FFA1 receptor modulation in experimental arthritis in male and female mice subjected to environmental enrichment

FFA1 receptor modulation in experimental arthritis in male and female mice subjected to environmental enrichment | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 19 December 2025 V1 Latest version Share on FFA1 receptor modulation in experimental arthritis in male and female mice subjected to environmental enrichment Authors : Marina Estrázulas , Gabriela W. Neculqueo , Ana B. S. Farias , and Maria Campos [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176614408.81867121/v1 136 views 73 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background and Purpose: Few animal studies consider sex dimorphism and the impact of environmental enrichment (EE) when assessing pharmacological strategies for inflammatory pain. The FFA1 receptor has demonstrated antinociceptive properties through enhanced neurogenesis on pain and inflammation. This study explored the interaction between EE and FFA1 receptor in a mouse model of inflammatory pain induced by complete Freund’s adjuvant (CFA) in both male and female mice. Experimental Approach: Inflammatory pain was induced in C57BL6/J mice through an intraplantar injection of CFA and were evaluated weekly with Von Frey filaments, hot-plate, and plethysmometer apparatuses, until 21 days. They received the FFA1 partial agonist GW9508 (2 mg kg -1 and 8 mg kg -1 ) or the selective antagonist GW1100 (4 mg kg -1 ) daily s.c., between days 7 and 14 after CFA, under different environmental conditions. Paw leukocyte migration was evaluated histologically. Cortical expression of brain-derived neurotrophic factor (BDNF) and FFA1 receptors were assessed by immunohistochemistry. Key Results: GW9508 reduced mechanical sensitivity at 8 mg kg -1 in males, and females with the 2 mg kg -1 dose. The thermal sensitivity had better outcomes with the lower dosage, with synergistic effects with the EE. Only females responded to the FFA1 modulation for the oedema assessments. GW9508 reduced the paw leukocyte migration in females, especially when reared in EE, while males had no differences between environments. GW9508 increased the cortical BDNF counts in females at 2 mg kg -1 , and at 8 mg kg -1 in males. Cortical FFA1 expression was decreased in the GW1100 groups irrespective of EE, with no effect for GW9508. In contrast, males showed increased expression in the EE groups, with a potentiated effect by the GW9508 8 mg kg -1 dosage. Conclusion and Implications: This study demonstrated the interaction between EE and FFA1 receptors, highlighting sex differences in central and peripheral responses to chronic inflammatory pain. FFA1 receptor modulation in experimental arthritis in male and female mice subjected to environmental enrichment Marina Estrázulas 1,2 , Gabriela W. Neculqueo 1,2 , Ana B. S. Farias 2,3 , Maria M. Campos 1,2 1 Programa de Pós-graduação em Medicina e Ciências da Saúde, Escola de Ciências da Saúde e da Vida, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil. 2 Grupo de Pesquisa em Farmacologia Aplicada: Caracterização de Mecanismos Envolvidos na Dor, Inflamação e Câncer, Escola de Ciências da Saúde e da Vida, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil. 3 Curso de Graduação em Psicologia, Escola de Ciências da Saúde e da Vida, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil. * Corresponding Author: Maria M. Campos, Escola de Ciências da Saúde e da Vida, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga, 6681, Partenon, Porto Alegre, RS 90619-900, Brazil. Tel: +55 51 33320 3677 Email address: [email protected] ; [email protected] ORCID 0000-0001-7738-9892 Abstract Background and Purpose: Few animal studies consider sex dimorphism and the impact of environmental enrichment (EE) when assessing pharmacological strategies for inflammatory pain. The FFA1 receptor has demonstrated antinociceptive properties through enhanced neurogenesis on pain and inflammation. This study explored the interaction between EE and FFA1 receptor in a mouse model of inflammatory pain induced by complete Freund’s adjuvant (CFA) in both male and female mice. Experimental Approach: Inflammatory pain was induced in C57BL6/J mice through an intraplantar injection of CFA and were evaluated weekly with Von Frey filaments, hot-plate, and plethysmometer apparatuses, until 21 days. They received the FFA1 partial agonist GW9508 (2 mg kg -1 and 8 mg kg -1 ) or the selective antagonist GW1100 (4 mg kg -1 ) daily s.c., between days 7 and 14 after CFA, under different environmental conditions. Paw leukocyte migration was evaluated histologically. Cortical expression of brain-derived neurotrophic factor (BDNF) and FFA1 receptors were assessed by immunohistochemistry. Key Results: GW9508 reduced mechanical sensitivity at 8 mg kg -1 in males, and females with the 2 mg kg -1 dose. The thermal sensitivity had better outcomes with the lower dosage, with synergistic effects with the EE. Only females responded to the FFA1 modulation for the oedema assessments. GW9508 reduced the paw leukocyte migration in females, especially when reared in EE, while males had no differences between environments. GW9508 increased the cortical BDNF counts in females at 2 mg kg -1 , and at 8 mg kg -1 in males. Cortical FFA1 expression was decreased in the GW1100 groups irrespective of EE, with no effect for GW9508. In contrast, males showed increased expression in the EE groups, with a potentiated effect by the GW9508 8 mg kg -1 dosage. Conclusion and Implications: This study demonstrated the interaction between EE and FFA1 receptors, highlighting sex differences in central and peripheral responses to chronic inflammatory pain. Keywords: FFA1 receptor, environmental enrichment, sex dimorphism, central BDNF, GW9508, GW1100 Bullet Point Summary: What is already known • There is insufficient translation from pre-clinical studies for new targets to treat chronic diseases. • Women are more affected and have different responses to pain medications for chronic conditions. • Environmental enrichment (EE) promotes beneficial physiological changes, such as neuromodulation