal sections where IOD was measured; \u003cstrong\u003e(B)\u003c/strong\u003e Representative photomicrographs of NeuN positive cells in the CA1 and CA3 subfields of the hippocampus; \u003cstrong\u003e(C and D) \u003c/strong\u003eIOD values of NeuN positive cells in the CA1 and CA3 subfields, respectively. BCCAO, bilateral common carotid artery occlusion; Veh, vehicle. Data are mean ± SEM, n = 4-5/group. *p \u0026lt; 0.05, **p \u0026lt; 0.001 and ***p \u0026lt; 0.0001 compared to sham + veh (one-way ANOVA).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6513240/v1/ef6ed4c5e8e3aa3d1231100c.png"},{"id":82647089,"identity":"e3e50925-441f-47c0-b44f-4d840db31ab4","added_by":"auto","created_at":"2025-05-13 16:18:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":20831697,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of BCCAO on the immunoreactivity (IR) of the Iba-1 (microglia) in the CA1 and CA3 subfields of the hippocampus. \u003cstrong\u003e(A)\u003c/strong\u003e Diagrams of hippocampal coronal sections where IOD was measured; \u003cstrong\u003e(B)\u003c/strong\u003eRepresentative photomicrographs of Iba-1-IR cells in the CA1 and CA3 subfields of the hippocampus; \u003cstrong\u003e(C and D)\u003c/strong\u003e IOD values of Iba-1-positive cells in the CA1 and CA3 subfields, respectively. BCCAO, bilateral common carotid artery occlusion; Veh, vehicle. Data are means ± SEM, n = 4-5/group. *p \u0026lt; 0.05, **p \u0026lt; 0.001 and ***p \u0026lt; 0.0001 as compared to sham + veh; #p\u0026lt;0.05 as compared to BCCAO + veh group (one-way ANOVA).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6513240/v1/10a5fd447d8ff2ace1834a84.png"},{"id":82647085,"identity":"eff9f34d-103a-4ffb-8dd4-366e58e8cb51","added_by":"auto","created_at":"2025-05-13 16:18:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":22340856,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of BCCAO on the immunoreactivity (IR) of the GFAP (astroglia) in the CA1 and CA3 subfields of the hippocampus. \u003cstrong\u003e(A)\u003c/strong\u003e Diagrams of hippocampal coronal sections where IOD was measured; \u003cstrong\u003e(B)\u003c/strong\u003e Representative photomicrographs of GFAP-IR (lower pannel) cells in the CA1 and CA3 subfields of the hippocampus; \u003cstrong\u003e(C and D)\u003c/strong\u003e IOD values of GFAP (lower) positive cells in the CA1 and CA3 subfields, respectively. More information in the legend of Figure 6.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6513240/v1/47a99bbba6e1a9e936d64d63.png"},{"id":90827997,"identity":"9a5b1769-6c29-47b4-93d0-8921f2015f82","added_by":"auto","created_at":"2025-09-08 16:04:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":57441219,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6513240/v1/a4a6b1ee-7df5-46e4-a6a5-4ad41635ff4e.pdf"},{"id":82647083,"identity":"d780c56b-1def-411c-9420-43f08a246977","added_by":"auto","created_at":"2025-05-13 16:18:34","extension":"jpg","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":351627,"visible":true,"origin":"","legend":"","description":"","filename":"GraphAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6513240/v1/5b06c2467c3f7bfe594afac2.jpg"}],"financialInterests":"","formattedTitle":"Dispersion of Brazilian green propolis extract in poloxamer 188 improves depression-like behavior and neuroinflammation in mice subjected to transient global cerebral ischemia","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIschemic brain disease is a leading cause of disability and death worldwide. Global cerebral ischemia (GCI), most often due to cardiac arrest, can also result from dysrhythmias, hypotensive shock, or medical procedures. Long-term survivors may suffer cognitive, emotional, and motor impairments linked to neuronal loss in the hippocampus, thalamus, and cortex (Anderson and Arciniegas \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Horstmann et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Sawyer et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Feigin et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These effects can be modeled in animals, enabling the study of neuroprotective agents in mice (Soares et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mori et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Aguiar et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and rats (Bacarin et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Godinho et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Bonato et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Despite advances, effective treatments remain unavailable, reinforcing the need for new neuroprotective strategies.\u003c/p\u003e \u003cp\u003ePropolis, a complex resin produced by \u003cem\u003eApis mellifera\u003c/em\u003e from plant sources, is used by bees to seal and reinforce hives (Burdock \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Marcucci \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Its composition varies by region and flora, contributing to diverse biological properties (Kumazawa et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Brazilian Green Propolis (BGP) exhibits antibacterial (Meccatti et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), antifungal (Kischkel et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), antiviral (Fiorini et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), immunomodulatory (Rebou\u0026ccedil;as-Silva et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), anti-inflammatory (Xu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), antioxidant (Costa et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and antitumor effects (Sep\u0026uacute;lveda et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). BGP also prevents cognitive impairments in Alzheimer\u0026rsquo;s disease models by reducing