Imunomodulação da ciclofosfamida na reação inflamatória em tilapias do Nilo (Oreochromis niloticus)
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Data
2017-12-09
Autores
Charlie-Silva, Ives [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O presente estudo investigou os efeitos da ciclofosfamida (CYP) na modulação de proteínas de fase aguda (APPs) e acúmulo celular em exsudatos presentes na bexiga natatória de tilápias (modelo de peixe de aerocistite) e o recrutamento de leucócitos na lamínula de vidro implantadas no tecido subcutâneo. Foram utilizadas 140 tilápias (Oreochromis niloticus) (± 150 g) distribuídas em 14 aquários com capacidade para 250 L de água (n = 10). Os animais foram distribuídos em dois ensaios: Experimento I: foram utilizados 80 animais distribuídos em quatro tratamentos: T1 - controle NAIVE; T2 - controle negativo + solução salina injetada NaCl; T3- controle positivo + injetado com Aeromonas hydrophila; T4- ciclofosfamida, Isopac ® Sigma (200 mg / kg, dose única, por via intraperitoneal) + injetado com A. hydrophila. As amostras de exsudato e sangue foram coletadas 6 e 24 horas após a infecção (HPI) para a contagem de células e determinação do fracionamento eletroforético das APPs por SDS-PAGE, digestão de proteínas em gel e identificação por espectrometria de massa. Experimento II: os peixes foram alocados (n=60) em 3 grupos experimentais: G1 - controle NAIVE; G2 – controle positivo + implante de lamínula; G3 - ciclofosfamida, Isopac ® Sigma (200 mg / kg, dose única, por via intraperitoneal) + implante de lamínula. Após serem anestesiados, foram submetidos ao implante da lamínula de vidro de 10mm de diâmetro no tecido subcutâneo. As amostras de sangue foram coletadas e as lamínulas foram cuidadosamente retiradas e lavadas com solução salina 0,9% após 74 e 144 horas. As amostras foram fixadas em solução de Bouin e coradas em hematoxilina-eosina. Em todas as lamínulas foram quantificados o total de macrófagos, células gigantes tipo corpo estranho e Langhans. Na evolução da aerocistite, a tilápia tratada com CYP apresentou diminuição nos valores circulantes de ceruloplasmina, complemento C3, α2macroglobulina e haptoglobina. A avaliação do exsudato revelou diminuição (p < 0,05) do número total de células acumuladas em peixes tratados com CYP quando comparada à resposta observada em peixes de controle positivo (T3). Em relação à contagem total de macrófagos e células gigantes no implante, observou-se aumento no acúmulo de macrófagos e formação de células gigantes ao longo do tempo (144 HPI). Ao mesmo tempo, houve diferença significativa (p < 0,05) nas contagens totais entre os grupos tratados CYP e controle negativo. Da mesma forma em mamíferos, vários estudos relataram os efeitos supressivos associados ao tratamento com este agente alquilante. O uso de 200 mg/kg de ciclofosfamida em O. niloticus, inibe a resposta inflamatória e induz leucopenia e trombocitopenia em processos inflamatórios agudos e crônicos.