through BDNF in central pain. • FFA1 receptors are valuable targets in pain and inflammation. What this study adds Males and females respond differently to the FFA1 modulation in pain and inflammation outcomes. Central FFA1 and BDNF act synergistically with EE in males. Clinical significance The environment and sex are key factors influencing the effects of FFA1 receptor modulation in inflammatory pain. Introduction There is ongoing discussion in pre-clinical research regarding how animal models can effectively replicate and provide insights into the complexities of human lifestyle. This debate highlights concerns about the limitations of rodent environments, which often do not allow these animals to behave naturally. Environmental enrichment (EE) is a multifactorial and multimodal paradigm, described as manipulating the animal’s macroenvironment with tools that ease the species’ behaviour. These modifications enable the rodents to explore and organise their cage, thereby reducing stress and enhancing their overall well-being. Moreover, these behavioural changes trigger morphological modifications, such as the modulation of brain-derived neurotrophic factor (BDNF) expression, which is responsible for the proliferation of progenitor cells in the hippocampus, increasing neuronal survival (Murakami et al., 2023; Atrooz et al., 2021). Kentner et al. (2021) showed that, despite the EE, these morphological changes are not phenotypically significant enough to mischaracterise a lineage or a study model. Numerous studies indicate that EE enhances resilience to neuropathic and inflammatory pain (Wang et al., 2019; Estrázulas et al., 2022; Falkowska et al., 2023). This topic is of significant interest, particularly given the scarcity of effective analgesic medications and the urgent need for new potential treatment targets, as the translation of findings from animal models to humans has been limited. The Free Fatty Acid Receptor 1 (FFA1) belongs to the superfamily of G protein-coupled receptors (GPCR) and is the target of medium and long-chain fatty acids. Studies have shown that it is related to a variety of painful and inflammatory conditions (Karki et al, 2015, Gong et al.,2021, Pyo et al, 2022), and the link between FFA1 and the neurogenesis in the hypothalamus, through the modulation of BDNF, can ameliorate pain sensitivity (Nakamoto et al, 2017, Sona et al, 2018, Engel et al, 2020). Chronic pain results from excessive neuronal stimulation, leading to central changes that lack protective or mending functions. This continuous activation of the central nervous system leads to increased sensitivity to pain, accompanied by sensory alterations such as hyperalgesia and allodynia, not only at the original injury site but also in other areas (Seifert 2021). Every year, 20% of the global population experiences chronic pain (Presto et al., 2023), with females significantly outnumbering males among patients, demonstrating a sex ratio of approximately 3:1 for arthritis (Choudhary et al., 2018). However, a review article examining research conducted over a decade (1996 to 2005) found that 79% of published studies focused solely on male rats and mice, while only 5% included both male and female rodents (Mogil and Bailey, 2010). The role of the FFA1 receptor in neuroinflammation is an important topic due to its implications in various contexts. This study aimed to evaluate the effects of pharmacologically modulating FFA1 receptors in a chronic arthritis model, comparing the outcomes in male and female mice under different environmental conditions. In addition to recognising the benefits of environmental enrichment (EE) on both peripheral and central pain and inflammation, the study also explored the correlation between EE and the activation of the FFA1 receptor in both sexes. Animals Animal models for assessing inflammatory pain are fundamental for the comprehension of the mechanisms behind diseases and the development of new therapies (Sadler et al, 2022). In this study, male and female C57BL/6J mice were used (N=200), with approximately 8 weeks and ±20 g at the beginning of the experiments, obtained from the Centre for Experimental Biological Models (PUCRS; CeMBE, Porto Alegre, Rio Grande do Sul, Brazil). All procedures followed the ARRIVE guidelines (NIH) and followed the Brazilian rules for animal procedures (Law 11.794, from October 9 th , 2008) and the Brazilian Guideline for the Care and Use of Animals for Scientific and Didactic Purposes (CONCEA, Brazil, 2014). The local Animal Ethics Committee evaluated and approved all the protocols (PUCRS/CEUA 11019). The experiments were performed in two separate cohorts of five animals per group, with a total of 10 animals per group. Animals were randomised firstly by a simple method to designate SE or EE, and then a block randomisation was made, determined by the number of controls and treatments. An analysis to measure the power of experiments was performed (G*Power 3.1.9.4 by Universität Kiel, Germany), considering a significance level of alpha 0.05; 2-tailed, revealing power values ranging from 75% to 95%. Animals were kept in microisolator cages (5 per cage), equipped with inlet/outlet air filters, under controlled temperature (22 ± 1˚C) and humidity (50% − 70%), and a light-dark cycle of 12 h (lights on at 7 AM, lights off at 7 PM). Cages were filled with autoclaved wood chip bedding and ad libitum pelleted food, and sterile water. During the experimental procedures, the laboratory temperature was kept at 22 ± 1 ◦ C, and animals were allowed to acclimatise to the room for one hour before the procedures. All experiments were conducted in the mornings to ensure the same behaviour. A total of 200 animals were used, 100 males and 100 females, which were distributed randomly in different experimental groups, according to treatment and housing conditions (10 animals/group), as it is shown in the experimental scheme depicted in Figure 1. During the behavioural assessments, the evaluator was only blind to the treatment, but because the animals were kept in their original cage, the macroenvironment factor could not be blinded. For the