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ENDOTOXAEMIA: RECENT ADVANCES IN PATHOPHYSIOLOGY AND TREATMENT
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J. vet. Anaesth. Vol. 21 (December 1994)
Hunt SP, Pin A & Evan G (1987) Induction of c-fos-like protein in spinal cord neurones following sensory stimulation. Nuture, 328, 632-634.
Morton DB & Griffiths PHM (1985) Guidelines on the recognition of pain, distress, and discomfort in experimental animals and a hypothesis for assessment. Veterinary Record, 116,431-436.
Hutchison GL, Crofts SL & Gray IG (1990) Preoperative piroxicam for postoperative analgesia in dental surgery. British Iournal of Anaesthesiu, 65, 500-503.
Rang HP, Bevan S & Dray A (1991) Chemical activation of nociceptive peripheral neurones. British Medical Bulletin, 47, 534-538. Woolf CJ (1983) Guidance for a central component of post-injury pain hypersensitivity. Nature, 306,686-688.
McQuay HJ, Carroll D & Moore RA (1988) Postoperative orthopaedic pain - the effect of opiate premedication and local anaesthetic blocks. Pain, 33,291-295.
Woolf CJ & Chang MS (1993) Pre-emptive analgesia -Treating postoperative pain by preventing the establishment of central sensitization. Anes thesia Analgesia, 77, 362-379.INTRODUCTION
Melzach R & Wall PD (1965) Pain mechanisms. A new theory. Science, 150,971-979.
ENDOTOXAEMIA: RECENT ADVANCES IN PATHOPHYSIOLOGY AND TREATMENT N. MOORE College of Veterinary Medicine, University of Georgia, Athens, Georgia
JAMES
occluded. The resultant ischaemia and intestinal distention cause disruption of the mucosal lining, thereby allowing the movement of intraluminal endotoxin into the peritoneal cavity and then into the systemic circulation (Moore and Morris, 1992). The clinical course of horses with intestinal strangulation obstruction is rapid, and the onset of circulatory shock is imminent, unless the damaged intestine is surgically removed within hours. Although surgical removal of the damaged intestine removes the primary site of mucosal disruption, the function of the remaining intestinal mucosal barrier may be suboptimal as a result of the initial cardiovascular responses to endotoxaemia. Consequently, horses with strangulating intestinal lesions commonly show acute evidence of endotoxaemia before surgical intervention and may exhibit more chronic effects of endotoxaemia after surgery. Colitis and proximal enteritis are devastating medical diseases of horses, often wrought with life threatening complications. The clinical signs of endotoxaemia which characterise these diseases are very pronounced and often persist longer than those associated with other causes of colic. Unlike surgically correctable causes of colic, the inflamed intestine can not be removed. Consequently, the systemic absorption of endotoxin most likely persists until the mucosa heals. As the detection of endotoxin in the plasma of horses with colic has been associated with a poor prognosis (King and Gerring, 1988), it is not surprising that inflammatory causes of colic have higher mortality rates than conditions causing non-strangulating intestinal obstruction (Morris, 1991). Once endotoxin migrates across the damaged intestinal wall, it may take one of two paths. Endotoxin absorbed by the portal system is transported to the liver where resident hepatic macrophages remove it from circulation. If the influx of endotoxin from the damaged intestine exceeds the ability of the liver to remove it, endotoxin spills over into the systemic circulation. Alternatively, endotoxin may move across the intestinal wall into the peritoneal cavity, be absorbed by the lymphatics, and reach the systemic circulation by entering the major veins at the thoracic duct (Deventer et al., 1988).
Gram-negative bacteria are enveloped by an outer lipopolysaccharide-rich membrane. This lipopolysaccharide, also termed "endotoxin", is comprised of three structural units: an outer polysaccharide component which affords lipopolysaccharide its antigenic properties, a core region composed of several monosaccharides, and the innermost portion, termed lipid A, which affords the molecule its potent proinflammatory biological activities (Moore et al., 1981; Moore, 1981; Moore and Morris, 1992). Circulatory shock secondary to either Gram-negative sepsis or endotoxaemia is associated with a high mortality rate in equine veterinary medicine. Septicaemia occurs all too often in equine neonates, which frequently are immuno-compromised by inadequate passive transfer of colostral immunoglobulins (Koterba et al., 1984). Gramnegative bacterial infections of the uterus or other soft tissues also can lead to circulatory shock due to movement of endotoxin from the tissues into the blood stream. Most commonly, however, circulatory shock in horses is the result of diseases involving the gastrointestinal system which lead to the systemic absorption of endotoxin (King and Gerring, 1988). The intestines of the horse harbour a very large population of Gram-negativebacteria and thus the intestinal milieu normally contains a large amount of endotoxin. Although the gastrointestinal tract has a very efficient mucosal barrier to restrict endotoxin to the intestinal lumen, gastrointestinal diseases which compromise this mucosal barrier result in the transmural movement of endotoxin (Meyers et al., 1982). Endotoxaemic horses exhibit abnormal behaviour ranging from depression to acute bouts of pain. Physical examination findings often include tachycardia, tachypnoea, hyperaemic mucous membranes, decreased gastrointestinal sounds, and dehydration. Diseases which most commonly result in the development of endotoxaemia in horses are intestinal strangulation obstruction and inflammatory intestinal diseases.