The present study investigated the effects of cyclophosphamide (CYP) on the modulation of acute phase proteins (APPs) and cellular accumulation in exudates present in the swim bladder of tilapia (aerocystitis fish model) and the recruitment of leukocytes on a glass cover slip implanted in the subcutaneous tissue. One hundred forty tilapia (Oreochromis niloticus) (± 150 g) were distributed in 14 aquariums with capacity of 250 L (n = 10), for two trials. Experiment I: For this purpose, 80 fish were distributed in four treatments: T1 – naïve control; T2 - negative control + injected with NaCl solution; T3- positive control + injected with A. hydrophila; and T4- cyclophosphamide (200 mg/kg) + injected with A. hydrophila. Samples of exudate and blood were collected 6 and 24 hours post-inoculation (HPI) for cell counts and determination of the APP electrophoretic fractionation by SDS-PAGE, in-gel protein digestion and mass spectrometric identification. Experiment II: Fish were allocated (n = 60) in 3 experimental groups: G1 – naive control; G2 - positive control + coverlet implant; and G3 - cyclophosphamide (200 mg/kg, single dose intraperitoneally) + cover slip implant. The glass coverslips (10 mm diameter) were implanted after anesthetization in the subcutaneous tissue. Blood samples were collected and the coverslips were carefully removed and washed with 0.9% saline 74 and 144 hours implantation. Then they were fixed in Bouin's solution and stained with hematoxylineosin. The totals of macrophages, foreign body type giant cells and Langhans cells on all coverslips were quantified. With respect to aerocystitis evolution, tilapia treated with CYP presented decrease in the circulating values of ceruloplasmin, complement C3, alpha2 macroglobulin and haptoglobin. The exudate evaluation revealed decrease (p <0.05) of the total number of cells accumulated in fish treated with CYP when compared the response observed in positive control fish (T3). The total count of macrophages and giant cells after implantation increased with time until 144 HPI. At the same time, there was a significant difference in the total counts between the CYP and negative control groups. Like for mammals, several studies have reported the suppressive effects associated with treatment with this alkylating agent in fish. The treatment with 200 mg of cyclophosphamide/kg of body weight in O. niloticus modulated acute phase protein responses and cell accumulation at the inflamed site, demonstrating the potential of this experimental model for pathophysiological studies during acute and chronic inflammatory reaction.
The present study investigated the effects of cyclophosphamide (CYP) on the modulation of acute phase proteins (APPs) and cellular accumulation in exudates present in the swim bladder of tilapia (aerocystitis fish model) and the recruitment of leukocytes on a glass cover slip implanted in the subcutaneous tissue. One hundred forty tilapia (Oreochromis niloticus) (± 150 g) were distributed in 14 aquariums with capacity of 250 L (n = 10), for two trials. Experiment I: For this purpose, 80 fish were distributed in four treatments: T1 – naïve control; T2 - negative control + injected with NaCl solution; T3- positive control + injected with A. hydrophila; and T4- cyclophosphamide (200 mg/kg) + injected with A. hydrophila. Samples of exudate and blood were collected 6 and 24 hours post-inoculation (HPI) for cell counts and determination of the APP electrophoretic fractionation by SDS-PAGE, in-gel protein digestion and mass spectrometric identification. Experiment II: Fish were allocated (n = 60) in 3 experimental groups: G1 – naive control; G2 - positive control + coverlet implant; and G3 - cyclophosphamide (200 mg/kg, single dose intraperitoneally) + cover slip implant. The glass coverslips (10 mm diameter) were implanted after anesthetization in the subcutaneous tissue. Blood samples were collected and the coverslips were carefully removed and washed with 0.9% saline 74 and 144 hours implantation. Then they were fixed in Bouin's solution and stained with hematoxylineosin. The totals of macrophages, foreign body type giant cells and Langhans cells on all coverslips were quantified. With respect to aerocystitis evolution, tilapia treated with CYP presented decrease in the circulating values of ceruloplasmin, complement C3, alpha2 macroglobulin and haptoglobin. The exudate evaluation revealed decrease (p <0.05) of the total number of cells accumulated in fish treated with CYP when compared the response observed in positive control fish (T3). The total count of macrophages and giant cells after implantation increased with time until 144 HPI. At the same time, there was a significant difference in the total counts between the CYP and negative control groups. Like for mammals, several studies have reported the suppressive effects associated with treatment with this alkylating agent in fish. The treatment with 200 mg of cyclophosphamide/kg of body weight in O. niloticus modulated acute phase protein responses and cell accumulation at the inflamed site, demonstrating the potential of this experimental model for pathophysiological studies during acute and chronic inflammatory reaction.
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Palavras-chave
Imunologia, Modelos experimentais, Proteína de fase aguda, Inflamação, Inflammation, Immunology, Experimental models, Acute-phase protein