Influência do enriquecimento ambiental sobre parâmetros nociceptivos e inflamatórios em um modelo de artrite em camundongos

Abstract

The present study aimed at investigating the effects of environmental enrichment (EE) on peripheral and central parameters in the mouse model of arthritis induced by CFA. Attempts have also been made to asses the effects of EE on the actions of the anti-inflammatory drug dexamethasone. The experimental protocols were approved by the Animal Ethics Committee of Pontificia Universidade Católica do Rio Grande do Sul (PUCRS; CEUA 9666). Ninety male C57BL/6-Junib specific-pathogen-free mice, one month-old at the beginning of experiments (20-25 g body weight) were divided into two main groups, maintained under standard conditions (SE) or exposed to EE protocols. The SE animals (n=45) were allocated in cages with a dimension of 19.5 cm × 36.5 cm × 16.0 cm. The mice of the EE groups (n=45) were kept in cages measuring 33.5 cm ×36.5 cm × 16.0 cm, being exposed to alternating stimuli every two days, i.e.: volunteer exercise wheel, cotton nests, PVC tunnels, nontoxic wooden toys and paper rolls. Seven days after the onset of EE, the arthritis model was induced by an intraplantar injection of complete Freund’s adjuvant (CFA), diluted in saline solution (0.9% NaCl), in a 1:1 ratio and a volume of 50 μL/paw (n=60; right paw). The animals in the control group (without arthritis; n=30) received the same volume of saline solution. The arthritis model was induced under anesthesia with a mixture of ketamine (50 mg/kg) and xylazine (5 mg/kg), intraperitoneally. Separate groups of animals that received CFA, kept in SE or EE, were treated with the glucocorticoid dexamethasone (0.5 mg/kg, subcutaneously – s.c.), every 48 h, from the first day of induction of arthritis (n=30). Therefore, six experimental groups with 15 animals/group were evaluated: SE control; SE CFA; SE CFA + dexamethasone; EE control; EE CFA and EE CFA + dexamethasone. The animals were analyzed in the acute (24, 48 and 72 h) and chronic (7, 14, 21 and 35 days) phases of arthritis, in experimental pain and inflammation paradigms. Mechanical allodynia and thermal sensitivity were measured using Von Frey filaments (0.4 g, applied 10 times) and in the hot plate apparatus, maintained at 50 oC, respectively. As an indicative of inflammation, paw edema was measured in a plethysmometer as the difference between the right and the left paw volume (in μL). Joint inflammation scores were also recorded, considering: 0, no evidence of hyperemia and/or inflammation; 1, hyperemia with little or no paw swelling; 2, swelling confined predominantly to the ankle region, with modest hyperemia; 3, increased paw swelling and hyperemia of the ankle and metatarsal regions, and 4, maximal paw swelling and hyperemia involving the ankle, metatarsal, and tarsal regions. At the end of the 35 days of evaluation, the animals were euthanized by inhalation of isoflurane. The serum, the paw tissue and the brain of the animals were collected. The serum was stored at -80 oC for further analysis of tumor necrosis factor (TNF) levels using the ELISA method. The plantar tissue and brains were fixed in 10% formaldehyde solution and processed for histochemical analysis, in four μm sections. The paw sections were stained with hematoxylin and eosin (HE) to assess the inflammatory infiltrate and the epidermis thickness. The brains were used to measure the activation of glial cells, with the use of IBA1 (prefrontal cortex) and GFAP (jagged girus), as markers for microglia and astrocyte activation, respectively. Brain-derived neurotrophic factor (BDNF) expression was evaluated in the hippocampus. The data were analyzed by ANOVA or Kruskal-Wallis tests, depending on the normal distribution. Values of P<0.05 were considered as indicative of significance. The results show that the EE intervention was not able to alter the mechanical allodynia or thermal hypersensitivity induced by CFA, compared with the SE group. Noteworthy, at 24 h after the application of CFA, the analgesic effects of dexamethasone were more pronounced in the EE group. Regarding inflammatory parameters, exposure to different environmental stimuli in the EE groups produced anti-inflammatory effects per se, as indicated by the evaluation of paw edema at 72 h, and inflammatory scores at 24 h and 72 h. The anti-inflammatory effects of dexamethasone were not altered by EE in either edema evaluation or arthritis scoring. Regarding the histological evaluation of the paw tissues, the EE protocols were able to reduce the thickness of the epidermis, with greater anti-inflammatory effects for dexamethasone, without altering the intensity of the inflammatory infiltrate. Serum TNF levels were not detectable in any of the experimental groups. Concerning the evaluation of cortical astrocyte activation, there were no significant effects for EE. However, the group exposed to EE presented a lower inhibitory effect for dexamethasone in this parameter. Interestingly, microglia activation was significantly lower in the EE groups, regardless of dexamethasone treatment. Finally, EE induced an increase of BDNF expression in the hippocampus of control and CFA groups, an effect that was reduced by dexamethasone treatment. Altogether, data of the present study demonstrate that EE was able to alter peripheral inflammatory parameters in the CFA mouse model, with changes in astrocyte activation and BDNF expression in the hippocampus. In addition, in some parameters, the EE modified dexamethasone responses. Our results clearly show that animal maintenance conditions and EE protocols can modify central and peripheral outcomes in inflammation models. These data are relevant for planning preclinical trials aimed at the development of analgesic and anti-inflammatory drugs.