Intestinal Diseases and Endotoxaernia Intestinal'strangulationobstruction occurs when both the intestinal lumen and the mesenteric blood vessels are 77
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Experimental Endotoxaemia
Of greater importance, however, is the interaction of endotoxin with lipopolysaccharide binding protein (Ulevitch et al., 1990; Tobias and Ulevitch, 1986; Tobias and Ulevitch, 1993). This protein, which is synthesized by hepatocytes, normally exists in trace amounts ( ~ 0 . 5 pg/ml) in circulation, but its concentration may increase up to 100-fold during the acute phase response. Lipopolysaccharide binding protein binds avidly to the lipid A component of endotoxins from all Gram-negative bacteria and serves as an opsonin for removal of these bacteria from the circulation. This protein also is unique as it dramatically increases the ability of endotoxin to activate cells of the mononuclear phagocyte system (Tobias and Ulevitch, 1993; Wright et al., 1990). Thus, rather than serving as a nonspecific natural detoxifying system for endotoxin, the interaction of endotoxin with lipopolysaccharide binding protein markedly enhances the ability of endotoxin to cause damage to the host.
The pathophysiological responses to the short term administration of endotoxin have been well characterised in a variety of animals including the horse. Initially, the cardiovascular response to endotoxin includes vasoconstriction in the systemic and pulmonary vascular beds. Accordingly, tachycardia, tachypnoea, hypoxaemia, and respiratory alkalosis ensue. This phase is followed by profound systemic hypotension and loss of vascular integrity. Ultimately, the repetitive inflammatory insults lead to progressive endothelial dysfunction which can culminate in multi-organ failure (Bone, 1991a; Bone, 1991b). The haematological effects of endotoxaemia in horses and other animals are typified by haemoconcentration, early leukopaenia followed by leukocytosis, and thrombocytopaenia. In horses haemoconcentration occurs due to splenic contraction and loss of fluid from the vascular space resulting from the altered vascular permeability. The leukopaenia is due to neutrophil margination (Moore, 1988).Dysfunction of haemostasis and fibrinolysis also has been documented during endotoxaemia in several species. In horses, coagulopathy has been characterised during experimental endotoxin infusions, experimental models of intestinal strangulation, and during clinical diseases (colitis, colic) associated with endotoxaemia (Duncan et al., 1985; Pablo et al., 1983; Welch et al., 1992). This coagulopathy can progress to fulminant disseminated intravascular coagulation (Welch et al., 1992). The metabolic derangements associated with endotoxaemia have been well characterized in other species, but not in horses (Burrows, 1981). In general, endotoxaemia induces a transient hyperglycaemia that is followed by hypoglycaemia. With progressing circulatory shock, tissue hypoperfusion leads to lactic acidosis coincident with the shift of cellular metabolism toward anaerobic glycolysis. Alterations in lipid metabolism also have been characterized during endotoxaemia. These include increased plasma triglycerides, hepatic fatty acid synthesis, and very low density lipoprotein production (Adi, 1992).
Cellular Recognition of Endotoxin In recent years, it has become evident that endotoxin alone has few direct inflammatory actions and that the deleterious effects of endotoxin depend on the synthesis of numerous proinflammatory substances by a variety of cells, including endothelial cells, neutrophils, platelets and lymphocytes. The mononuclear phagocytethe is the cell responsible for many of the inflammatory responses during endotoxaemia (Mathison, et al., 1991; Mathison et al., 1992). Cell culture techniques have been used to characterize the responses of isolated equine peripheral blood monocytes, peritoneal, alveolar and mammary macrophages to endotoxin (Morris, 1991; Henry and Moore, 1988; MacKay and Lester, 1992; MacKay et al., 1991; Henry and Moore, 1991; Barton et al., in press; Morris, et al., 1990; Morris and Moore, 1987). The results of several recent studies indicate that recognition of endotoxin by mononuclear phagocytes occurs through both receptor and non-receptor mediated events (Morrison, 1985).The study of interactions of synthetic lipid A with macrophage membranes has led to the discovery of endotoxin scavenging receptors believed to function in the removal of endotoxin from the circulation (Hampton et al., 1991). Similarly, the leukocyte antigen CD18 binds endotoxin and may have an important function in the phagocytosis and degradation of endotoxin (Wright and Tong, 1986). Of more clinical importance, however, is the fact that the proinflammatory effects of endotoxin are fundamentally linked to the interaction of the lipopolysaccharide binding protein endotoxin complex with the mononuclear cell receptor known as CD14. This interaction leads to activation of the mononuclear cell and synthesis of the inflammatory mediators that cause the clinical signs of endotoxaemia (Ulevitch et al., 1990).
Interaction of Endotoxin with Plasma Proteins There is a large body of evidence indicating that endotoxin interacts both with circulating high density lipoproteins and a unique protein termed lipopolysaccharide binding protein (Ulevitch et al., 1990; Tobias and Ulevitch, 1986). Because of its marked hydrophobic chemical nature, endotoxin exists in aggregates when it first comes in contact with blood. These aggregates then disaggregate and the free endotoxin forms stable complexes with the high density lipoproteins. Although the formation of these complexes prolongs the half-life of endotoxin in the blood stream, the binding of endotoxin to the high density lipoproteins also markedly reduces the ability of the endotoxin to interact with cells. Presumably this reduction in the endotoxic activity of endotoxin is due to a nonspecific interaction of the lipid A component of endotoxin with the phospholipid portion of the high density lipoproteins.
