Putative Neuroprotective Compound, AM-36, but not Minocycline, attenuates Neutrophil Migration into Mouse Dorsal Skin Air Pouch

Bevyn Jarrott1, Hannah Culver2, Stella O’Donnell2. 1Florey Neuroscience Institutes, Parkville, Victoria, Australia, 2School of Medical Science, Griffith University, Gold Coast, Queensland, Australia.

It has been well established, in studies on inflammation in peripheral tissues, that neutrophils migrate from the circulation, become activated and initiate secondary injury due to the secretion of, for example, free radicals as well as myeloperoxidase and NADPH oxidase enzymes. Neutrophil-induced damage has been found to occur in the brain during ischemic events, such as stroke (Weston et al. 2006) and can occur many hours/days after the initial ischemic event as demonstrated qualitatively by immunohistochemistry. Hence, it can be hypothesised that prevention of neutrophil entry into brain might be a potential mechanism for neuroprotective drugs. In this study, two compounds with different mechanisms of action which have been shown to be neuroprotective in rodents, viz AM-36 and minocycline, have been studied for their ability to attenuate neutrophil migration. As quantification of neutrophils in brain parenchyma is not feasible, this preliminary study has used the approach of Perretti & Flower (1993) to quantify the effect of compounds on neutrophil migration. Dorsal skin air pouches were prepared in groups of BALB/c mice and, at least six days later, either 20 ng murine rIL-1 or vehicle (carboxymethylcellulose 0.5%) was injected into the pouch. Groups of mice were given a test compound (or normal saline) i.v. 2 hours before the IL-1. Four hours later, cells in the pouch lavage fluid were spun onto a cytospin slide, stained and the number of neutrophils counted using standard criteria. IL-1 caused a highly significant increase in pouch neutrophils compared with controls (mean ± SEM cells (106), 3.31 ± 0.423, n= 17 cf 0.588 ± 0.1103, n=15, P<0.001). Mice treated with either dexamethasone (50 μg/mouse) or fucoidin (3mg/mouse), compounds known to attenuate neutrophil migration, had approximately 50 % reductions (P<0.05) in pouch neutrophils. Mice given AM-36 (10 mg/kg) (a sodium channel blocker/antioxidant - Weston et al. 2006), had a significant reduction in pouch neutrophils (2.10 ± 0.324 ×106, n=12) compared with IL-1 controls. But minocycline (50 mg/kg) (a matrix metalloproteinase inhibitor/antioxidant) did not reduce neutrophil migration at this or double the dose.

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Weston R.M. et al. (2006) Br. J. Pharmacol., 812, 849-852.