Histologic and inflammatory lamellar changes in horses with oligofructose-induced laminitis treated with a CXCR1/2 antagonist

Pesquisa Veterinária Brasileira

Soriani, Frederico M.¹  Alves, Geraldo Eleno S.¹  Faleiros, Rafael R.¹  Mendes, Heloisa M.F.¹  Lima, Leonardo R. de¹  Teixeira, Mauro M.¹  Souza, Danielle G. de¹ 

[1] Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

关键词:  Neutrophil;    chemokine;    CXC receptors;    horse;    laminitis;   

DOI  :  10.1590/S0100-736X2016000100002

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 摘 要 】

:With the hypothesis that blocking chemokine signaling can ameliorate acute laminitis, the aim was to evaluate the therapeutic effect of intravenous DF1681B, a selective antagonist for CXCR1 and CXCR2 (chemokine receptors), in an oligofructose equine laminitis model. To twelve mixed breed clinically healthy hoses with no previous history of hoof-related lameness was administered oligofructose (10g/kg given by nasogastric tube) and divided into two groups: treated (intravenous DF1681B at 30mg/kg 6, 12, 18, and 24h after oligofructose) and non-treated groups. Laminar biopsies were performed before and 12, 36, and 72h after administering oligofructose. Samples were stained with periodic acid-Schiff (PAS) and scored from 0 to 6 according to epidermal cell and basal membrane changes. The IL-1β, IL-6, and CXCL1 RNA expressions were determined by RT-PCR. Parametric and non-parametric tests were used to compare times within each group (P<0.05). The PAS grades and IL-1β and IL-6 RNA expression increased in the non-treated group, but remained constant in the treated horses. In conclusion, DF1681B therapy reduced laminar inflammation and epidermal deterioration in treated horses. CXCR1/2 blockage should be considered therapeutically for equine acute laminitis.Index Terms: Neutrophil; chemokine; CXC receptors; horse; laminitisResumo:A expressão de quimiocinas e a infiltração de leucócitos no tecido laminar são característicos de laminite aguda de equinos. O presente estudo avaliou o efeito terapêutico da administração intravenosa de DF1681B , um antagonista seletivo para CXCR1 e CXCR2 (receptores de quimiocinas), em um modelo de laminite equina por oligofrutose. Utilizaram-se doze cavalos sem raça definida, compreendendo quatro machos e oito fêmeas não gestantes, com idade (média ±SD) 7±3,5 anos, pesando 305±35kg e com uma pontuação média de condição corporal de 5±1/9. Os indivíduos elegíveis eram clinicamente saudáveis, sem história prévia de claudicação relacionados ao casco. Após administração de oligofrutose (10g/kg por sonda nasogástrica), os animais foram divididos em dois grupos: tratado (30mg/kg de DF1681B intravenosa, 6, 12, 18 e 24h após a oligofrutose) e não tratado, que recebeu placebo. Biópsias laminares foram realizadas antes e 12, 36 e 72h após a administração de oligofrutose. As amostras foram coradas com ácido periódico de Schiff (PAS) e classificadas de 0-6 de acordo com alterações nas células epidérmicas e na membrana basal. Também determinaram-se as expressões gênicas de IL-1β, CXCL1 e IL-6 por RT-PCR. Testes paramétricos e não paramétricos foram utilizados para comparar os momentos em cada grupo (P<0,05). Estatisticamente, os graus PAS e as expressões de IL-1β e IL-6 se elevaram após a indução no grupo não tratado, mas se mantiveram constantes nos cavalos tratados. Em conclusão, a terapia por DF1681B reduziu a inflamação laminar e a deterioração epidérmica em equinos submetidos ao modelo de intoxicação por oligofructose. O bloqueio de receptores CXCR1/2 deve ser considerado como uma opção terapêutica para prevenção da laminite aguda de equinos.Termos de Indexação: Neutrófilos; quimiocinas; receptores CXC; cavalo; laminiteIntroductionThe equine hoof is extremely resistant to the demanding external environment; however, the destruction of its microstructure by laminitis has catastrophic consequences for the welfare and even survival of the affected horse. The damaged dermal-epidermal junction destroys adherence between the hoof and distal phalanx, preventing weight bearing and locomotion. A strong correlation between microstructural damage and the severity of clinical signs was established (Pollitt 1996).Dermal-epidermal junction collapse is common in laminitis of numerous etiologies, but the initial event triggering the structural failure remains poorly understood (Katz & Bailey 2012). Multiple studies propose resident leukocyte activation in the lamella and leukocytic infiltration from the bloodstream, associated with systemic inflammatory response syndrome (SIRS), as probable mechanisms that initiate