neuroinflammation and modulating hippocampal calcium signaling (Ito et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Inagaki et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and supports cognitive function in elderly populations at high altitudes (Zhu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnder ischemic conditions, BGP shows neuroprotective effects. It inhibits oxidative stress in retinal ganglion cells exposed to oxygen-glucose deprivation/reoxygenation and reduces retinal damage induced by N-methyl-D-aspartate in mice (Nakajima et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). \u003cem\u003eIn vitro\u003c/em\u003e, BGP prevents lipid peroxidation in mouse forebrain homogenates and scavenges DPPH-induced free radicals. BGP (30 or 100 mg/kg) reduced brain infarction 24 h after transient middle cerebral artery occlusion (tMCAO) in mice (Shimazawa et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Similarly, commercial propolis showed protective effects after tMCAO in rats, linked to increased TGF-β1 and reduced MMP9 mRNA expression (Abdel-Rahman et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), suggesting anti-inflammatory action. However, BGP's impact on ischemia-induced functional deficits remains underexplored.\u003c/p\u003e \u003cp\u003eDespite its therapeutic potential, BGP has a complex, plant-dependent chemical composition and poor aqueous solubility, limiting its bioavailability (Burdock \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The most common pharmaceutical formulation of BGP is the hydroethanolic extract (HE) which can be used as final or intermediary dosage form (Burdock \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Bruschi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). However, the HE has some disadvantages like aromatic odor, strong and unpleasant taste, high ethanol concentration, and poor aqueous solubility as well (Bruschi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Pereira et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, poloxamer 188 (P188), a non-ionic, nontoxic biocompatible copolymer, has been found promising for its surfactant action (Alexandridis and Hatton \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), it is commonly utilized for its adsorption properties in applications like cosmetics and emulsion formulations (Chen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, this study examined the effects of the HE of BGP dispersed (HE-D) in P188 on adult C57BL/6 mice subjected to BCCAO. Functional impairments were assessed using a comprehensive battery of behavioral tests, including the open field test (OFT), elevated zero maze (EZM), and tail suspension test (TST). Hippocampal neurodegeneration was evaluated using NeuN as a neuronal marker, while the expression of neuroinflammatory markers, including Iba-1 (microglial marker) and GFAP (astrocytic marker), were also analyzed.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eMale C57BL/6J mice (2\u0026ndash;3 months old, 25\u0026ndash;30 g) were housed in polyethylene cages under controlled temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) and a 12/12 h light/dark cycle, with free access to food (Nutrilab-CR1; Nuvital Nutrients) and water. All procedures were approved by the Ethics Committee of the State University of Maring\u0026aacute; (CEUA 3943160921).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation and evaluation of HE\u003c/h2\u003e \u003cp\u003eBGP sample was obtained from \u003cem\u003eApis mellifera L\u003c/em\u003e. bee hives at an apiary of the Iguatemi Experimental Farm, the State University of Maring\u0026aacute;, located in the city of Maring\u0026aacute; (PR, Brazil) (latitude: 23\u0026deg; 25' 38\" South, longitude: 51\u0026deg; 56' 15\" West). The extraction of HE was obtained by turbo extraction, using BGP/ethanol ratio 30/70 (w/w) (Bruschi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). HE was filtered though filter paper and the initial weight was made using ethanol. The quality control of 30% (w/w) HE of the BGP was evaluated as previously described by Pereira et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), where all results were expressed as percentages (%, w/w), representing the average of at least three assays (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The work was registered in Brazil with authorization from the National System for the Management of Genetic Heritage (SISGEN No. AC7A2F5).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical analysis for HE of BGP\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhysicochemical analysis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResult (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRelative density (g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.8743\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDryness residue (%, w/w)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e18.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthanol content (%, w/w)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e67.