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On-line version ISSN 1678-4162
Arq. Bras. Med. Vet. Zootec. vol.55 no.3 Belo Horizonte June 2003
Efeitos do diclofenaco e da dexametasona na endotoxemia experimental em eqüinos
P.C.S. RosaI; J.R. PeiróII; R.C. CampebellI; C.A.A. ValadãoIII; G.H. BecharaIII, *
IEstudante de pós-graduação da Faculdade de Ciências Agrárias e Veterinária da UNESP Jaboticabal, SP
IICurso de Medicina Veterinária da UNESP Araçatuba, SP
IIIFaculdade de Ciências Agrárias e Veterinárias da UNESP Via de Acesso Prof. Paulo D. Castellane, s/n 14884-900 - Jaboticabal, SP
Fifteen healthy Mangalarga horses, aged two to three years were used to evaluate the possible beneficial effects of dexamethasone and sodium diclofenac administration during experimental endotoxemia in horses. They were divided into three groups with five animals each: control (C), sodium diclofenac (SD) and dexamethasone (DM). All groups were given 0.1µg of Escherichia coli O55:B5 endotoxin/kg of body weight, intravenous, over 15 minutes, and one of the following preparations: group C 20ml of 0.9% saline intravenous, 30 minutes before endoxin infusion; group SD - 2.2mg/kg, per os, 60 minutes before endotoxin infusion and group DM 1.1 mg/kg, intravenous, 30 minutes before endotoxin infusion. No increase in rectal temperature was observed in the SD or DM treated groups. SD did not prevent the significant leukopenia, neutropenia and lymphopenia induced three hours after LPS injection, but DM attenuated these changes. No significant changes in plasma and peritoneal fluid total protein, inorganic phosphorus or glucose concentrations and in total nucleated cell count in peritoneal fluid were observed. SD was effective to prevent the fever and changes in intestinal borborygmi and DM blocked the cellular changes induced by experimental endotoxemia.
Keywords: endotoxemia, equine, peritoneal fluid, sodium diclofenac, dexamethasone
Quinze eqüinos machos, da raça Mangalarga, com idades entre dois e três anos, foram utilizados para se avaliar os possíveis efeitos clínicos benéficos da administração de dexametasona ou diclofenaco sódico durante a endotoxemia experimental em eqüinos. Os animais foram divididos em três grupos de cinco animais cada: controle (C), diclofenaco sódico (SD) e dexametasona (DM). Todos os grupos receberam 0,1µg/kg de lipopolissarídeo de Escherichia coli 055:B5, durante 15 minutos, por via intravenosa mais: grupo SD 2,2mg/kg de SD, por via oral, 60 minutos antes da infusão da endotoxina; grupo DM 1,1mg/kg, por via intravenosa, 30 minutos antes da endotoxina; grupo C 20ml de NaCl 0,9%, por via intravenosa, 30 minutos antes da endotoxina. O SD não preveniu a leucopenia, neutropenia e linfopenia ocorridas três horas após a indução da endotoxemia, porém a DM atenuou essas alterações. As taxas de proteínas plasmática e peritoneal, a concentração de glicose e de fósforo inorgânico e a contagem de células nucleadas totais peritoneais mantiveram-se inalteradas. O diclofenaco foi eficaz na prevenção da febre e alterações nos borborigmos intestinais enquanto que a dexametasona bloqueou as alterações no número de células inflamatórias em relação ao grupo controle.
Palavras-chave: endotoxemia, eqüino, líquido peritoneal, diclofenaco sódico, dexametasona
Endotoxin, the lipopolysaccharide (LPS) component of the outer membrane of Gram-negative bacteria, normally does not cause pathologic changes despite high concentrations in the intestinal lumen because the intact intestinal mucosal barrier prevents systemic absorption (Bayston, Cohen, 1990). However, disruption of this barrier allows absorption which initiates a cascade of events leading to septic/endotoxic shock (Ochalski et al., 1993) or circulatory shock (Moore, 1995). In comparison with other species, horses are more sensitive to the effects of parenterally administered LPS and it is likely that endotoxin plays a pivotal role in the pathogenesis of a number of important equine diseases (Moore et al., 1995).
Approximately 80µg/ml of endotoxin has been reported to be present in the cecal fluid of clinically normal horses. As little as 1µg of LPS in the systemic circulation is sufficient to induce fever and leukopenia. Endotoxin is detected in the peritoneal fluid of horses with acute gastrointestinal tract disease (MacKay, 1992). Once endotoxin escapes from the intestinal lumen owing to disruption of the mucosal barrier secondary to ischemia or inflammation, it exerts its local and systemic effects by inducing the host's cells to synthesize effector molecules, which subsequently are responsible for mediating the pathological effects of endotoxemia (Moore, 1995).