Mediators of Endotoxaemia Mononuclear phagocytes (peripheral blood monocytes and macrophages) synthesize and release numerous inflammatory mediators which are responsible for the majority of pathophysiological responses to endotox78
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tions have been measured in adult horses and neonatal foals administered endotoxin (MacKay and Lester, 1992; Robinson et al., 1993). Tissue factor (thromboplastin) is a glycoprotein synthesised by monocytes and macrophages in response to endotoxin. Unlike most of the other mediators involved in the pathogenesis of endotoxaemia, tissue factor remains closely associated with its cell of origin. In fact, approximately 90% of the tissue factor remains on the surface of the cell. When exposed to coagulation factor VII, tissue factor initiates the extrinsic arm of the coagulation cascade. Consequently, tissue factor activity can be measured indirectly using a whole blood recalcification time assay or directly by measuring the ability of cell lysates to shorten the recalcification time of pooled plasma. Using these assays, increases in tissue factor activity have been identified in horses administered endotoxin and in horses with naturally occurring abdominal diseases characterised by endotoxaemia (Henry and Moore, 1991). The metabolites of arachidonic acid have received a great deal of attention over the past 10 years in research on endotoxaemia. Arachidonic acid is an important component of the phospholipid layer of cellular membranes. Damage to the membrane results in activation of phospholipase A,, which cleaves arachidonic acid from the phospholipids. The free arachidonic acid then is converted by cyclo-oxygenase to the very labile endoperoxide intermediates, and then to prostaglandin I,, prostaglandin E,, prostaglandin F,, or thromboxane A,. In general prostaglandins E, and I, relax vascular smooth muscle by increasing cAMP concentrations. In contrast, prostaglandin F,, and thromboxane A, decrease cAMP and cause vasoconstriction. Many of the early cardiopulmonary and behavioural effects of endotoxaemia in horses appear to be mediated by arachidonic acid metabolites. In particular, the dyspnoea, hypoxaemia, and pulmonary hypertension that characterize the early response to endotoxin administration are accompanied by a profound increase in the plasma concentration of the stable metabolite of thromboxane A,. Subsequently, the discoloration of the mucous membranes, prolongation of the capillary refill time, hypotension, and intermittent signs of abdominal pain are accompanied by increases in plasma concentrations of the stable metabolite of prostaglandin I,. Therefore, it appears that many of the initial responses to endotoxaemia are influenced by the generation and release of vasoconstrictive metabolites and the later effects are associated with metabolites that induce vasodilation.
aemia. Of the multitude of inflammatory mediators synthesized in response to endotoxin, several have been studied extensively in the equine species. These include tumour necrosis factor, interleukins 1 and 6, tissue factor, and the arachidonic acid metabolites. One of the earliest events subsequent to the interaction of the lipopolysaccharide binding protein endotoxin complex with CD14, is the synthesis of the cytokine known as tumour necrosis factor (Heumann et al., 1992). Tumour necrosis factor is a 17KD polypeptide which plays a pivotal role in endotoxic shock. Serum concentrations of tumour necrosis factor are increased in both clinical endotoxic shock and in experimental endotoxaemia in people and animals (Moore and Morris, 1992; Morris, 1991; Morris et al., 1990; Morris et al., 1991; Michie et al., 1988). In addition, the systemic administration of recombinant tumour necrosis factor to animals causes the same pathophysiological consequences as does administration of endotoxin (Tracey et al., 1986). Furthermore, the administration of anti-tumour necrosis factor antibodies to animals prior to inducing endotoxic or septic shock leads to a dramatic reduction in mortality rate (Beutler and Cerami, 1987). The biological activity of tumour necrosis factor is quite diverse. Animals given tumour necrosis factor intravenously develop shock characterized by progressive hypotension, haemoconcentration, metabolic acidosis, and disseminated intravascular coagulation which can eventually lead to death (Turner et al., 1989). Other properties attributed to this polypeptide include stimulation of interleukin-1 and interleukin-6 synthesis, the acute phase response, fever, prostaglandin synthesis, endothelial leukocyte adhesion, molecule expression and procoagulant activity. Interleukin-1 is the name now associated with a group of inflammatory mediators previously known as "endogenous pyrogen". Interleulun-1 is responsible for the febrile response to endotoxin and initiates arachidonic acid metabolism by stimulating phospholipase A, activity on the cell surface. Furthermore, interleukin-1 causes systemic hypotension, initiates the wastage of muscle mass for gluconeogenesis and synthesis of acute phase proteins by the liver, reduces food intake, and increases the stickiness of vascular endothelial cells. There is evidence that the hypotensive effect of interleukin-1 is mediated in part by increased nitric oxide production (Beasely et al., 1991). Circulating concentrations of interleukin-1B have been documented after administration of endotoxin to healthy human volunteers (Cannon et al., 1990)and rabbits (Wakabayashi et al., 1991).The biological effects of interleukin-1 and tumour necrosis factor are remarkably similar and these cytokines often have synergistic effects. Interleukin-6 is another cytokine produced primarily by monocytes and macrophages. Interleukin-6 is very important in the acute phase reaction to inflammatory stimuli and its synthesis is increased by tumour necrosis factor and interleukin-1. Although there is little evidence indicating that interleukin-6 produces deleterious effects on the host, circulating concentrations of this cytokine increase during endotoxaemia and have been used as a prognostic indicator in clinical cases. Serum concentra-
Treatment of Endotoxaernia Although our knowledge regarding the pathophysiol-
ogy of endotoxaemia has increased dramatically in the past 10 years, treatment of this problem in horses has changed more slowly. This delay may be due to the inherent complexity of the processes involved in clinical endotoxaemia, the exquisite sensitivity of the horse to endotoxin, and the cost of using certain therapeutic agents on patients weighing 500 kg. The more commonly used methods of treatment include non-steroidal anti79