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal polyphenol content (%, w/w)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Surgery\u003c/h2\u003e \u003cp\u003eCerebral ischemia was induced by BCCAO as described previously (Mori et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The animals were placed under anesthesia with a mixture of isoflurane/ oxygen (1.3\u0026ndash;1.5% isoflurane in 100% oxygen, Isoforine\u0026reg;, Crist\u0026aacute;lia) delivered through a universal vaporizer (Oxigel). An incision was made in the ventral neck to expose the common carotid arteries. Next, the arteries were occluded for 20 minutes using aneurysm clips (ADCA, Belo Horizonte, Brazil). After the 20 minutes occlusion time, the aneurism clips were carefully removed, and the arteries were visually inspected for reperfusion. The incision was then closed with sutures. In the sham-operated animals, the carotid arteries were exposed but not occluded and the animals were designated as controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental design\u003c/h2\u003e \u003cp\u003eHE was dispersed in 5% P188 (veh; Sigma-Aldrich, USA) to create the HE-D formulation, administered by gavage. C57BL/6J mice were divided into five groups: Sham\u0026thinsp;+\u0026thinsp;veh (n\u0026thinsp;=\u0026thinsp;19), BCCAO\u0026thinsp;+\u0026thinsp;veh (n\u0026thinsp;=\u0026thinsp;15), and BCCAO\u0026thinsp;+\u0026thinsp;HE-D at 50 (n\u0026thinsp;=\u0026thinsp;20), 100 (n\u0026thinsp;=\u0026thinsp;17), or 150 mg/kg (n\u0026thinsp;=\u0026thinsp;19). Treatments began 1 h after BCCAO and continued once daily for 7 days. Mice underwent behavioral tests as following Open field test (OF) and elevated zero maze (EZM) on day 7, and tail suspension test (TST) on day 15 post-surgery. Apparatuses were cleaned with 70% ethanol between tests. After the final test, mice were euthanized with sodium thiopental (Thiopentax, Cristalia, Brazil), and brains were collected for histological and molecular analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Behavioral tests\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 The open field test\u003c/h2\u003e \u003cp\u003eThe open field (OF) test is an experimental model used to evaluate the locomotor activity in mice and rats (Prut and Belzung \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). It consists in a circular arena that was made of transparent polyvinyl chloride (43 cm in diameter and 40 cm high wall). The animal was put into the OF apparatus and its exploratory activity was recorded for 5 min, by measuring the distance traveled (in meters, ANY-maze software).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. The elevated zero maze\u003c/h2\u003e \u003cp\u003eThe EZM has been used to assess anxious-like behavior in rodents (Carola et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The apparatus consists of a circular arena (46 cm in diameter, 5.5 cm wide) made of gray plastic, elevated 20 cm above the ground. The arena is divided into 4 quadrants: two open quadrants with small side walls (3 mm high), and two closed quadrants with higher side walls (11 cm high). Each animal was placed individually in one of the open quadrants and allowed to explore the arena for 6 min under low light conditions. The time spent in the open quadrants and the number of entries into the open quadrants of the maze were recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. The tail suspension test\u003c/h2\u003e \u003cp\u003eThis test is based on the premise that in situations of inescapable stress, animals adopt a posture of immobility, which is reduced by drugs with antidepressant activity (Pitzer et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), where longer immobility time is considered depressive-like behavior. Mice were suspended 50 cm above the ground using adhesive tape, and the test lasted for 6 min. The parameters analyzed were latency to the first immobility episode and immobility time during the test period.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eThe animals were anesthetized with an intraperitoneal injection of sodium thiopental (Thiopentax, Crist\u0026aacute;lia, SP, Brazil) and were transcardially perfused with 0.1 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.2 M phosphate buffer. Brains were removed and subsequently fixed in 4% PFA for 24 hours. The brains were cryoprotected by immersion in 30% sucrose in 0.1 M sodium phosphate buffer pH 7.4. Then, the brains were frozen and then sectioned using a cryostat (Criocut 1800, Reichert-Jung, Heidelberg, Germany), into serial coronal slices of 40 \u0026micro;m, spanning from bregma coordinates \u0026minus;\u0026thinsp;3.14 mm to -4.30 mm (Franklin and Paxinos 2019).\u003c/p\u003e \u003cp\u003eFree floating sections were washed with 0.01 M PBS and the endogenous peroxidase activity blocked in 0.3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in PBS. The sections were incubated with 2% bovine serum albumin (BSA) and subsequently incubated with the following polyclonal antibodies: rabbit anti-NeuN (neuronal specific nuclear, 1:500; Abcam, Cambridge, USA), rabbit anti-Iba-1 (ionized calcium-binding adapter molecule 1, 1:1500; Wako Chemicals, Cambridge, USA), rabbit anti-GFAP (glial fibrillary acidic protein, 1:2000; Abcam, Santa Cruz, CA, USA). Then, the sections were incubated with respective biotinylated secondary antibodies (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 hours. Next, the incubation in ABC solution (Vectastain Elite ABC Kit, Vector Laboratories, Burlingame, CA, USA) for 2 hours at room temperature. The peroxidase colorimetric reaction was performed using 3,3'-diaminobenzidine (DAB; Sigma) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Nickel sulfate II was added to the DAB solution to enhance color contrast.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Immunohistochemistry analysis\u003c/h2\u003e \u003cp\u003eImmunohistochemical analyses were conducted using an Olympus BX41 microscope (Tokyo, Japan) coupled with a high-performance color camera (QColor3, Ontario, Canada). Camera settings and microscope parameters were meticulously maintained constant. ImageJ software (NIH, Bethesda, MD, USA) was used to calculate the number of cells and integrated optical density (IOD) when indicated. The IOD measurements (Iba-1, GFAP and NeuN) were determined in the prefixed areas of interest in the hippocampus (CA1\u0026thinsp;=\u0026thinsp;0.12 mm\u003csup\u003e2\u003c/sup\u003e; CA3\u0026thinsp;=\u0026thinsp;0.15 mm\u003csup\u003e2\u003c/sup\u003e 0.04). For IOD measurements, selected images were converted to 32-bit grayscale, and the background was subtracted. The threshold for a positive signal was predefined, and IOD was calculated.