Endotoxin has been shown to activate arachdonic acid metabolism, yielding prostaglandins and thromboxanes by the cyclooxygenase enzyme system. Presently, two cyclooxygenase enzymes are known, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). COX-1 is a constitutive enzyme which synthesizes prostaglandins for normal activity. COX-2 regulates release of prostaglandins important for the inflammatory response (Cannon et al., 2000). Whereas COX-1 expression is largely unaffected by inflammatory signals, expression of COX-2 is increased substantially after exposure of cells to stimuli such as lipopolysaccharide (LPS) or cytokines such as interleukin-1 (IL-1) and IL-6 (Lyons-Giordano et al., 1993). The pattern of distribution and induction of COX-2 suggests it is the isoform primarily responsible for the synthesis of prostanoids that mediate responses to pathologic processes such as inflammation, pain, and fever (Schwartz et al., 1999).
Diclofenac, a nonsteroidal anti-inflammatory drug which inhibits both COX-1 and COX-2, with a predominant effect on COX-2, reduces inflammation, swelling, and arthritic pain by inhibiting the production of prostaglandins (Brooks et al., 1980; Todd, Sorkin, 1988; Skoutakis et al., 1988). Also, it has shown strong antipyretic and analgesic activities (Todd, Sorkin, 1988; Ortiz et al., 2002).
Both corticosteroids, such as dexamethasone, and nonsteroidal anti-inflammatory drugs have been used in the management of equine abdominal disorders. Despite the controversy over mechanisms of action, the therapeutic effectiveness of large doses of glucocorticoids has been shown for many species including ponies (Templeton et al., 1987).
The effects of corticosteroids have been assessed in endotoxemia in dogs and human beings (White et al., 1978). The effects include stabilization of cellular and subcellular membranes, increased aerobic energy production, minimization of plasma lactate values, and improved peripheral microcirculation, providing a means for modifying the detrimental cellular effects of endotoxins (Fraunenfelder et al., 1982).
There is substantial evidence that endotoxemia is associated with an increase in arachidonic acid metabolites in blood and tissues. This increase was associated with synthesis of the COX-2 (Leach et al., 1998). Ruetten and Thiemermann (1997) have recently demonstrated that the expression of COX-2 caused by LPS in the rat in vivo is prevented by dexamethasone. Interestingly, prevention by dexamethasone of the expression of COX-2 was associated with beneficial effects on the circulatory and multiple organ failure caused by endotoxin (Leach et al., 1998).
This study was performed to determine the clinical effects and cellular and biochemical changes in both the blood and peritoneal fluid after intravenous injection (IV) of LPS in horses pretreated with sodium diclofenac (SD) and dexamethasone (DM).
MATERIAL AND METHODS
This study was approved by the FCAV-UNESP Animal Care Committee. Fifteen adult intact male Mangalarga horses, aged two to three years, were given alfalfa hay and water ad libitum and dewormed with ivermectin, 30 days before the study. Complete clinical examination was performed before start this study. The horses were divided into three groups of five animals each: control (C), sodium diclofenac (SD) and dexamethasone (DM).
Sublethal endotoxemia was induced by IV infusion of 0.1µg/kg of E. coli 055:B5 endotoxin (Sigma Chemical Co., St Louis, MO, USA.) given in 250ml of 0.9% saline over 15 minutes. Horses in control group received 0.9% saline solution administered IV 30 minutes before the LPS injection. Horses in SD group were given sodium diclofenac (Farmavida, Bragança Paulista, SP, Brazil.) (2.2 mg/mg) per os one hour before LPS. In DM group, horses received dexamethasone (Schering-Plough, Rio de Janeiro, RJ, Brazil.) (1.1 mg/kg) IV 30 minutes before endotoxin injection. Peripheral blood and peritoneal fluid were collected at 0, 1 ¼, 3, 6, 12 and 24 hours after LPS injection (PI). Plasma and peritoneal fluid total protein concentrations were measured by the Biuret method (Labtest Diagnóstica S/A, Belo Horizonte, MG, Brazil), and glucose and inorganic phosphorus concentrations were determined, using the enzymatic and Lustosa-Basques (Labtest Diagnóstica S/A, Belo Horizonte, MG, Brazil) methods, respectively. Complete peripheral blood and peritoneal white blood cell counts were performed and the clinical signs of endotoxemia, rectal temperature, respiratory and heart rates, capillary refill time, mucous membrane color, intestinal borborygmi were, also, recorded at each time as mentioned above.