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inflammatory drugs, corticosteroids, anti-endotoxin serum, and cardiosupportive agents. Non-steroidal anti-inflammatory drugs, which are effective by inhibiting cyclo-oxygenase, are the most common types of drugs used to combat endotoxaemia in horses. Of the large number of non-steroidal anti-inflammatory drugs presently available, flunixin meglumine, ketoprofen and phenylbutazone have received most attention. Of these, flunixin and ketoprofen appear to be most efficacious and are often used at doses less than those recommended by the manufacturers. Based on the results of one experimental study (Semrad et al., 1987), many clinicians elect to administer flunixin at 0.25 mg/kg TID. Although it has been suggested that ketoprofen inhibits both cyclo-oxygenase and lipoxygenase, data documenting the effect of ketoprofen on synthesis of lipoxygenase products in horses are lacking. Corticosteroids have been used in the treatment of endotoxic shock for decades and their use is as controversial now as it was in the past. Corticosteroids should be effective for two reasons: 1) they inhibit phospholipase A, activity and thus should prevent the generation of prostaglandins, thromboxanes, leukotrienes and platelet activating factor (Cohn, 1991) and 2) they prevent the synthesis of tumour necrosis factor (Morris et al., 1991). However, compared with flunixin in a model of lethal endotoxic shock in anaesthetised ponies, the corticosteroids were less effective at improving metabolic status and prolonging survival than flunixin (Ewert et al., 1985). Treatment of endotoxaemia has changed markedly in recent years with the availability of antibodies against the endotoxin molecule. These antibodies are directed against the conserved regions of endotoxin (lipid-A and the core polysaccharides) and thus provide protection against endotoxins from different bacterial species. To be maximally effective, these antibodies must bind endotoxin before it can cause inflammatory cells to synthesize and release the various mediators; thus, these antibodies should be gven early in the clinical course of disease. The results of clinical trials and experimental studies using anti-endotoxin antibodies have been conflicting. In a double-blind clinical study, horses treated with antiendotoxin plasma had an increased survival rate (87% vs. 53%), improved clinical appearance, and a shorter duration of hospitalization when compared to horses administered normal plasma (Spier et al., 1989). In an experimental study of endotoxaemia, treatment with antiendotoxin plasma failed to improve either clinical or clinicopathological parameters (Morris et al., 1986). Furthermore, in one experimental study young foals treated with the anti-endotoxin antibodies before initiation of endotoxaemia had higher serum concentrations of tumour necrosis factor and interleukin-6, lower neutrophil counts and more severe clinical signs than foals receiving only endotoxin (Durando and MacKay, 1992). Systemic hypotension occurs periodically in horses anaesthetised for emergency abdominal surgery. Presumably this problem is associated with the development, or worsening of, endotoxaemia. Treatment of hypotension under such circumstances usually involves the administration of either dopamine or dobutamine to
increase cardiac output and systemic arterial pressure. These drugs are selected because of their rapid onset and the fact that they have proved to be efficacious in horses (Trim et al., 1985; Swanson and Muir, 1986).
Furure Therapeutic Approaches With the substantial amount of data implicating cytokines, particularly tumour necrosis factor, in mediating the ill effects of endotoxin, there is a considerable amount of effort being expended to develop therapeutic agents directed against these cytokines. Consequently, monoclonal antibodies and soluble receptors are being evaluated as methods of preventing the deleterious effects of tumour necrosis factor. Two other promising areas of interest are the CD14 endotoxin receptor and lipopolysaccharide binding protein. If it were feasible to interfere with the binding of endotoxin with lipopolysaccharide binding protein or prevent the interaction of the endotoxin-lipopolysaccharide binding protein complex with CD14, it might be possible to remove the initial signal that stimulates the mononuclear phagocyte to synthesize and release the inflammatory mediators that cause the clinical syndrome of endotoxaemia.
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Morris DD, Moore JN, Fischer K & Tarleton RL (1990). Endotoxin-induced tumor necrosis factor activity production by equine peritoneal macrophages. Circ. Shock, 30,229-236.
Henry M & Moore J (1988). Endotoxin-induced procoagulant activity in equine peripheral blood monocytes. Circ. Shock, 26,297-309.
Morris D, Whitlock R & Corbeil L (1986). Endotoxemia in horses: Protection provided by antiserum to core lipopolysaccharide. American Journal of Veterinary Research, 47, 544-550.
Henry MM & Moore JN (1991). Clinical relevance of monocyte procoagulant activity in horses with colic. Journal of the American Veterinary Medical Association, 198,843-848.
Momson DC (1985) Handbook of endotoxin: Cellular biology of endotoxin. L. J. Berry, Eds. Elsevier publishing Co., Amsterdam, pp. 2555.
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Koterba AM, Brewer BD & Tarplee FA (1984). Clinical and clinicopathological characteristics of the septicaemic neonatal foal: review of 38 cases. Equine Veterinary Journal, 16,376.
Semrad S, Hardee G, Hardee M & Moore J (1987). Low dose flunixin meglumine: Effects on eicosanoid production and clinical signs induced by experimental endotoxaemia in horses. Equine Veterinary Journal, 19,201-206.
MacKay RJ, King RR, Dankert JR, Reis KJ & Skelley LA (1991). Cytotoxic tumor necrosis factor activity produced by equine alveolar macrophages: preliminary characterization. Veterinary Immunology and Zmmunopathology, 29,15-30.
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Swanson C & Muir W (1986). Dobutamine-induced augmentation of cardiac output does not enhance respiratory gas exchange in anesthetized recumbent healthy horses. American Journal of Veterinary Research, 47,1573-1576.
Mathison JC, Tobias PS, Wolfson E & Ulevitch RJ (1991). Regulatory mechanisms of host responsiveness to endotoxin (lipopolysaccharide). Pathobiology, 59, 185-188. Mathison JC, Tobias PS, Wolfson E & Ulevitch RJ (1992). Plasma lipopolysaccharide (LPS)-binding protein: A key component in macrophage recognition of gram-negative LPS. Journal of Immunology, 149,200-206.