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism-9 software was used. One-way ANOVA followed by Dunnett\u0026rsquo;s multiple comparison test was used to evaluate the effects of HE-D or vehicle on the behavioral and immunohistochemical changes caused by BCCAO. Shapiro-Wilk\u0026rsquo;s and Brown-Forsythe\u0026rsquo;s or Bartlett\u0026rsquo;s tests were used to test the assumption of normality and homoscedasticity, respectively. In the case where the homoscedasticity assumptions was not found, Welch\u0026rsquo;s ANOVA was used, as recommended by the statistic package. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and differences were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Open field test\u003c/h2\u003e \u003cp\u003eThe distance traveled (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), the number of entries (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and the time spent in the center (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) of the OF were not changed by BCCAO (F\u003csub\u003e4.83\u003c/sub\u003e = 0.67, p\u0026thinsp;\u0026gt;\u0026thinsp;0.5, BCCAO\u0026thinsp;+\u0026thinsp;veh vs. sham\u0026thinsp;+\u0026thinsp;veh). Compared to the sham group, the BCCAO groups that received the HE-D formulation at the doses of 100 mg/kg or 150 mg/kg exhibited a greater number of entries (F\u003csub\u003e4.80\u003c/sub\u003e = 3.07, p\u0026thinsp;=\u0026thinsp;0.01\u0026ndash;0.04 and spent more time in the center (C, W\u0026thinsp;=\u0026thinsp;5.76, p\u0026thinsp;=\u0026thinsp;0.008\u0026ndash;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Elevated zero maze test\u003c/h2\u003e \u003cp\u003eANOVA detected the main effect of groups for both the parameter number of entries (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and the time spent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) in the open arm (F\u003csub\u003e4,79\u0026minus;81\u003c/sub\u003e = 3.66\u0026ndash;5.35, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u0026ndash;0.01). The sham-operated mice preferred the closed quadrant over the open quadrant of the EZM, indicating their anxiety-like behavior toward open places. This anxiety-like behavior was not changed by BCCAO (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 vs. sham). However, the number of entries and the time spent in the open arm increased significantly in the BCCAO group that received HE-D 150 mg/kg compared to the sham-operated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u0026ndash;0.01). Further, compared to the BCCAO\u0026thinsp;+\u0026thinsp;veh group, the time spent in the open arm also increased significantly in the BCCAO\u0026thinsp;+\u0026thinsp;HE-D 150 mg/kg group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These data indicate that HE-D can improve emotional performance in the EZM test.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Tail-suspension test\u003c/h2\u003e \u003cp\u003eA highly significant main effect of groups appeared for both the parameters of latency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, F\u003csub\u003e4,76\u003c/sub\u003e = 9.36, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and immobility time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, W\u003csub\u003e4,37.31\u003c/sub\u003e = 6.23; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Compared to the sham group, BCCAO increased the immobility time (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) but not the latency (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that cerebral ischemia caused depressive-like behavior. Compared to the BCCAO\u0026thinsp;+\u0026thinsp;veh group, the latency to immobility increased after treatment with HE-D at doses of 100 or 150 mg/kg (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e panel A, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u0026ndash;0.01); in contrast, the immobility time was significantly reduced after HE-D 150 mg/kg (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e panel B, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These data indicate that HE-D can mitigate depressive-like behavior that was induced by BCCAO in the TST.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Immunohistochemistry\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows that Ischemia-induced neuronal loss in the hippocampus was significantly expressed in the CA3 (F\u003csub\u003e4.20\u003c/sub\u003e = 7.064, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u0026ndash;0.05), but not in the CA1 subfield. This neurodegenerative effect in the CA3 region was not mitigated after the HE-D treatments, independently of the dose used (BCCAO\u0026thinsp;+\u0026thinsp;HE-D vs. BCCAO\u0026thinsp;+\u0026thinsp;veh, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e show the impact of BCCAO on the immunoreactivity (IR) of the Iba-1 (microglia) and GFAP (astrocyte) markers in the CA1 and CA3 subfields of the hippocampus, and the effect of the HE-D formulation thereon. Either in the CA1 and CA3, a main effect of groups was revealed by ANOVA for both Iba-1 and GFAP (F\u003csub\u003e4.23\u0026minus;25\u003c/sub\u003e = 6.46\u0026ndash;10.19, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u0026ndash;0.01). Compared to sham operation, BCCAO increased the IOD of the Iba-1 and GFAP in both hippocampal regions examined (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u0026ndash;0.05), indicating an inflammatory effect of BCCAO. Compared to the vehicle-treated group, treatments with the HE-D formulation reduced the IOD for both Iba-1 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and GFAP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u0026ndash;0.05), either in the CA1 or CA3. Although this effect varied with the doses used, these results indicate the HE-D formulation exerted an anti-inflammatory effect after BCCAO.