microscopic analysis, the evaluator was completely blind, and a second blinded evaluator was summoned to verify outlier results. Reagents Complete Freund’s Adjuvant (CFA) was obtained from Sigma Aldrich Chemical Company (St. Louis, MO, USA). The FFA1 partial agonist, GW9508, and the antagonist, GW1100, were obtained from Cayman Chemical Company (Michigan, USA). Environmental Enrichment Protocol Animals were divided into the Standard Environment (SE) and Enriched Environment (EE) major groups. Cage sizes were 19.5 cm x 36.5 cm x 16 cm for SE housing, and 33.5 cm x 36.5 cm x 16 cm for EE. In addition, animals kept in EE cages received a rotation of tools every two days: (1) cotton balls, for nesting; (2) wood blocks, for the chewing habit; (3) plastic tunnels, for the compartmentalization of the cage; (4) Lego® blocks, for colour novelty experience and interaction with different objects and textures; and (5) cardboard paper, for chewing and nesting, with two of them always being provided at the same time, and the rotation removed one of them for another of the list. Every two days, the previous tool was removed and replaced with a new one. Animals started the EE rotation 7 days before the arthritis induction. The decision to use the rotation is based on previous literature, which shows mice prefer novelty and complexity in their cage, instead of a constant complex cage (Bohn et al, 2023). Arthritis Model After one week of adaptation and the beginning of EE, the mice were anaesthetised with a mixture of ketamine (50 mg kg -1 ) and xylazine (5 mg kg -1 ) through intraperitoneal administration. The CFA, consisting of a solution containing heat-killed and dried 0,1% Mycobacterium tuberculosis , was injected, in a volume of 50 µl, in the right hindpaw of each animal, as described by Estrázulas et al (2022). Control groups received the same volume of 0,9% NaCl. Pain and inflammation tests were performed on days –1, 7, 14 and 21 after the arthritis induction. Assessments on day –1 were made to establish baseline behaviour and check for possible individual deviations, and on day 7 to evaluate the progression of pain and inflammation parameters before the treatment with the FFA1 modulators. The protocol timeline is depicted in Figure 1. Treatment with GW9508 and GW1100 The protocol consisted of the administration of the FFA1 modulators through the subcutaneous route, every 24 h, for 7 days, between days 7 and 14 post-CFA. The time of injection was selected based on previous literature (Estrázulas et al, 2022), consisting of the period between the acute and chronic phases of the arthritis model. The dosages were 8 mg kg -1 and 2 mg kg -1 for the GW9508, and 4 mg kg -1 for the GW1100, based on previous literature, as well as the chosen route of administration (Freitas et al, 2020). Mechanical Sensitivity Animals were placed individually on a Plexiglass (9 x 7 x 11 cm) platform (Evonik Performance Materials GmbH, Essen, Germany), comprising a 0.6 mm wire mesh, with an adaptation period of one hour before testing. Von Frey’s monofilaments of 0.4 g were applied 10 times perpendicularly to the plantar surface of the right hindpaw, with 2 to 5 s between applications. The frequency of withdrawal response was evaluated and expressed as a percentage, being considered a positive response when the animal removed the paw from the mesh. A significant increase in the response frequency, compared with the control groups, was considered indicative of mechanical sensitivity. The test was performed on days -1, 7, 14, and 21 after arthritis induction. Thermal Sensitivity The hot plate apparatus (Ugo Basile®, Model 7280, Varese, Italy) was used. The temperature was adjusted to 50 ± 1ºC, as described by Dagnino et al (2019). A positive response, such as paw elevation or paw retraction in the ipsi- or contralateral paw; licking, stomping, jumping, scratching, or leaning reactions was considered, and the time interval was evaluated. If there was no response after 20 seconds, the animal was removed from the apparatus to prevent tissue damage. Oedema Assessment The oedema was measured with a plethysmometer apparatus (Ugo Basile®) and expressed as the mean difference between the right and left hindpaws of each animal. After the gentle restriction of the animal, each paw was immersed three times in the liquid, and the volume displaced was measured in microliters (μl). Euthanasia and Sample Collection After 21 days, the animals were euthanised with an overdose of isoflurane (5%). A blood sample was collected through intracardiac puncture, centrifuged later at 1,500 rpm for 20 min, and the serum supernatant was stored in a -80ºC freezer for further analysis. The brain was removed and kept in a 10 % formaldehyde solution for immunohistochemistry analysis. A sample of the right hindpaw skin was stored in 10% formaldehyde for haematoxylin and eosin staining. Biochemical and Histochemical Assessments Twenty-four h after tissue fixation procedures, the samples were dehydrated and embedded in paraffin. Tissue sections of 4 μm were obtained, and the evaluation of the leukocyte infiltration in the papillary layer of the dermis was carried out using haematoxylin-eosin (HE)-stained slides. The brain slices were used for immunohistochemistry analysis to assess the expression of BDNF at a dilution of 1:500 (Boster Biological Technology, Pleasanton, California, Catalogue Number PB9075) and FFA1 receptor at a dilution of 1:100 (Cayman Chemicals, MI, Item No. 10007205). The analysis was conducted across the cortex, using a Zeiss Axio Imager M2 light microscope (Carl Zeiss, Gottingen, Germany) with a magnification of x400. Serum was used for Elisa analysis, according to the manufacturer (R&D Systems, Minneapolis, MN) of each cytokine: 1) mouse IL-17 DuoSet ELISA, Catalogue Number DY4221, Lot 1328852, detection range between 15.625 e 1000 pg mL -1 , and 2) mouse IFN-γ DuoSet ELISA, Catalogue Number DY485, Lot 1367142, detection range between 31.3 a 2000 pg mL -1 . The analysis was expressed in picograms/millilitre. Statistical Analysis Data was presented as mean ± SEM. The sample size was measured using the G* Power 3.1 