Tobias PS Soldau K & Ulevitch RJ (1986).Isolation of a lipopolysaccharide binding acute phase reactant from rabbit serum. Journal of Experimental Medicine, 164,777-793. Tobias PS & Ulevitch RJ (1993). Lipopolysaccharide binding protein and CD14 in LPS dependent macrophage activation. Zmmunobiology, 187, 227-232.
Meyers KM, Reed SM & Keck M (1982). Circulating endotoxin-like substance(s) and altered hemostasis in horses with gastrointestinal disorders. American Journal of Veterinary Research, 43,2233-2235.
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Trim C, Moore J & White N (1985). Cardiopulmonary effects of dopamine hydrochloride in anaesthetized horses. Equine Veterinary Journal, 17,41-44.
Moore JN (1981). Endotoxemia: Definition, characterization, and host defenses. Compendium of Continuing Education, 3,355-359. Moore JN (1988). Recognition and treatment of endotoxemia. Veterinary Clinics of North America, 4, 105-113.
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Moore JN & Morris DD (1992). Endotoxemia and sepsis in horses: Experimental and clinical correlates. Journal of the American Veterinary Medical Association, 200, 1903-1914. Morris DD (1991).Endotoxemia in horses: A review of cellular and humoral mediators involved in its pathogenesis. Journal of Veterinary Znternal Medicine, 5,167-181.
Wakabayashi G, Gelfand J, Jung W, Connolly R, Burke J & Dinarello C (1991). Staphylococcus epidermidis induces complement activation, tumor necrosis factor and interleukin-1, a shock-like state and tissue injury in rabbits without endotoxemia. Journal of Clinical Investigation, 87, 1925.1935.
Moms D & Moore J (1987). Endotoxin-induced production of thromboxane and prostacyclin by equine peritoneal macrophages. Circ. Shock, 23,295-303.
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Morris DD, Moore JN & Crowe N (1991). Serum tumor necrosis factor activity in neonatal foals with presumed septicemia. Journal of the American Veterinary Medical Association, 199, 1584-1591.
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Hunt SP, Pin A & Evan G (1987) Induction of c-fos-like protein in spinal cord neurones following sensory stimulation. Nuture, 328, 632-634.
Morton DB & Griffiths PHM (1985) Guidelines on the recognition of pain, distress, and discomfort in experimental animals and a hypothesis for assessment. Veterinary Record, 116,431-436.
Hutchison GL, Crofts SL & Gray IG (1990) Preoperative piroxicam for postoperative analgesia in dental surgery. British Iournal of Anaesthesiu, 65, 500-503.
Rang HP, Bevan S & Dray A (1991) Chemical activation of nociceptive peripheral neurones. British Medical Bulletin, 47, 534-538. Woolf CJ (1983) Guidance for a central component of post-injury pain hypersensitivity. Nature, 306,686-688.
McQuay HJ, Carroll D & Moore RA (1988) Postoperative orthopaedic pain - the effect of opiate premedication and local anaesthetic blocks. Pain, 33,291-295.
Woolf CJ & Chang MS (1993) Pre-emptive analgesia -Treating postoperative pain by preventing the establishment of central sensitization. Anes thesia Analgesia, 77, 362-379.INTRODUCTION
Melzach R & Wall PD (1965) Pain mechanisms. A new theory. Science, 150,971-979.
ENDOTOXAEMIA: RECENT ADVANCES IN PATHOPHYSIOLOGY AND TREATMENT N. MOORE College of Veterinary Medicine, University of Georgia, Athens, Georgia
JAMES
occluded. The resultant ischaemia and intestinal distention cause disruption of the mucosal lining, thereby allowing the movement of intraluminal endotoxin into the peritoneal cavity and then into the systemic circulation (Moore and Morris, 1992). The clinical course of horses with intestinal strangulation obstruction is rapid, and the onset of circulatory shock is imminent, unless the damaged intestine is surgically removed within hours. Although surgical removal of the damaged intestine removes the primary site of mucosal disruption, the function of the remaining intestinal mucosal barrier may be suboptimal as a result of the initial cardiovascular responses to endotoxaemia. Consequently, horses with strangulating intestinal lesions commonly show acute evidence of endotoxaemia before surgical intervention and may exhibit more chronic effects of endotoxaemia after surgery. Colitis and proximal enteritis are devastating medical diseases of horses, often wrought with life threatening complications. The clinical signs of endotoxaemia which characterise these diseases are very pronounced and often persist longer than those associated with other causes of colic. Unlike surgically correctable causes of colic, the inflamed intestine can not be removed. Consequently, the systemic absorption of endotoxin most likely persists until the mucosa heals. As the detection of endotoxin in the plasma of horses with colic has been associated with a poor prognosis (King and Gerring, 1988), it is not surprising that inflammatory causes of colic have higher mortality rates than conditions causing non-strangulating intestinal obstruction (Morris, 1991). Once endotoxin migrates across the damaged intestinal wall, it may take one of two paths. Endotoxin absorbed by the portal system is transported to the liver where resident hepatic macrophages remove it from circulation. If the influx of endotoxin from the damaged intestine exceeds the ability of the liver to remove it, endotoxin spills over into the systemic circulation. Alternatively, endotoxin may move across the intestinal wall into the peritoneal cavity, be absorbed by the lymphatics, and reach the systemic circulation by entering the major veins at the thoracic duct (Deventer et al., 1988).