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study explored the effects of the HE-D formulation administered post-ischemia in C57BL/6J mice. In those animals, HE-D reduced both anxiety and depressant-like behaviors. Although HE-D did not prevent ischemia-induced neurodegeneration in the hippocampus, it effectively mitigated hippocampal neuroinflammation, as evidenced by a reduction in the expression of both Iba-1 and GFAP-IR.\u003c/p\u003e \u003cp\u003eBCCAO leads to significant emotional and cognitive impairments. Twenty minutes of BCCAO has been shown to increase anxiety- and despair-like behaviors in mice (Soares et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Mori et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Aguiar et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, unexpectedly, we did not observe significant behavioral differences when comparing ischemic animals to their sham-operated controls. However, the control animals in this study received P188 as a vehicle. Upon investigating the pharmacological effects of P188, we observed that P188 may help to alleviate anxiety-like behaviors even in healthy animals. In pilot studies, our group observed the anxiolytic-like effects of P188 in healthy animals compared to those that received saline (data not shown). Consistent with these findings, the inclusion of P188 with curcumin reduced anxiety-like behavior in mice tested in the elevated plus maze test, as well as despair-like behaviors measured in the TST and forced swim test (Rubab et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The authors of this related study proposed that some of the observed effects may be attributable to the surfactant action of P188 (Rubab et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Beyond ischemia/reperfusion, the protective effects of P188 have recently been reviewed for models of traumatic brain injury or mechanical injury in cells, neurotoxicity, Parkinson\u0026rsquo;s disease, and amyotrophic lateral sclerosis (Chen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, it can be hypothesized that P188 may have exerted neuroprotective effects \u003cem\u003eper se\u003c/em\u003e, preventing the differences between sham and ischemic animals from manifesting in the behavioral tests employed.\u003c/p\u003e \u003cp\u003eThe OFT assessed locomotor activity and anxiety-related behaviors in mice. While ischemic animals did not exhibit significant changes in anxiety-related behaviors compared to sham animals, BCCAO mice treated with HE-D (100 and 150 mg/kg) demonstrated increased exploration in the center of the open field, suggesting anxiolytic-like effects. This finding was consistent with the results obtained from the EZM test. HE-D (150 mg/Kg) resulted in an anxiolytic-like effect in ischemic animals compared to their controls. In agreement, Reis et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) reported that BGP obtained using edible vegetable oil increased open-arm entries in both the OFT and the elevated plus maze tests. Similarly, Li et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) showed that propolis essential oil effectively alleviated anxiety-like behaviors and inhibited the hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis in rats.\u003c/p\u003e \u003cp\u003eIschemic mice presented an increase in the immobility time in the TST, reflecting despair-like behaviors. HE-D (150 mg/kg) produced an antidepressant-like effect in BCCAO mice. Similar effects were observed by others (Reis et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In a recent study, it has been demonstrated that Turkish propolis decreased depression-like behaviors linked to chronic unpredictable stress in rats through its anti-inflammatory and antioxidant effects (Celebi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present study confirmed significant cell loss in the CA3 hippocampal region of BCCAO mice, as indicated by reduced NeuN expression (Soares et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mori et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, treatment with HE-D did not protect hippocampal neurons from ischemic damage. This contrasts with the findings of Abdel-Rahman et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who reported neuroprotective effects of commercial propolis extract in the hippocampus of mice subjected to transient, focal cerebral ischemia (tMCAO model). Using hematoxylin and eosin staining, the authors demonstrated that propolis exerted neuroprotective effects, potentially through increased TGF-β1 expression and decreased MMP9 expression (Abdel-Rahman et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is important to note, however, that functional recovery does not always align with cellular protection, including neuronal preservation. Subcellular ultrastructural changes, such as dendritic remodeling, reactive synaptogenesis, and growth-promoting processes, may contribute significantly to functional recovery after cerebral ischemia (Garc\u0026iacute;a-Ch\u0026aacute;vez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Bacarin et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Godinho et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe hallmark of neuroinflammation is the activation of glial cells, accompanied by the infiltration and recruitment of peripheral inflammatory cells (Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). We observed an increase in brain inflammation, evidenced by elevated Iba-1-IR and GFAP-IR in the hippocampus. Treatment with HE-D (100 mg/kg) alleviated neuroinflammation in the CA1 and CA3 