software, based on a previous publication (Estrázulas et al, 2022). Protocols consisted of 10 animals per group, with a total of 20 experimental groups, with a side effect of 0,2: α = 0.05 and statistical strength 0.8. Brown-Forsythe and Bartlett’s tests were used for checking data normality. Kruskal–Wallis (nonparametric data) and one-way analysis of variance (ANOVA) (parametric data) were used for histochemical and biochemical data. Two-way ANOVA was used for data from experiments evaluating multiple time points (-1, 7, 14, and 21 days after CFA induction). Data from days -1 and 7 can be found in the supplementary material. When the interaction of factors was statistically significant (P < 0.05), pairwise comparisons were conducted using Tukey’s post hoc test after two-way ANOVA. All the tests were performed using GraphPad Software version 10.5.0 (GraphPad Software, Inc., San Diego, CA). Results After CFA injection, both females and males presented mechanical hypersensitivity, as indicated by an increase in the paw withdrawal frequency after applying the Von Frey filaments of 0.4 g. (Figure 2) The agonist, on the 8 mg kg -1 dosage, applied subcutaneously every 24 h, between days 7 and 14, did not affect mechanical hypersensitivity in females (F (5, 54) = 43.83) (Figure 2A), but displayed significant effects on males (F (5, 104) = 98.45) (Figure 2B). On day 21 after arthritis induction, the pain threshold was lowered between the groups SE CFA vs SE CFA + GW9508 (p = 0.0033) and EE CFA vs EE CFA + GW9508 (p = 0.0224). The 2 mg kg -1 dosage presented a difference in females (F (5, 53) = 55.17) at 14 days on the SE + GW9508 group (p = 0.0202), when compared to its control, and at 21 days, this analgesic effect became clearer in SE CFA vs SE CFA + GW9508 (p < 0.0001) and EE CFA vs EE CFA + GW9508 (p = 0.0008) groups (Figure 2C). Males (F 5, 96) = 39.86) had no significant effect with the treatment over time (Figure 2D). The FFA1 antagonist, GW1100, at a dosage of 4 mg kg -1 , also applied s.c. every 24 hours, between days 7 and 14 after arthritis induction. showed an increase in the sensitivity to the Von Frey filaments. Females (Figure 2E), on the 14 th day, in both SE and EE treated groups, showed an increase in hypersensitivity when compared to their respective CFA groups (F (5, 104) = 148.6; with p = 0.0071 and p < 0.0001, respectively). In males (Figure 2F), only the SE group showed an increase in the paw withdrawals on days 14 and 21(F (5, 104) = 126.6; p = 0.0117 and p = 0.0478, respectively). Supplementary Figure 1 shows baseline behaviour and 7 days of mechanical sensitivity after CFA (A and B = females and males with GW9508 mg kg -1 ; C and D = females and males with GW9508 mg kg -1 ) and E and F= females and males with GW1100 mg kg -1 ). The arthritis induction increased the thermal sensitivity of both female and male animals, after the CFA injection, according to assessment in the Hot Plate apparatus (Figure 3). The agonist GW9508, when tested at the higher dosage in females (Figure 3A), showed an increase in thermal tolerance on day 14, when comparing the groups EE CFA + GW9508 and SE CFA + GW9508 (F (5, 54) = 40.28; p = 0.0011). For males (Figure 3B), the treated group on EE conditions had an increase in thermal sensitivity at day 14, when compared to its positive control (F (5, 106) = 49.39; p = 0.0462), and the effect persisted on day 21 (p = 0.0086). The lower dosage showed an expressive improvement in females (Figure 3C) when comparing the SE CFA + GW9508 vs. its positive control, at 14 days (F (5, 54) = 36.29; p < 0.0001). At the same time point, the EE demonstrated higher thermal tolerance when comparing EE CFA vs SE CFA (p =0.0311). On day 21, the treatment with this dosage showed a protective effect on both experimental groups, when compared to their positive controls (SE CFA + GW9508 vs SE CFA (p < 0.0001); and EE CFA + GW9508 vs EE CFA (p < 0.0001). In males, the 2 mg kg -1 dosage (Figure 3D) produced thermal analgesia in the SE groups at 14 and 21 days (F (5, 96) = 36.65; p = 0.0109 and p = 0.0340, respectively). EE males also had a positive outcome at 14 and 21 days (p = 0.0128 and p < 0.0001). Besides, the EE showed a synergistic effect (p = 0.0094) when treated groups were compared at 21 days. Finally, the antagonist GW1100 increased the thermal sensitivity of females (Figure 3E), at days 14 and 21, in the EE group (F (5, 104) = 148.6; p = 0.0019 and p < 0.0001, respectively), and in the SE-treated group on day 21, when compared to its positive controls (p = 0.0071). Similarly, males (Figure 3F) on the 21st day showed a lowered pain threshold in the EE group (F (5,90) = 69.68; p = 0.0098). Additionally, a synergistic effect was observed when comparing the EE CFA + GW1100 and SE CFA + GW1100 groups (p = 0.0106). Supplementary Figure 2 shows baseline behaviour and 7 days thermal sensitivity after CFA (A and B = females and males with GW9508 8 mg kg -1 ; C and D = females and males with GW9508 mg kg -1 ) and E and F= females and males with GW1100 mg kg -1 ). When evaluating the oedema formation using a plethysmometer apparatus, the CFA injection led to an increased paw volume, as expected (Figure 3). The treatment with the FFA1 agonist, GW9508, at the 8 mg kg -1 dosage, did not show beneficial action on the oedema formation, neither in females (F (5, 54) = 74.71) nor males (F (5, 102) = 96.75) (Figure 4 A and B, respectively). On the lower dosage of 2 mg kg -1 , the agonist exhibited anti-inflammatory effects in EE females (Figure 4C), when compared to its inflamed control, on day 14 (F (5, 52) = 120.1; p = 0.0231). Furthermore, a synergistic effect of the agonist and EE can be seen, with an even more pronounced reduction in oedema when comparing the SE CFA + GW9508 and the EE CFA + GW9508 (p = 0.0030) groups. Males also displayed a synergistic effect between EE and SE treated groups with the 2 mg kg -1 dosage (F (5, 96) = 58.14; p = 0.0357) (Figure 4D). Finally, with the antagonist, GW1100, females in EE groups showed a marked increase in oedema on days 14 and 21 (F (5, 94) = 121.8; p = 0.0019 and p < 0.0001, respectively) (Figure 4E). Also, the