The complement system is thought to be involved in the pathogenesis of numerous neurological diseases, although its precise role remains controversial. In this study we used orally active C5a receptor antagonists (PMX53 and PMX205) developed in our laboratories in a rat model of 3-nitropropionic acid (3-NP) -induced Huntington's disease. Administration of the C5a antagonists (10 mg/kg/day, oral) either 48 h pre- or 48 h post-toxin significantly reduced body weight loss, anorexia, and behavioral and motor deficits associated with 3-NP intoxication. Striatal lesion size, apoptosis, neutrophil infiltration, and hemorrhage were also significantly reduced in C5a antagonist-treated rats. Immunohistochemical analysis demonstrated marked deposition of C3 and C9, and up-regulation of C5a receptors on neuronal cells at the time of lesion formation. Inhibition of prostaglandins or TNF-alpha with ibuprofen or infliximab had no effect in this model. The C5a antagonists did not affect 3-NP-induced cell death when added directly to rat striatal neuronal cultures, indicating a secondary mechanism of action in vivo. Our findings demonstrate for the first time that complement activation in the brain, particularly C5a, is a key event in the pathogenesis of this disease model, and suggest a future role for inhibitors of C5a in the treatment of neurodegenerative diseases.
We have previously shown that complement factor 5a (C5a) plays a role in the pathogenesis of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats by using the selective,
Glucose consumption by the hoof explants was inhibited by 2-deoxyglucose and aminophenylmercuric acetate. The explants consumed relatively large amounts of glucose during the first 2 days of incubation and then little over the next 6 days.Despite the reduced glucose consumption, the hoof explants did not separate over 8 days of incubation. The results indicated that the integrity of the hoof explants was initially dependent on consumption of glucose and provide a possible explanation for the development of laminitis caused by conditions such as carbohydrate overload, acute inflammatory conditions, corticosteroid therapy and hyperlipidaemia. It would be expected that these conditions would induce a major hormonally-mediated metabolic shift away from glucose consumption by many peripheral tissues. It is suggested, therefore, that if the metabolic change occurred faster than the hoof tissue could adapt to an alternative energy substrate, then hoof separation and laminitis would occur.
Summary
A method for culturing explants of lamellar hoof was developed to investigate the process of lamellar separation that occurs in laminitis. Explants, consisting of hoof wall, dermal and epidermal lamellae and the adjacent sub‐lamellar connective tissue remained intact when cultured in tissue culture medium for 2 days. However, when cultured in the presence of the matrix metalloproteinase (MMP) activator aminophenylmercuric acetate (APMA), the lamellae separated when tension was applied by pulling the hoof wall in an opposite direction to the connective tissue. The separation occurred between the epidermal basal cells and the basement membrane therefore mimicking the lesion of laminitis. Electrophoresis of culture medium from control hoof explants into gradient polyacrylamide gels co‐polymerised with gelatin revealed that the explants had produced 2 gelatinases of molecular weight 92 and 72 kDa corresponding to EqMMP‐9 and EqMMP‐2 respectively. Minor bands of lower molecular weight were the active forms of these enzymes. The zymograms of culture medium from APMA treated explants revealed an increase in the amount of active MMPs. Equine polymorphs cultured for 2 days produced only EqMMP‐9. Lamellar explant medium from horses with acute laminitis contained increased amounts of zymogen and active EqMMP‐2 and EqMMP‐9 particularly in explants from the fore hooves. Zymography of homogenates of normal lamellar hoof tissue revealed only EqMMP‐2 and a minor active band. However, homogenates of lamellar tissue from horses with laminitis showed that EqMMP‐9 was present as well as increased EqMMP‐2 in both zymogen and active forms. Addition of the MMP inhibitor batimastat (BB‐94) to the culture medium of APMA treated explants prevented lamellar separation. BB‐94 incubated with polyacrylamide strips containing the MMPs from laminitis affected lamellar explants inhibited enzymatic activity at a concentration of 1 mmol/l. It is concluded that activation of MMPs may be responsible for the lamellar separation seen in laminitis and that MMP inhibitors may be useful clinically for preventing this process.
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