subfields. These findings align with those of Ito et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Using an Alzheimer's disease model, the authors demonstrated that BGP prevented changes in the gene expression of microglial and astrocytic markers, suggesting that prophylactic administration of propolis may effectively suppress inflammation and modulate immune responses in glial cells (Ito et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, the administration of commercial propolis attenuated astrogliosis, and microgliosis in the brains of mice subjected to tMCAO (Abdel-Rahman et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although evidence regarding the mechanisms underlying the improvement of neuroinflammation remains limited, anti-inflammatory effects of propolis may be mediated by the inhibition of nuclear factor-κB (NF-κB) activation in microglia (Wu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conclusion, post-treatment with HE-D exerts both functional and structural positive effects in mice following BCCAO. HE-D induced anxiolytic-like and antidepressant-like actions, while also mitigating neuroinflammation associated with BCCAO. These findings suggest that the observed neuroprotective effects of HE-D may be attributed to its anti-inflammatory properties.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe local Ethics Committee on Animal Experimentation of the State University of Maring\u0026aacute; approved the experimental procedures in accordance with the guidelines of the U.S. National Institutes of Health and Brazilian College for Animal Experimentation (CEUA no. 3943160921). All efforts were made to minimize the number of animals used and reduce their suffering.\u003cbr\u003e\u0026nbsp;\u003cbr\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no competing or conflict of interests. All authors contribute for this study. G Candido and NA Kohara performed the animals\u0026rsquo; surgeries. GC and RS dos Santos performed the extract dilution tests. GC performed behavioral testing and histological experiments. CA Terdardi and PP Quini analyzed the histological tests. ML Bruschi provided the propolis hydroalcoholic extract. GC wrote the first draft of the manuscript. H Milani analyzed the data and helped with statistical analysis. RMW Oliveira conceived the experimental design, planned the experiments, and performed the data workup. All authors discussed the results and commented on the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received finantial support from the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) and State University of Maring\u0026aacute;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the experimental data will be made available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdel-Rahman RF et al. (2020). Propolis ameliorates cerebral injury in focal cerebral ischemia/reperfusion (I/R) rat model via upregulation of TGF-\u0026beta;1. Saudi Pharm J 28:116\u0026ndash;126. https://doi.org/10.1016/j.jsps.2019.11.013\u003c/li\u003e\n\u003cli\u003eAguiar RP et al. (2020). Activation of 5-HT1A postsynaptic receptors by NLX-101 results in functional recovery and an increase in neuroplasticity in mice with brain ischemia. Prog Neuro-Psychopharmacol Biol Psychiatry 99:109832. https://doi.org/10.1016/j.pnpbp.2019.109832\u003c/li\u003e\n\u003cli\u003eAlexandridis P and Hatton TA (1995). Poly(ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces: thermodynamics, structure, dynamics, and modeling. Colloids Surf A: Physicochem Eng Asp 96(1-2):1-46. https://doi.org/10.1016/0927-7757(94)03028-X\u003c/li\u003e\n\u003cli\u003eAnderson CA and Arciniegas DB (2010). Cognitive sequelae of hypoxic-ischemic brain injury: a review. NeuroRehabilitation 26(1):47\u0026ndash;63. https://doi.org/10.3233/NRE-2010-0535\u003c/li\u003e\n\u003cli\u003eBacarin CC et al. (2016). Postischemic fish oil treatment restores long-term retrograde memory and dendritic density: An analysis of the time window of efficacy. Behav Brain Res 311: 425\u0026ndash;439. https://doi.org/10.1016/j.bbr.2016.05.047.\u003c/li\u003e\n\u003cli\u003eBonato JM et al. (2021). Roflumilast protects against spatial memory impairments and exerts anti-inflammatory effects after transient global cerebral ischemia. Eur J Neurosci 53(4):1171\u0026ndash;1188. https://doi.org/10.1111/ejn.15089\u003c/li\u003e\n\u003cli\u003eBruschi ML et al. (2003). Gelatin microparticles containing propolis obtained by spray-drying technique: preparation and characterization. Int J Pharmac 264(1-2): 45\u0026ndash;55. https://doi.org/10.1016/s0378-5173(03)00386-7\u003c/li\u003e\n\u003cli\u003eBruschi ML et al. (2007). Semisolid systems containing propolis for the treatment of periodontal disease: in vitro release kinetics, syringeability, rheological, textural, and mucoadhesive properties. J Pharmac Sci 96(8): 2074\u0026ndash;2089. https://doi.org/10.1002/jps.20843\u003c/li\u003e\n\u003cli\u003eBurdock GA (1998). Review of the biological properties and toxicity of bee propolis (propolis). Food Chem Toxicol 36:347\u0026ndash;363. https://doi.org/10.1016/S0278-6915(97)00145-2\u003c/li\u003e\n\u003cli\u003eCarola V et al. (2002). Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behavior in inbred mice. Behav Brain Res 134: 49\u0026ndash;57. https://doi.org/10.1016/S0166-4328(01)00452-1\u003c/li\u003e\n\u003cli\u003eCelebi G et al. (2023). Propolis prevents vascular endothelial dysfunction by attenuating inflammation and oxidative damage in the chronic unpredictable stress model of depression in rats. J Pharm Pharmacol 75:1418\u0026ndash;1429. https://doi.org/10.1093/jpp/rgad071\u003c/li\u003e\n\u003cli\u003eChen WN et al. (2022). Poloxamer 188 (P188), a potential polymeric protective agent for central nervous system disorders: A systematic Review. Curr Neuropharmacol 20(4):799\u0026ndash;808. https://doi.org/10.2174/1570159X19666210528155801\u003c/li\u003e\n\u003cli\u003eCosta P et al. (2020). Role of the antioxidant properties in the gastroprotective and gastric healing activity promoted by Brazilian green propolis and the healing efficacy of Artepillin C. Inflammopharmacology 28:1009\u0026ndash;1025. https://doi.org/10.1007/s10787-019-00649-7\u003c/li\u003e\n\u003cli\u003eFeigin VL et al. (2022). World Stroke Organization (WSO): global stroke fact sheet 2022. Int J Stroke 17:18\u0026ndash;29. https://doi.org/10.1177/17474930211065917\u003c/li\u003e\n\u003cli\u003eFiorini AC et al. (2021). Antiviral activity of Brazilian green propolis extract against SARS-CoV-2 (Severe acute respiratory syndrome-coronavirus 2) infection: Case report and review. Clinics 76:e2357. https://doi.org/10.6061/clinics/2021/e2357\u003c/li\u003e\n\u003cli\u003eGarc\u0026iacute;a-Ch\u0026aacute;vez D et al. (2008). Long-term evaluation of cytoarchitectonic characteristics of prefrontal cortex pyramidal neurons, following global cerebral ischemia and neuroprotective melatonin treatment, in rats. Neurosci letters 448(1):148\u0026ndash;152. https://doi.org/10.1016/j.neulet.2008.10.043\u003c/li\u003e\n\u003cli\u003eGodinho J et al. (2018). Ethyl-acetate fraction of Trichilia catigua restores long-term retrograde memory and reduces oxidative stress and inflammation after global cerebral ischemia in rats. Behav Brain Res 337:173\u0026ndash;182. https://doi.org/10.1016/j.bbr.2017.08.050\u003c/li\u003e\n\u003cli\u003eHorstmann A et al (2010). Resuscitating the heart but losing the brain: brain atrophy in the aftermath of cardiac arrest. Neurology 74(4):306\u0026ndash;312. https://doi.org/10.1212/WNL.0b013e3181cbcd6f\u003c/li\u003e\n\u003cli\u003eInagaki R et al. (2024). Preventive effect of propolis on cognitive decline in Alzheimer\u0026apos;s disease model mice. Neurobiol Aging 139:20\u0026ndash;29. https://doi.org/10.1016/j.neurobiolaging.2024.03.002\u003c/li\u003e\n\u003cli\u003eIto T et al. (2023). Brazilian green propolis prevents Alzheimer\u0026rsquo;s disease-like cognitive impairment induced by amyloid beta in mice. BMC Complement Med Ther 23:416. https://doi.org/10.1186/s12906-023-04247-7\u003c/li\u003e\n\u003cli\u003eKischkel B et al. (2020). Silver nanoparticles stabilized with propolis show reduced toxicity and potential activity against fungal infections. Futur Microbiol 15:521\u0026ndash;539. https://doi.org/10.2217/fmb-2019-0173\u003c/li\u003e\n\u003cli\u003eKumazawa S, Hamasaka T, Nakayama T (2004). Antioxidant activity of propolis of various geographic origins. Food Chem 84:329\u0026ndash;339. https://doi.org/10.1016/S0308-8146(03)00216-4\u003c/li\u003e\n\u003cli\u003eLi YJ et al. (2012). Therapeutic effects of propolis essential oil on anxiety of restraint-stressed mice. Hum Exp Toxicol 31:157\u0026ndash;165. https://doi.org/10.1177/0960327111412805\u003c/li\u003e\n\u003cli\u003eMarcucci MC (1996). Biological and therapeutic properties of chemical propolis constituents. Quim Nova 19:529\u0026ndash;536. https://doi.org/10.20396/chemkeys.v0i10.9640\u003c/li\u003e\n\u003cli\u003eMeccatti VM et al. (2023). Synergistic antibiofilm action of Cinnamomum verum and Brazilian green propolis hydroethanolic extracts against multidrug-resistant strains of Acinetobacter baumannii and Pseudomonas aeruginosa and their biocompatibility on human keratinocytes. Molecules 28:6904. https://doi.org/10.3390/molecules2819690\u003c/li\u003e\n\u003cli\u003eMori MA et al. (2017). Cannabidiol reduces neuroinflammation and promotes neuroplasticity and functional recovery after brain ischemia. Prog Neuro-Psychopharmacol Biol Psychiatry 75:94\u0026ndash;105. https://doi.org/10.1016/j.pnpbp.2016.11.005\u003c/li\u003e\n\u003cli\u003eMori MA et al (2021). Differential contribution of CB1, CB2, 5‐HT1A, and PPAR‐\u0026gamma; receptors to cannabidiol effects on ischemia‐induced emotional and cognitive impairments. Eur J Neurosci 53:1738\u0026ndash;1751. https://doi.org/10.1111/ejn.15134\u003c/li\u003e\n\u003cli\u003eNakajima Y et al. (2009). Neuroprotective effects of Brazilian green propolis and its main constituents against oxygen‐glucose deprivation stress, with a gene-expression analysis. Phytother Res 23:1431\u0026ndash;1438. https://doi.org/10.1002/ptr.2797\u003c/li\u003e\n\u003cli\u003ePereira RRA et al. (2013). Preparation and characterization of mucoadhesive thermoresponsive systems containing propolis for the treatment of vulvovaginal candidiasis. J Pharm Sci 102:1222\u0026ndash;1234. https://doi.org/10.1002/jps.23451\u003c/li\u003e\n\u003cli\u003ePitzer C, Kurpiers B, Eltokhi A (2022). Sex differences in depression-like behaviors in adult mice depend on endophenotype and strain. Front Behav Neurosci 16:1\u0026ndash;12. https://doi.org/10.3389/fnbeh.2022.838122\u003c/li\u003e\n\u003cli\u003ePrut L and Belzung C (2003). The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 463:3\u0026ndash;33. https://doi.org/10.1016/S0014-2999(03)01272-X\u003c/li\u003e\n\u003cli\u003eRebou\u0026ccedil;as-Silva J et al. (2023). Leishmanicidal and immunomodulatory properties of Brazilian green propolis extract (EPP-AF\u0026reg;) and a gel formulation in a pre-clinical model. Front Pharmacol 14:1013376. https://doi.org/10.3389/fphar.2023.1013376\u003c/li\u003e\n\u003cli\u003eReis JSS et al. (2014). Antidepressant-and anxiolytic-like activities of an oil extract of propolis in rats. Phytomedicine 21:1466\u0026ndash;1472. https://doi.org/10.1016/j.phymed.2014.06.001\u003c/li\u003e\n\u003cli\u003eRubab S et al. (2021). Enhanced neuroprotective and antidepressant activity of curcumin-loaded nanostructured lipid carriers in lipopolysaccharide-induced depression and anxiety rat model. Int J Pharm 603:120670. https://doi.org/10.1016/j.ijpharm.2021.120670\u003c/li\u003e\n\u003cli\u003eSawyer KN et al. (2020). Relationship