SE-treated group had an increased response when compared to its positive control (p = 0.0071) on the 21st day. Interestingly, males did not demonstrate any changes in the oedema formation when the antagonist was tested (F (5, 90) = 95.07) (Figure 4F). Supplementary Figure 3 shows baseline results and 7 days of oedema formation after CFA (A and B = females and males with GW9508 8 mg kg -1 ; C and D = females and males with GW9508 2 mg kg -1 ) and E and F= females and males with GW1100 4 mg kg -1 ). The inflammatory infiltrate was evaluated through H&E staining by quantifying leukocyte migration on the granular layer of the epidermis, from the right hindpaw (Figure 5). Negative control groups SE and EE were used for calculating basal percentages. As expected, the CFA injections increased the leukocyte counts in both SE and EE groups (F (2.126, 19.14) = 85.26, p < 0.0001 on females; F (1.699, 15.29) = 43.84, p < 0.0001 on males). In the SE females, the agonist GW9508 decreased the cell migration with both agonist dosages, when compared to the CFA group (SE GW9508 8 mg kg -1 p = 0.00012; SE GW9508 2 mg kg -1 p = 0.006), but the antagonist GW1100 did not evoke any significant effect (p = 0.8119). The EE groups also had lower leukocyte counts when the agonist was administered at 8 mg kg -1 (p = 0.0043) or 2 mg kg -1 (p = 0.0002), while the antagonist had no effect (p = 0.4388). When comparing the environmental conditions, the EE GW9508 8 mg kg -1 and EE GW1100 groups had higher cell migration vs. the SE equivalent groups (p = 0.0005 and p = 0.0019, respectively). Males had a decrease in the leukocyte migration in the SE GW9508 8 mg kg -1 group (F (1.699, 15.29) = 43.84; p = 0.0052) and an increase in the GW1100 group (p =0.0315) when compared to the SE arthritis group. The EE GW9508 8 mg kg -1 showed a small improvement in cell count (p = 0.0438), while the other groups had no response to the treatments when compared to the EE CFA group (EE CFA + GW9508 2 mg kg -1 , p = 0.9804; EE CFA + GW1100, p = 0.3201). To gain further insights into systemic inflammation in our experimental paradigm, the proinflammatory cytokines IL-17 and IFN-γ were measured in the serum of animals, but no detectable levels were observed in any experimental group (Supplementary material). The BDNF positive cell counts were evaluated through immunohistochemistry labelling, focusing on the prefrontal cortex, as it is shown in Figure 6 A (females) and B (males). Negative and positive control females had no significant difference between groups in neither SE nor EE paradigm (F (1.343, 6.713) = 53.08, p > 0.05), but the FFA1 modulation shows improvement with the agonist, in both dosages in the SE protocol (GW9508 8 mg kg -1 p = 0.002, and GW9508 2 mg kg -1 p = 0.0056), while EE only showed a small improvement with the GW9508 2 mg kg -1 p = 0.047). In contrast, GW1100 had a significant response in both environments (SE control vs SE GW1100 p= 0.0001; SE CFA vs SE GW1100 p= 0.0098; EE control vs EE GW1100 p= 0.97; EE CFA vs EE GW1100 p= 0.0127). Males displayed a similar response (F (1.456, 7.28) = 44.98, p< 0.0001) with a difference count between negative control groups and CFA group only in the SE groups (p = 0.0012), and improved count between environments without the FFA1 modulation (p = 0.0003). GW9508 (8 mg kg -1 ) increased the BDNF immunopositivity when compared to the SE control (p= 0.0263) and the CFA group (p = 0.0136), while EE only had a significant response against the CFA group (EE negative control p = 0.9148 and EE CFA p< 0.0348). The 2 mg kg -1 dosage had a slight increase in the SE group, when compared to the arthritis group (p= 0,0427), and in the EE group, compared to the negative control (p= 0.0008) and EE CFA (p= 0.0017). There was also an improvement when comparing the different environments with the GW9508 2 mg kg -1 dosage (p=0.0102). The antagonist had a response only on the EE protocol (SE control and SE CFA p > 0.05; EE control vs EE GW1100 p= 0.0334; EE CFA vs EE GW1100 p= 0.0065). It can also be seen a synergistic effect between the antagonist and the enriched environment (SE GW1100 vs EE GW1100 p = 0.0065). Lastly, the FFA1-positive cell counts were also evaluated through immunohistochemistry labelling on the cortex. Females (Figure 7C) had no differences between groups in any environment protocol when using the FFA1 agonist GW9508 (p > 0.05). Interestingly, the antagonist GW1100 showed reduced count in both SE and EE groups, when compared with their respective negative (SE p = 0.0012, EE p = 0.0392) and positive (SE p = 0.0276, EE, p = 0.0098) control groups. Males (Figure 7D) reared in standard conditions showed a significant increase with the 8 mg kg -1 agonist GW9508 (F (1.891, 9.456) = 19.76) when compared to the SE control group (p = 0.0220)0, and the CFA group (p = 0.0301), while no response was observed with the lower dosage (2 mg kg -1 ) with p > 0,05. In the enriched environment, the agonist showed no increase in FFA1 counts when compared to the control and CFA groups (for GW9508 8 mg kg -1 p 0.05 in both comparisons). The antagonist GW1100 depicted a decrease in FFA1 labelling in the EE group compared to the negative control and the CFA groups (p= 0.0262 and p= 0.0047, respectively). Interestingly, the EE by itself showed a significant increase in the FFA1 levels, when analysing SE control vs EE control (p= 0.0073), SE CFA vs EE CFA (p= 0.0008), and SE GW9508 8 mg kg -1 vs EE GW9508 8 mg kg -1 (p = 0.0055) and SE GW1100 4 mg kg -1 vs EE GW1100 4 mg kg -1 (p = 0.0021). Discussion The present study explored for the first time whether the pharmacological modulation of FFA1 receptors could affect the painful and inflammatory conditions in CFA-injected mice, considering the influence of sex and environmental factors. We sought to investigate the complex interactions between environmental conditions, sex, and FFA1 receptor activation, specifically looking at their effects on central BDNF levels and FFA1 receptor expression across the different experimental conditions. Scientific research has focused on environmental enrichment (EE) to enhance animal well-being, simulate a complex