A sterile teat cannula was inserted into the abdominal cavity through a small skin incision made in an appropriately prepared area on the ventral abdomen. The peritoneal fluid was collected into sterile tubes containing EDTA. Blood was obtained by jugular venipuncture and placed into sterile tubes containing EDTA. One sample was used for a complete hemogram; plasma was used for determination of glucose and total protein concentrations.
Data were analyzed by split-plots in time, using a completely randomized design with whole plot (groups) and subunits (time). Post-hoc comparisons were made with Tukey's test. Differences were considered significant at P< 0.05 (Statistical Analysis System v.6.12 - USA). Data are reported as means ± SEM.
The clinical aspects associated with LPS infusion (one to three hours PI) were characterized by depression, signs of mild abdominal pain (occasionally looking at the flank, pawing) with borborygmi decrease and a significant increase in the rectal temperature in C horses from 1 ¼ hour to 12 hours compared with baseline. Hyperemic mucous membranes and increased capillary refill time were observed from one to three hours PI in C, SD and DM, but no difference was observed among groups. Rectal temperature was significantly lower in SD horses than in C horses from 1 ¼ hour through six hours, but did not differ from DM horses at the same times (Fig.1). DM horses had a significant lower temperature at three hours when compared with C horses. No changes in intestinal borborygmi were observed in the SD or DM horses. No changes in heart and respiratory rates among groups over time were found.
It was observed a decrease in total white blood cell count (WBC) during the first three hours after LPS injection, mainly due to a decrease in neutrophil and lymphocyte counts in C and SD (Fig.2 to 4). Dexamethasone attenuated this neutropenia 1 ¼ hour after the LPS injection and evident increased leukocyte count was sustained till 24 hours. Leukocytosis by neutrophilia was observed in the DM horses with counts significantly higher from three to 24 hours PI than those for horses of all other groups.
The SD horses showed a marked decrease in neutrophils counting during the first 1 ¼ hour, followed by a gradual increase up to six hours PI. DM attenuated and SD did not interfere in the leukopenia due to neutropenia (Fig. 2 and 3).
A significant lymphopenia was observed during the first six hours in all groups (Fig.4). A statistical difference in these cells countings was observed between DM horses and the other two groups at 12 and 24 hours.
No significant changes among groups concerning plasma and peritoneal fluid total protein, inorganic phosphorus or glucose concentrations were observed. No changes in total nucleated cell counts in peritoneal fluid were found among groups.
The pathophysiologic effects of live Gram-negative organisms are similar to those of bacterial endotoxin and experimental endotoxemia is a recognized model for the study of Gram-negative septic shock (Ward et al., 1986). Because of these similarities, the administration of sublethal amounts of endotoxin to horses permits the study of possible therapeutic agents for equine colic (Lochner, 1986). The LPS dose used in this study was similar to that used in previous studies in horses (Moore, 1988; Henry, Moore 1990).
Several endogenous pyrogens have been identified including the proinflammatory cytokines interleukin (IL)-1a, IL-1b, tumor necrosis factor (TNF)-a, and IL-6 (Henry, Moore, 1990; Cargile et al., 1995; Zetterström et al., 1998). In bacterial endotoxin-mediated fever, all of these cytokines have been implicated (Zetterström et al., 1998). Sundgren-Anderson et al. (1998) suggest that a central, rather than a peripheral, pool of IL-6 is important in the TNF-a-induced frebrile response. This is in accordance with other reports showing that hypothalamic concentrations of IL-6 are increased during LPS-induced fever and that IL-6 administered centrally produces fever (Rothwell et al., 1991). Interleukin-1 and IL-6 activate COX, which generate PGE2 in the central nervous system. The net result is a resetting of the hypothalamic temperature set point, which is manifested clinically as fever (Schwartz et al., 1999). In this study, the febrile response to endotoxin was similar to that observed by others after IV injection of LPS in horses (Morris et al., 1990; Valadão et al., 1995). Diclofenac reduction of the febrile response could be explained by its more selective activity against the type-2 cyclooxygenase (Wanecek et al., 1997; Kawai 1998; Schwartz et al., 1999; Cannon et al., 2000), as this is the isoform primarily responsible for the synthesis of prostanoids that mediate responses to pathologic processes such as inflammation, pain, and fever (Schwartz et al., 1999). COX inhibitors, such as diclofenac, likely suppress the production of prostaglandins (Martinez et al., 1999), including PGE2, in brain regions critical to the generation of fever in humans, as shown in animals (Schwartz et al., 1999). Diclofenac also inhibited LPS fever completely and for the duration of the febrile response in rabbits (Mabika, Laburn, 1999).