between duration of targeted temperature management, ischemic interval, and good functional outcome from out-of-hospital cardiac arrest. Crit Care Med 48(3):370\u0026ndash;377. https://doi.org/10.1097/CCM.0000000000004160\u003c/li\u003e\n\u003cli\u003eSep\u0026uacute;lveda C et al. (2020). Antitumor activity of propolis: Recent advances in cellular perspectives, animal models and possible applications. Food Rev Int 36:429\u0026ndash;455. https://doi.org/10.1080/87559129.2019.1649692\u003c/li\u003e\n\u003cli\u003eShimazawa M et al. (2005). Neuroprotection by Brazilian green propolis against in vitro and in vivo ischemic neuronal damage. Evid Based Complement Altern Med 2:201\u0026ndash;207. https://doi.org/10.1093/ecam/neh078\u003c/li\u003e\n\u003cli\u003eSoares LM et al. (2016). Rolipram improves cognition, reduces anxiety- and despair-like behaviors and impacts hippocampal neuroplasticity after transient global cerebral ischemia. Neuroscience 326:69\u0026ndash;83. https://doi.org/10.1016/j.neuroscience.2016.03.062\u003c/li\u003e\n\u003cli\u003eSoares LM et al. (2013). Cognitive impairment and persistent anxiety-related responses following bilateral common carotid artery occlusion in mice. Behav Brain Res 249:28\u0026ndash;37. https://doi.org/10.1016/j.bbr.2013.04.010\u003c/li\u003e\n\u003cli\u003eWu Z et al. (2013). Brazilian green propolis suppresses the hypoxia‐induced neuroinflammatory responses by inhibiting NF‐\u0026kappa;B activation in microglia. Oxid Med Cell Longev 2013:906726. https://doi.org/10.1155/2013/906726\u003c/li\u003e\n\u003cli\u003eXu X et al. (2020). The chemical composition of Brazilian green propolis and its protective effects on mouse aortic endothelial cells against inflammatory injury. Molecules 25:4612. https://doi.org/10.3390/molecules25204612\u003c/li\u003e\n\u003cli\u003eZhang L et al. (2010). Escin attenuates cognitive deficits and hippocampal injury after transient global cerebral ischemia in mice via regulating certain inflammatory genes. Neurochem Int 57:119\u0026ndash;127. https://doi.org/10.1016/j.neuint.2010.05.001\u003c/li\u003e\n\u003cli\u003eZhu A et al. (2018). Brazilian green propolis prevents cognitive decline into mild cognitive impairment in elderly people living at high altitude. J Alzheimers Dis 63:551\u0026ndash;560. https://doi.org/10.3233/JAD-170630\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bilateral common carotid arteries occlusion, behavior, P188, Brazilian Green Propolis, neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-6513240/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6513240/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBrazilian green propolis (BGP) has notable anti-inflammatory and neuroprotective properties, making it a potential candidate for treating neurological conditions. However, its impact on the sequelae of cerebral ischemia remains insufficiently explored. This study aimed to evaluate the effects of the hydroalcoholic extract (HE) of BGP dispersed (HE-D) in Poloxamer 188 (P188) on adult C57BL/6 mice subjected to bilateral common carotid artery occlusion (BCCAO), a model of transient global cerebral ischemia. P188 was selected as the surfactant due to the low solubility of HE, which could compromise its pharmacokinetic profile, reducing bioavailability and potentially limiting its neuroprotective effects. Male C57BL/6J mice were subjected to BCCAO for 20 minutes. P188 or the HE-D at doses of 50, 100, and 150 mg/kg was administered orally once daily for seven consecutive days. The animals were assessed for locomotor activity, anxiety-related and antidepressant-like behaviors of the treatments. Following the behavioral evaluations, the brains were collected to investigate the impact of HE-D on neuronal death and neuroinflammation induced by BCCAO. HE-D (150 mg/Kg) decreased anxiety-like behavior induced by BCCAO in mice. Also, HE-D induced antidepressant-like effects in mice with BCCAO. HE-D mitigated hippocampal neuroinflammation induced by BCCAO, as seen by a decreased immunoreactivity to both inflammatory markers, Iba-1 and GFAP. These results suggest that HE-D may present beneficial effects on the consequences of BCCAO by decreasing hippocampal neuroinflammation.\u003c/p\u003e","manuscriptTitle":"Dispersion of Brazilian green propolis extract in poloxamer 188 improves depression-like behavior and neuroinflammation in mice subjected to transient global cerebral ischemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 16:18:29","doi":"10.21203/rs.3.rs-6513240/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-06-23T04:01:31+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-06-02T14:07:04+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-09T07:59:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Revista Brasileira de Farmacognosia","date":"2025-04-29T14:24:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-25T02:27:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Revista Brasileira de Farmacognosia","date":"2025-04-24T07:34:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"874ca7b2-258e-4a9b-9e5b-f6d5a5582ee8","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-08T16:00:45+00:00","versionOfRecord":{"articleIdentity":"rs-6513240","link":"https://doi.org/10.1007/s43450-025-00698-w","journal":{"identity":"revista-brasileira-de-farmacognosia","isVorOnly":false,"title":"Revista Brasileira de Farmacognosia"},"publishedOn":"2025-09-06 15:57:04","publishedOnDateReadable":"September 6th, 2025"},"versionCreatedAt":"2025-05-13 16:18:29","video":"","vorDoi":"10.1007/s43450-025-00698-w","vorDoiUrl":"https://doi.org/10.1007/s43450-025-00698-w","workflowStages":[]},"version":"v1","identity":"rs-6513240","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6513240","identity":"rs-6513240","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}