lifestyle, and provide insights into how the environment influences drug effects (Rojas-Carjaval et al., 2022). In this study, we chose not to use a running wheel as a form of enrichment to differentiate the effects of EE from those of physical activity. However, research indicates that EE can still increase physical activity levels without a running wheel, even when the enrichment includes minimal elements or is of short duration (Mo et al., 2016). Evidence suggests that EE, through physical activity, social interactions, and sensory stimulation, triggers various neurobehavioral effects that induce genetic changes and improve the immune system. Brod et al. (2017) found that mice living in EE conditions exhibited a higher percentage of neutrophils and lower levels of lymphocytes in their peripheral blood. It is well-documented that CFA increases mechanical and thermal hyperalgesia during the first week post-injection in both male and female mice (Chillingworth and Donaldson, 2003). Interestingly, the 8 mg kg -1 dosage of the GW9508 only improved mechanical hypersensitivity in male mice, while female mice exhibited a more significant analgesic effect with the lower dosage (2 mg kg -1 ). Patterns of sex dimorphism are becoming evident in chronic models, and this may be one such instance. Female response appears to be predominantly mediated by cytokines originating from T cells and macrophage mobilisation, whereas male physiology relies more on the microglia system (Smith, 2024). Moreover, GW9508 is a dual agonist that activates FFA1 and FFA4 receptors. Despite having a higher affinity for the FFA1, this duality can justify why females respond to a lower dosage. FFA4 is highly expressed in macrophages, being involved in anti-inflammatory outcomes, through calcium mobilisation and promoting a pro-resolution inflammatory state (Hidalgo et al, 2021). Another possible explanation for the better effects with the lower dosage is the sex hormone oestradiol. Females have higher oestrogen concentrations than males, with abundant oestrogen receptors (ER) in several immune cells, where studies demonstrate that the ER signalling decreases inflammation, in part, affecting neutrophils directly (Hoffman et al, 2023). Also, ER reduce the p65 activity, preventing the inflammatory cascade of the NF-kB pathway (Ghisletti et al, 2005). In microglia, ER signalling promotes neuroprotective anti-inflammatory responses on CNS injury (Johann and Beyer, 2013). Rabadán et al (2019) observed that mice kept in larger cages, with 4 or 8 littermates, presented a lower thermal threshold than animals kept in regular cages provided only with the exercise wheel, with higher locomotor activity. Herein, the antagonist GW1100 increased mechanical pain in both SE and EE female groups, on day 14, after receiving the 7 days treatment, although this hypersensitivity was not maintained on day 21. Male in the SE GW1100-treated groups showed lower tolerance to the Von Frey filaments in a continuous form, as it can be seen in days 14 and 21. Interestingly, EE prevented this effect in males. It is well known that EE improves neuropathic pain resilience, through increased central BDNF and microglia, shifting to an anti-inflammatory state (Estrázulas et al, 2022; Cutuli et al, 2022). Some studies suggest that females are more resistant to external factors, such as EE or drugs that act on GPCR. One study demonstrated that female kept in short-term EE had no response on neonatal stress, when compared to male responses (Ji et al., 2022), while a review pointed that female rats are also resistant to stress-induced analgesia (SIA), and female mice had fewer analgesic effects when compared to males to drugs that act on GPCRs (Wiesenfeld-Hallin, 2005). Interestingly, the thermal pain response on animals receiving GW1100 demonstrate a synergistic effect of EE and the FFA1 antagonist response, in both females and males, on the 21st day. Males might display increased anxiety due to more interactions with the objects and larger cages, considering there is no consensus between EE studies in regards of EE improving anxiety and depression in neuropathic pain induced in mice. Females, as mentioned above (Ji et al., 2022), require more time under EE to show positive effects in pain. In a study with CCI mice model, EE alleviated both mechanical and thermal pain (Medeiros et al, 2025). However, it is important to point that the EE was introduced 21 days prior to the CCI injury and kept for more 21 days. The positive effects can be due to the longer exposure, either prior to the induction of pain and for the total time of the experiment. Regarding inflammation, the oedema persisted on CFA-injected animals throughout the 21 days, with minimal responses to the partial agonist in males and a slight response in females kept in EE with the 2 mg kg -1 dosage. In a rat model of arthritis with CIA, paw volume was higher in ovariectomized rats when compared to castrated rats, indicating that oestrogen is responsible for the joint protection, which was not seen with testosterone or progesterone (Ganesan et al, 2008). As for cell influx, the antagonist GW1100 significantly increased the percentage of leukocyte infiltration in the paw tissue, in both males and females, in the SE or EE paradigm. A possible explanation for the increase in the inflammatory infiltrate, as well as the differences in thermal hypersensitivity and oedema formation with the FFA1 antagonist, is the modulation of serotonin (5-HT). Wan et al (2020) points out that serotonin plays a role in the innate and adaptive immune responses, where the deficit of 5-HT leads to the imbalance of Th and Treg, exacerbating the progression of arthritis in a CIA mice model. If GW1100 blocks the serotonin pathway, animals have increased anxiety, justifying the increased thermal hypersensitivity and oedema. In fact, another EE study showed that mice reared in groups of 8 individuals had higher sensitivity to the hot plate apparatus (Rabadán et al, 2019). These results suggest that social interaction and physical activity from EE can influence mechanical and thermal pain in a sex-dependent