structural failure (Black et al. 2006, Hurley et al. 2006, Belknap et al. 2007, Loftus et al. 2007, Stewart et al. 2009, Faleiros et al. 2009b, Faleiros et al. 2011a). Increased pro-inflammatory cytokine expression (mainly IL-1β and IL-6), as well as CXC and CCR chemokines, during the developmental stage, indicates that these molecules may play an important role in leukocyte migration in the early events of laminitis (Belknap et al. 2007, Faleiros et al. 2009a, Leise et al. 2011, Faleiros et al. 2011b, Visser et al. 2011).CXCR1 and CXCR2 are G protein receptors and have a high affinity for chemokines that are glutamine-leucine-arginine positive (Mukaida 2003, Kim et al. 2011). These chemokines have been detected in several human and equine tissues affected by inflammatory conditions associated with severe neutrophil infiltration (Franchini et al. 2000, Giustizieri et al. 2001, Ainsworth et al. 2006, Woodman et al. 2006). Selective pharmacologic blockade of CXCR1/2 is a promising strategy for controlling inflammation and could potentially treat and prevent equine laminitis (Bizzarri et al. 2006, Coelho et al. 2008).DF1681B is a CXCR1/2 noncompetitive, allosteric antagonist and potentially inhibits a variety of chemokine-induced biological activities (Souza et al. 2004, Bizzarri et al. 2006, Gorio et al. 2007, Zarbock et al. 2008, Marsh et al. 2011). DF1681B reduced secondary spinal cord degeneration in a rat model of trauma, as well as reduced oligodendrocyte apoptosis and neutrophil infiltration of ED-2 positive cells. In addition, the recovery rate for hind limb movement was higher in treated animals (Gorio et al. 2007). In another study, DF1681B inhibited neutrophil adhesion to synovial endothelium, reducing neutrophilic recruitment into the synovial membrane, and alleviating tissue damage in a rat model of arthritis (Coelho et al. 2008). DF1681B was also shown to reduce ischemic and intestinal reperfusion injury in mice by decreasing vascular permeability and neutrophil influx (Souza et al. 2004).Despite recent evidence indicating leukocyte involvement in laminitis development (Black et al. 2006, Hurley et al. 2006, Belknap et al. 2007, Loftus et al. 2007, Stewart et al. 2009, Faleiros et al. 2009b, Faleiros et al. 2011a), and the therapeutic potential of DF1681B for neutrophil migration in rodents, this drug has not yet been studied in horses.The present study evaluated the effect of DF1681B, a CXCR1/2 antagonist, on histological lesions, and the IL-1β, IL-6, and CXCL1 expressions in equine lamellar tissue following oligofructose-induced laminitis.Materials and MethodsTwelve mixed breed horses, comprising four castrated males and eight non-pregnant females, aged (mean±SD) 7±3.5 years, weighing 305±35 kg, and with a mean body condition score of 5±1/9 were evaluated. Horses were considered clinically normal based on physical and hematological exams and had no previous history of hoof-related lameness. After a 2-week adaptation period of consuming only grass hay (Cynodum sp.), the horses were randomly divided into two groups: treated (n=6) and non-treated (n=6). This protocol was approved by the Ethics Committee on Animal Use and Experimentation of UFMG (CEUA /UFMG 225/2009).To induce laminitis, all horses were administered 10g/kg of oligofructose (Raftilose P95, Embrafarma Pharmaceutical Expertise, São Paulo/SP, Brazil) diluted in 3L of water through a nasogastric tube. At 6, 12, 18, and 24h after oligofructose administration, horses in the treated group received DF1681B (Dompé Pharma SpA Research Center, L'Aquila, Italy), intravenously, at 30mg/kg diluted in 500mL of sterile 0.9% saline. The solution was filtered through a 0.22 μm bacteriological filter (Biofil syringe filter, Jet Bio-Filtration Products Co., Guangzhou, China) before administration.Before (T0) and 12, 36, and 72h after oligofructose administration, biopsies were taken sequentially from the left pelvic (T0), right pelvic (T12), right thoracic (T36), and left thoracic (T72) hooves of each horse. The horses were sedated with 10% xylazine (1mg/kg intravenously), and perineural anesthesia (5mL of 2% lidocaine at each branch of the palmar or plantar nerve) was administered at the abaxial surface of the proximal sesamoid bones in each hoof. At each biopsy site, three unaligned holes were generated across the dorsal hoof wall. Using aseptic technique, an 8 mm drill mounted in an electric driller was used to remove the hoof wall until the white soft tissue preceding the lamella was reached. The drill bit was then replaced with a 6 mm diameter

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