Gram-negative bacteria are enveloped by an outer lipopolysaccharide-rich membrane. This lipopolysaccharide, also termed "endotoxin", is comprised of three structural units: an outer polysaccharide component which affords lipopolysaccharide its antigenic properties, a core region composed of several monosaccharides, and the innermost portion, termed lipid A, which affords the molecule its potent proinflammatory biological activities (Moore et al., 1981; Moore, 1981; Moore and Morris, 1992). Circulatory shock secondary to either Gram-negative sepsis or endotoxaemia is associated with a high mortality rate in equine veterinary medicine. Septicaemia occurs all too often in equine neonates, which frequently are immuno-compromised by inadequate passive transfer of colostral immunoglobulins (Koterba et al., 1984). Gramnegative bacterial infections of the uterus or other soft tissues also can lead to circulatory shock due to movement of endotoxin from the tissues into the blood stream. Most commonly, however, circulatory shock in horses is the result of diseases involving the gastrointestinal system which lead to the systemic absorption of endotoxin (King and Gerring, 1988). The intestines of the horse harbour a very large population of Gram-negativebacteria and thus the intestinal milieu normally contains a large amount of endotoxin. Although the gastrointestinal tract has a very efficient mucosal barrier to restrict endotoxin to the intestinal lumen, gastrointestinal diseases which compromise this mucosal barrier result in the transmural movement of endotoxin (Meyers et al., 1982). Endotoxaemic horses exhibit abnormal behaviour ranging from depression to acute bouts of pain. Physical examination findings often include tachycardia, tachypnoea, hyperaemic mucous membranes, decreased gastrointestinal sounds, and dehydration. Diseases which most commonly result in the development of endotoxaemia in horses are intestinal strangulation obstruction and inflammatory intestinal diseases.
Intestinal Diseases and Endotoxaernia Intestinal'strangulationobstruction occurs when both the intestinal lumen and the mesenteric blood vessels are 77
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Of greater importance, however, is the interaction of endotoxin with lipopolysaccharide binding protein (Ulevitch et al., 1990; Tobias and Ulevitch, 1986; Tobias and Ulevitch, 1993). This protein, which is synthesized by hepatocytes, normally exists in trace amounts ( ~ 0 . 5 pg/ml) in circulation, but its concentration may increase up to 100-fold during the acute phase response. Lipopolysaccharide binding protein binds avidly to the lipid A component of endotoxins from all Gram-negative bacteria and serves as an opsonin for removal of these bacteria from the circulation. This protein also is unique as it dramatically increases the ability of endotoxin to activate cells of the mononuclear phagocyte system (Tobias and Ulevitch, 1993; Wright et al., 1990). Thus, rather than serving as a nonspecific natural detoxifying system for endotoxin, the interaction of endotoxin with lipopolysaccharide binding protein markedly enhances the ability of endotoxin to cause damage to the host.
The pathophysiological responses to the short term administration of endotoxin have been well characterised in a variety of animals including the horse. Initially, the cardiovascular response to endotoxin includes vasoconstriction in the systemic and pulmonary vascular beds. Accordingly, tachycardia, tachypnoea, hypoxaemia, and respiratory alkalosis ensue. This phase is followed by profound systemic hypotension and loss of vascular integrity. Ultimately, the repetitive inflammatory insults lead to progressive endothelial dysfunction which can culminate in multi-organ failure (Bone, 1991a; Bone, 1991b). The haematological effects of endotoxaemia in horses and other animals are typified by haemoconcentration, early leukopaenia followed by leukocytosis, and thrombocytopaenia. In horses haemoconcentration occurs due to splenic contraction and loss of fluid from the vascular space resulting from the altered vascular permeability. The leukopaenia is due to neutrophil margination (Moore, 1988).Dysfunction of haemostasis and fibrinolysis also has been documented during endotoxaemia in several species. In horses, coagulopathy has been characterised during experimental endotoxin infusions, experimental models of intestinal strangulation, and during clinical diseases (colitis, colic) associated with endotoxaemia (Duncan et al., 1985; Pablo et al., 1983; Welch et al., 1992). This coagulopathy can progress to fulminant disseminated intravascular coagulation (Welch et al., 1992). The metabolic derangements associated with endotoxaemia have been well characterized in other species, but not in horses (Burrows, 1981). In general, endotoxaemia induces a transient hyperglycaemia that is followed by hypoglycaemia. With progressing circulatory shock, tissue hypoperfusion leads to lactic acidosis coincident with the shift of cellular metabolism toward anaerobic glycolysis. Alterations in lipid metabolism also have been characterized during endotoxaemia. These include increased plasma triglycerides, hepatic fatty acid synthesis, and very low density lipoprotein production (Adi, 1992).
Cellular Recognition of Endotoxin In recent years, it has become evident that endotoxin alone has few direct inflammatory actions and that the deleterious effects of endotoxin depend on the synthesis of numerous proinflammatory substances by a variety of cells, including endothelial cells, neutrophils, platelets and lymphocytes. The mononuclear phagocytethe is the cell responsible for many of the inflammatory responses during endotoxaemia (Mathison, et al., 1991; Mathison et al., 1992). Cell culture techniques have been used to characterize the responses of isolated equine peripheral blood monocytes, peritoneal, alveolar and mammary macrophages to endotoxin (Morris, 1991; Henry and Moore, 1988; MacKay and Lester, 1992; MacKay et al., 1991; Henry and Moore, 1991; Barton et al., in press; Morris, et al., 1990; Morris and Moore, 1987). The results of several recent studies indicate that recognition of endotoxin by mononuclear phagocytes occurs through both receptor and non-receptor mediated events (Morrison, 1985).The study of interactions of synthetic lipid A with macrophage membranes has led to the discovery of endotoxin scavenging receptors believed to function in the removal of endotoxin from the circulation (Hampton et al., 1991). Similarly, the leukocyte antigen CD18 binds endotoxin and may have an important function in the phagocytosis and degradation of endotoxin (Wright and Tong, 1986). Of more clinical importance, however, is the fact that the proinflammatory effects of endotoxin are fundamentally linked to the interaction of the lipopolysaccharide binding protein endotoxin complex with the mononuclear cell receptor known as CD14. This interaction leads to activation of the mononuclear cell and synthesis of the inflammatory mediators that cause the clinical signs of endotoxaemia (Ulevitch et al., 1990).