manner. IL-17 and IFN-γ were undetectable in serum, unlike studies using paw homogenates (Sarkar et al., 2009; El-Tanbouly & Abdelrahman, 2022) or the CIA model, where Tang et al. (2021) detected both in serum and paw homogenate. This can be due to model differences and sample collection timing – their samples were collected on day 42 post-arthritis. The central BDNF is known for modulating synaptic plasticity in an activity-dependent manner, affecting NMDA receptor expression, dendritic spine density, and neurogenesis. Also, a review shows that EE distinctly modulates the expression of BDNF transcripts (Cutuli et al, 2022), increasing the hippocampus BDNF in most of the studies evaluated, independent on the complexity of the EE. One hypothesis is that EE can preserve the noradrenaline signalling, activating the β 2 -adrenoreceptors, shifting the microglia activation towards an anti-inflammatory state (Qian et al, 2011). In fact, a study with BDNF blockage proved that BDNF has a neuroprotective role, especially before the onset of a mouse model of autoimmune encephalomyelitis (Lee et al, 2012). This modulation is evident in the present study, especially in males, where not only does the FFA1 agonist increase the BDNF-positive cells, but it also has a synergistic effect with the EE. Females rely more on using the adaptive immune system to respond to peripheral injury, recruiting macrophages and T-lymphocytes, not depending on the BDNF (Smith, 2024), and this can explain why the same synergistic effect of FFA1 modulation and EE is not seen in the females’ BDNF immunolabeling of this study. Regarding the response to FFA1 modulators, research indicated that FFA1 is directly associated with the phosphorylation of CREB, which serves as a precursor for neurotrophic factors such as BDNF. A study demonstrated that the increase in BDNF levels with GW9508 is dose-dependent in a mouse model of Alzheimer’s disease (Khan et al., 2016). The superfamily of G protein-coupled receptors (GPCRs) consists of receptors that are closely linked to pain, influencing both sensitisation and alleviation depending on the ligand and its effects. The most significant actions of these receptors include the formation of cyclic adenosine monophosphate (cAMP) and the activation of phospholipase C, responsible for producing inositol trisphosphate (IP3), diacylglycerol (DAG), and modulating several ion channels (Hermann et al., 2024). FFA1 couples all four isoforms: G s , G i/o , G q/11 , and G 12/13 and recruits β-arrestin upon activation (Grundmann et al, 2021). This highlights its connection to inflammatory processes. In our study, the agonist GW9508 (8 mg kg -1 ) determined an increase in BDNF-positively labelled cells in the cortex of male mice in both SE and EE groups. The EE showed a marked response in all BDNF immunopositivity groups, even in the negative control group, compared to the SE groups. Additionally, FFA1 immunolabeling also demonstrated increased counts in all EE male groups, with significant effects with the 8 mg kg -1 dosage, while the SE group showed a less expressive increment in FFA1. Hence, it is tempting to suggest that the FFA1 agonism act synergistically with BDNF via microRNA expression. EE increases brain VEGF and BDNF levels through augmented norepinephrine release and β1/2 receptors availability, and shifts the microglia profile to the M2 type, amplifying TrkB activity and IP3, MAPK/ERK, and PI3/AKT pathways. This leads to CREB and gene modulation, producing more BDNF and GPCRs. Likewise, the FFA1 agonist modulation improves IP3 and DAG pathways, also leading to gene expression (Figure 7). As mentioned before, females mobilise macrophages originating from T cells (Smith, 2024), where these macrophages increase the expression of COX-2, an enzyme known to potentiate pain and inflammation in chronic diseases, such as rheumatoid arthritis (Sakurai et al, 2019). A study showed the differences in mechanical pain on a CFA mouse model, with sex differences and route of administration. Females had an analgesic effect for 11 days, while males kept it until 40 days, when the COX-2 inhibitor drug was administered through nanotheranostic agents, while the same drug, freely administered, had no effect (Liu et al, 2020). Once again, considering that the agonist GW9508 also activates FFA4, the s.c. route can be responsible for the low response seen in both SE and EE female groups, contrasting with the response to the selective antagonist GW1100. In conclusion, this study provides evidence that the environment of preclinical research plays a crucial role in physiological outcomes. Although there are no standardised protocols for EE across studies, it is consistently associated with beneficial effects, offering complex settings in which animals can adapt to challenges, reducing stress-related bias, and leading one step closer to the human lifestyle condition. Here, we demonstrated the benefits of EE with the modulation of the FFA1 receptors, which are possible targets for pain and inflammation therapies. The summary of the results can be seen in Figure 8. While some hypotheses presented here require direct experimental validation, this study offers a broader perspective on the critical role that both sex and environmental factors play in preclinical drug testing, besides reinforcing the pivotal role of FFA1 receptors for inflammatory pain, via central and peripheral mechanisms. Data Availability Statement Raw data is available upon request. Funding Statement This study was supported by Coordenação de Aperfeiçoamento de Nível Superior (CAPES; Financial Code 001) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). MMC is a research career awardee of CNPq (304205/2022-2). The sponsor had no role in the design or conduct of this research. GWN and ME received PhD scholarships from CAPES. ME also received Institutional Support. ABSF is an undergraduate student in Psychology receiving institutional and CNPq support (PUCRSBIC and PIBIC programs, respectively). Conflict of Interest Statement The authors declare that they have no conflicts of interest. Statement for generative artificial intelligence assistance During the preparation of this work, the authors employed Grammarly (v1.2.169.1689) to enhance the clarity and readability of the content. Following the use of this tool, the authors conducted a thorough review and made necessary revisions to ensure the quality of the final document. 