Previous reports have demonstrated that leukopenia and neutropenia in response to LPS injection were induced by cytokines that caused margination of neutrophils (Ward et al., 1987; Lavoie et al., 1990) followed by leukocytosis (Moore, 1988; Henry, Moore, 1990).
Mild increase in rectal temperature with absence of changes in intestinal borborygmi and white cells counts in the DM horses were presumably due to the DM inhibition of the release of many biologically active substances, including neutrophil chemoattractants (Cecilio et al., 1997). Corticosteroids interfere with the chemotactic mediators blocking the final migration step, retaining the neutrophils between the endothelium and the basal membrane (Cecilio et al., 1997). Lymphopenia rapidly occurs in horses after endogenous release of corticosteroids (Valadão et al., 1995) or by prostaglandin E2 release that supresses the imune response as well inhibits lymphocyte proliferation (Peiró et al., 1999). Nevertheless, dexamethasone did not inhibit in vivo the different stages of neutrophil migration, rolling or adhesions to the venular endothelium induced by leukotriene B4 in hamsters (Cecilio et al., 1997).
Steroids are known to reduce the synthesis of prostaglandins by stimulating the formation of an inhibitor molecule lipocortin, which inhibits phospholipase A2 and prevent the release of arachidonic acid from membrane phospholipids (Olson, Brown Jr., 1986). This takes longer (two to three hours) before a reduction of prostaglandin synthesis (Templeton et al., 1987). The increased neutrophil counts observed in animals treated with dexamethasone from 3 hours to the end of this study is in accordance to others (Templeton et al., 1987) and may be explained by those mechanisms of steroids which help to prevent neutrophil adhesions to the endothelium (Ewert et al., 1985).
Glucocorticoids are widely used for immunosuppressive and anti-inflammatory effects. They have an inhibitory effect on cytokine production and action (Morresey, 2001). Glucocorticoids have little or no effect on constitutive prostaglandin production as they only inhibit COX-2 gene expression (Inoue et al., 1999). So, the lower temperature observed in DM group compared with the control one could be occurred due to the decrease or inhibition of PGE2 production. This decreased temperature values could also be due to the inhibition TNF synthesis (Morris et al., 1991). Unfortunately, glucocorticoids have untoward side effects such as laminitis caused by the potentiation of catecholamines action on vascular tone and immunosuppression, which preclude their clinical use in the treatment of endotoxemia in horses. With these concerns in mind, corticosteroids should be used with caution (Holbrook, Moore, 1994).
Increases in heart and respiratory rates have been shown to be coincident with the onset of increased body temperature following endotoxin infusion in horses (Moore, 1988; Cargile et al., 1995; Valadão et al., 1995). None of the drugs administered were effective in preventing the heart and respiratory effects after the LPS injection.
It is now well stablished that IV administration of LPS blocks cells migration to an inflamed site and prevents the formation of exudate, edema and pain (Rocha, Ferreira, 1986). The reduction in leukocytes number occurs in parallel with the reduction in vascular inflammatory response. The inhibition observed in the neutrophil migration might be due to the presence of a neutrophil inhibitory factor (NIF) in the circulation, known to be released in vitro by neutrophils or mononuclear cells incubated with LPS. However, other factors may also contribute, since LPS directly affected neutrophil chemotaxis (Rocha, Ferreira, 1986). On the other hand, studies using intraperitoneal injection of LPS alone (Burrows, 1979; Cunha, Ferreira, 1986; Cecilio et al., 1997; Peiró et al., 1999) reported a significant dose and time dependent neutrophil migration with a bell-shaped dose-response curve for mice and horses. This may be due to the liposolubility of the LPS and the fact that when it is injected in high concentrations in the extravascular tissue, the amount that enters the circulatory system is sufficient to block neutrophil migration (Cecilio et al., 1997; Peiró et al., 1999).
It is concluded that diclofenac was effective in preventing the fever and changes in intestinal borborygmi in horse LPS-induced experimental endotoxemia whereas dexamethasone did not exert these effects but blocked the cellular changes.
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Recebido para publicação em 24 de outubro de 2002
Recebido para publicação, após modificações, em 3 de abril de 2003