Interaction of Endotoxin with Plasma Proteins There is a large body of evidence indicating that endotoxin interacts both with circulating high density lipoproteins and a unique protein termed lipopolysaccharide binding protein (Ulevitch et al., 1990; Tobias and Ulevitch, 1986). Because of its marked hydrophobic chemical nature, endotoxin exists in aggregates when it first comes in contact with blood. These aggregates then disaggregate and the free endotoxin forms stable complexes with the high density lipoproteins. Although the formation of these complexes prolongs the half-life of endotoxin in the blood stream, the binding of endotoxin to the high density lipoproteins also markedly reduces the ability of the endotoxin to interact with cells. Presumably this reduction in the endotoxic activity of endotoxin is due to a nonspecific interaction of the lipid A component of endotoxin with the phospholipid portion of the high density lipoproteins.
Mediators of Endotoxaemia Mononuclear phagocytes (peripheral blood monocytes and macrophages) synthesize and release numerous inflammatory mediators which are responsible for the majority of pathophysiological responses to endotox78
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tions have been measured in adult horses and neonatal foals administered endotoxin (MacKay and Lester, 1992; Robinson et al., 1993). Tissue factor (thromboplastin) is a glycoprotein synthesised by monocytes and macrophages in response to endotoxin. Unlike most of the other mediators involved in the pathogenesis of endotoxaemia, tissue factor remains closely associated with its cell of origin. In fact, approximately 90% of the tissue factor remains on the surface of the cell. When exposed to coagulation factor VII, tissue factor initiates the extrinsic arm of the coagulation cascade. Consequently, tissue factor activity can be measured indirectly using a whole blood recalcification time assay or directly by measuring the ability of cell lysates to shorten the recalcification time of pooled plasma. Using these assays, increases in tissue factor activity have been identified in horses administered endotoxin and in horses with naturally occurring abdominal diseases characterised by endotoxaemia (Henry and Moore, 1991). The metabolites of arachidonic acid have received a great deal of attention over the past 10 years in research on endotoxaemia. Arachidonic acid is an important component of the phospholipid layer of cellular membranes. Damage to the membrane results in activation of phospholipase A,, which cleaves arachidonic acid from the phospholipids. The free arachidonic acid then is converted by cyclo-oxygenase to the very labile endoperoxide intermediates, and then to prostaglandin I,, prostaglandin E,, prostaglandin F,, or thromboxane A,. In general prostaglandins E, and I, relax vascular smooth muscle by increasing cAMP concentrations. In contrast, prostaglandin F,, and thromboxane A, decrease cAMP and cause vasoconstriction. Many of the early cardiopulmonary and behavioural effects of endotoxaemia in horses appear to be mediated by arachidonic acid metabolites. In particular, the dyspnoea, hypoxaemia, and pulmonary hypertension that characterize the early response to endotoxin administration are accompanied by a profound increase in the plasma concentration of the stable metabolite of thromboxane A,. Subsequently, the discoloration of the mucous membranes, prolongation of the capillary refill time, hypotension, and intermittent signs of abdominal pain are accompanied by increases in plasma concentrations of the stable metabolite of prostaglandin I,. Therefore, it appears that many of the initial responses to endotoxaemia are influenced by the generation and release of vasoconstrictive metabolites and the later effects are associated with metabolites that induce vasodilation.