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Schematic representation of group divisions, depicting timeline, treatment period, nociceptive and inflammatory tests and samples collected at the end of the experiments. Each cohort had arthritis and treatments randomized throughout the groups. SE = standard environment; EE = enriched environment. Figure 2 . Mechanical hypersensitivity to Von Frey filaments, with 10 0,4 g applications, with an interval of 5-10 seconds between tries. The percentage was calculated according to the number of times the animal showed discomfort in the right hindpaw. Females (A) and males (B) treated with the agonist, GW9508, 8 mg kg -1 ; Females (C) and males (D) treated with GW9508, 2 mg kg -1 ; and females (E) and males (F) treated with GW1100, 4 mg kg -1 . N= 10/group. * indicates significant statistics (p < 0,05) in comparison to the same environment Control group; # indicates significant difference between treatment and the same environmental CFA group. SE= standard environment; EE= enriched environment. Figure 3. Thermal hypersensitivity to the hot plate apparatus, set to 50 ± 1ºC. Time, counted in seconds, during where animals showed discomfort behaviour. Females (A) and males (B) treated with the agonist, GW9508, 8 mg kg -1 ; Females (C) and males (D) treated with GW9508 2 mg kg -1 ; and females (E) and males (F) treated with GW1100, 4 mg kg -1 . N= 10/group. * indicates significant statistics (p < 0,05) in comparison to the same environment Control group; # indicates significant statistics between treatment and CFA of the same environment; ● indicates differences between SE and EE CFA groups; ▲ indicates significant differences between SE and EE treatments. SE= standard environment; EE= enriched environment Figure 4. Oedema formation, measured with the plethysmometer apparatus. Volume is depicted in µl. Females (A) and males (B) treated with the agonist, GW9508, 8 mg kg -1 ; Females (C) and males (D) treated with GW9508, 2 mg kg -1 ; and females (E) and males (F) treated with GW1100, 4 mg kg -1 . N= 10/group. * indicates significant statistics (p < 0,05) in comparison to the same environment Control group; # indicates significant statistics between treatment and CFA of the same environment; ▲ indicates significant differences between SE and EE treatments. SE= standard environment; EE= enriched environment. Figure 5. Inflammatory Count (%) of the quantified leukocyte migration on the granular layer of the epidermis, from the right hindpaw. SE and EE control groups means were calculated and considered as 100%, for analysis purposes. N = 10/group * indicates significant statistics (p < 0,05). SE= standard environment; EE= enriched environment. Figure 6. Positive BDNF (A and B) and FFA1 (C and D) label count through immunohistochemistry staining in female (A and C) and male (B and D) brain cortex. N = 6/group. * indicates significant statistics (p < 0,05). SE= standard environment; EE= enriched environment. Figure 7. Environmental enrichment (EE) enhances BDNF expression in male mice, which in turn activates the PI3K/AKT and MAPK/ERK signalling pathways via TrkB receptors, ultimately leading to CREB activation. TrkB also stimulates the IP3 pathway, promoting calcium influx, which further reinforces CREB activation, modulates microRNA expression, and supports BDNF bioavailability. In parallel, activation of FFA1 receptors increases the production of DAG and IP3, also contributing to calcium influx. Together, BDNF and FFA1 are proposed to act synergistically in response to EE. Solid arrows indicate direct pathways; dashed arrows represent indirect pathways. Figure 8. Summary of the effects of the FFA1 modulation in the outcomes of pain (mechanical and thermal hypersensitivity), Inflammation (oedema and leukocyte infiltration), and the Cortical BDNF and FFA1 receptors, according to the environment and the differences between females and males. SE = Standard environment; EE = Enriched environment. ↓ indicates lower levels when compared to the standard CFA groups; ↑ indicates increased levels when compared to the standard CFA group; = means no difference to the environmental effects. GW9508 8 mg/kg GW9508 2 mg/kg GW1100 4 mg/kg Female SE = ↓ ↑ EE = ↓ ↑ Male SE ↓ ↓ ↑ EE ↓ = = Thermal Hypersensitivity GW9508 8 mg/kg GW9508 2 mg/kg GW1100 4 mg/kg Female SE = ↓ = EE = ↓ ↑ Male SE = ↓ = EE ↓ ↓ ↑ Paw Oedema GW9508 8 mg/kg GW9508 2 mg/kg GW1100 4 mg/kg Female SE = = ↑ EE = ↓ (trend) ↑ Male SE = = = EE = ↑ (trend) = Leukocyte Infiltrate GW9508 8 mg/kg GW9508 2 mg/kg GW1100 4 mg/kg Female SE ↓ ↓ = EE ↓ ↓ ↓ Male SE ↓ = ↑ EE ↓ = = Cortical BDNF GW9508 8 mg/kg GW9508 2 mg/kg GW1100 4 mg/kg Female SE ↑ ↑ ↓ EE = ↑ ↓ Male SE ↑ = = EE ↑ ↑ ↓ Cortical FFA1 GW9508 8 mg/kg GW9508 2 mg/kg GW1100 4 mg/kg Female SE = = ↓ EE = = ↓ Male SE ↑ = = EE = = ↓ Information & Authors Information Version history V1 Version 1 19 December 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Marina Estrázulas Pontificia Universidade Catolica do Rio Grande do Sul View all articles by this author Gabriela W. Neculqueo Pontificia Universidade Catolica do Rio Grande do Sul View all articles by this author Ana B. S. Farias Caracterização de Mecanismos Envolvidos na Dor View all articles by this author Maria Campos [email protected] Pontificia Universidade Catolica do Rio Grande do Sul View all articles by this author Metrics & Citations Metrics Article Usage 136 views 73 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Marina Estrázulas, Gabriela W. Neculqueo, Ana B. S. Farias, et al. FFA1 receptor modulation in experimental arthritis in male and female mice subjected to environmental enrichment. Authorea . 19 December 2025. DOI: https://doi.org/10.22541/au.176614408.81867121/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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