aemia. Of the multitude of inflammatory mediators synthesized in response to endotoxin, several have been studied extensively in the equine species. These include tumour necrosis factor, interleukins 1 and 6, tissue factor, and the arachidonic acid metabolites. One of the earliest events subsequent to the interaction of the lipopolysaccharide binding protein endotoxin complex with CD14, is the synthesis of the cytokine known as tumour necrosis factor (Heumann et al., 1992). Tumour necrosis factor is a 17KD polypeptide which plays a pivotal role in endotoxic shock. Serum concentrations of tumour necrosis factor are increased in both clinical endotoxic shock and in experimental endotoxaemia in people and animals (Moore and Morris, 1992; Morris, 1991; Morris et al., 1990; Morris et al., 1991; Michie et al., 1988). In addition, the systemic administration of recombinant tumour necrosis factor to animals causes the same pathophysiological consequences as does administration of endotoxin (Tracey et al., 1986). Furthermore, the administration of anti-tumour necrosis factor antibodies to animals prior to inducing endotoxic or septic shock leads to a dramatic reduction in mortality rate (Beutler and Cerami, 1987). The biological activity of tumour necrosis factor is quite diverse. Animals given tumour necrosis factor intravenously develop shock characterized by progressive hypotension, haemoconcentration, metabolic acidosis, and disseminated intravascular coagulation which can eventually lead to death (Turner et al., 1989). Other properties attributed to this polypeptide include stimulation of interleukin-1 and interleukin-6 synthesis, the acute phase response, fever, prostaglandin synthesis, endothelial leukocyte adhesion, molecule expression and procoagulant activity. Interleukin-1 is the name now associated with a group of inflammatory mediators previously known as "endogenous pyrogen". Interleulun-1 is responsible for the febrile response to endotoxin and initiates arachidonic acid metabolism by stimulating phospholipase A, activity on the cell surface. Furthermore, interleukin-1 causes systemic hypotension, initiates the wastage of muscle mass for gluconeogenesis and synthesis of acute phase proteins by the liver, reduces food intake, and increases the stickiness of vascular endothelial cells. There is evidence that the hypotensive effect of interleukin-1 is mediated in part by increased nitric oxide production (Beasely et al., 1991). Circulating concentrations of interleukin-1B have been documented after administration of endotoxin to healthy human volunteers (Cannon et al., 1990)and rabbits (Wakabayashi et al., 1991).The biological effects of interleukin-1 and tumour necrosis factor are remarkably similar and these cytokines often have synergistic effects. Interleukin-6 is another cytokine produced primarily by monocytes and macrophages. Interleukin-6 is very important in the acute phase reaction to inflammatory stimuli and its synthesis is increased by tumour necrosis factor and interleukin-1. Although there is little evidence indicating that interleukin-6 produces deleterious effects on the host, circulating concentrations of this cytokine increase during endotoxaemia and have been used as a prognostic indicator in clinical cases. Serum concentra-
Treatment of Endotoxaernia Although our knowledge regarding the pathophysiol-
ogy of endotoxaemia has increased dramatically in the past 10 years, treatment of this problem in horses has changed more slowly. This delay may be due to the inherent complexity of the processes involved in clinical endotoxaemia, the exquisite sensitivity of the horse to endotoxin, and the cost of using certain therapeutic agents on patients weighing 500 kg. The more commonly used methods of treatment include non-steroidal anti79
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inflammatory drugs, corticosteroids, anti-endotoxin serum, and cardiosupportive agents. Non-steroidal anti-inflammatory drugs, which are effective by inhibiting cyclo-oxygenase, are the most common types of drugs used to combat endotoxaemia in horses. Of the large number of non-steroidal anti-inflammatory drugs presently available, flunixin meglumine, ketoprofen and phenylbutazone have received most attention. Of these, flunixin and ketoprofen appear to be most efficacious and are often used at doses less than those recommended by the manufacturers. Based on the results of one experimental study (Semrad et al., 1987), many clinicians elect to administer flunixin at 0.25 mg/kg TID. Although it has been suggested that ketoprofen inhibits both cyclo-oxygenase and lipoxygenase, data documenting the effect of ketoprofen on synthesis of lipoxygenase products in horses are lacking. Corticosteroids have been used in the treatment of endotoxic shock for decades and their use is as controversial now as it was in the past. Corticosteroids should be effective for two reasons: 1) they inhibit phospholipase A, activity and thus should prevent the generation of prostaglandins, thromboxanes, leukotrienes and platelet activating factor (Cohn, 1991) and 2) they prevent the synthesis of tumour necrosis factor (Morris et al., 1991). However, compared with flunixin in a model of lethal endotoxic shock in anaesthetised ponies, the corticosteroids were less effective at improving metabolic status and prolonging survival than flunixin (Ewert et al., 1985). Treatment of endotoxaemia has changed markedly in recent years with the availability of antibodies against the endotoxin molecule. These antibodies are directed against the conserved regions of endotoxin (lipid-A and the core polysaccharides) and thus provide protection against endotoxins from different bacterial species. To be maximally effective, these antibodies must bind endotoxin before it can cause inflammatory cells to synthesize and release the various mediators; thus, these antibodies should be gven early in the clinical course of disease. The results of clinical trials and experimental studies using anti-endotoxin antibodies have been conflicting. In a double-blind clinical study, horses treated with antiendotoxin plasma had an increased survival rate (87% vs. 53%), improved clinical appearance, and a shorter duration of hospitalization when compared to horses administered normal plasma (Spier et al., 1989). In an experimental study of endotoxaemia, treatment with antiendotoxin plasma failed to improve either clinical or clinicopathological parameters (Morris et al., 1986). Furthermore, in one experimental study young foals treated with the anti-endotoxin antibodies before initiation of endotoxaemia had higher serum concentrations of tumour necrosis factor and interleukin-6, lower neutrophil counts and more severe clinical signs than foals receiving only endotoxin (Durando and MacKay, 1992). Systemic hypotension occurs periodically in horses anaesthetised for emergency abdominal surgery. Presumably this problem is associated with the development, or worsening of, endotoxaemia. Treatment of hypotension under such circumstances usually involves the administration of either dopamine or dobutamine to
increase cardiac output and systemic arterial pressure. These drugs are selected because of their rapid onset and the fact that they have proved to be efficacious in horses (Trim et al., 1985; Swanson and Muir, 1986).
Furure Therapeutic Approaches With the substantial amount of data implicating cytokines, particularly tumour necrosis factor, in mediating the ill effects of endotoxin, there is a considerable amount of effort being expended to develop therapeutic agents directed against these cytokines. Consequently, monoclonal antibodies and soluble receptors are being evaluated as methods of preventing the deleterious effects of tumour necrosis factor. Two other promising areas of interest are the CD14 endotoxin receptor and lipopolysaccharide binding protein. If it were feasible to interfere with the binding of endotoxin with lipopolysaccharide binding protein or prevent the interaction of the endotoxin-lipopolysaccharide binding protein complex with CD14, it might be possible to remove the initial signal that stimulates the mononuclear phagocyte to synthesize and release the inflammatory mediators that cause the clinical syndrome of endotoxaemia.
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