DY-9760e, a Calmodulin Antagonist, Reduces Brain Damage after Permanent Focal Cerebral Ischemia in Cats

Abstract

Although the cellular pathophysiological mechanisms underlying ischemic brain damage remain to be fully clarified, accumulating evidence suggests that an excessive elevation of intracellular Ca 2ϩ in neurons is a primary mechanism by which cells become injured following cerebral ischemia. 1-3)An overload of Ca 2ϩ in neurons disrupts the ionic balance and activates various Ca 2ϩ -dependent enzymes. Treatment of ischemic animals with compounds that block Ca 2ϩ entry through Ca 2ϩ -permeable channels has been shown to provide protection against the consequences of ischemia in laboratory animals, 4-6) but these treatments have not been found suitable for use in humans. 7-9)Calmodulin is a major Ca 2ϩ -binding protein found mainly in the central nervous system. 10,11) Since many of the pathways through which Ca 2ϩ acts involve Ca 2ϩ /calmodulin signaling systems, including calmodulin-dependent enzymes, inhibition of calmodulin may be a possible alternative approach towards the treatment and prevention of post-ischemic injury. In fact, it has been reported that Ca 2ϩ -bound calmodulin, which is an active form, is increased following ischemia and regional changes correlate with regions of ischemic neuronal damage.12) Furthermore, calmodulin antagonists protect cultured neurons from cell death induced by glutamate, an excitatory amino acid, and protect hippocampal CA1 neurons against hypoxia/hypoglycemia in organotypic cultures. 13,14) DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) is a calmodulin antagonist that is 2 to 70 times more potent than W-7, a well-known calmodulin antagonist, and can protect neurons against neuronal cell death elicited by Ca 2ϩ overload. [15][16][17] This compound ameliorates brain injury that occurs after transient or permanent focal ischemia and microembolization in rats. [18][19][20][21][22] Although these studies in various rodent models show a hopeful characteristic of DY-9760e, it is subsequently important to assess if it exerts a protective effect in a large animal model of focal cerebral ischemia. Cats, a gyrencephalic species in common with humans, have long been used both in pathophysiological studies for cerebral ischemia and in investigations for the protective efficacy of agents. [23][24][25][26][27] Therefore, the present study was designed to evaluate the potential acute therapeutic effect of DY-9760e on ischemic injury resulting from permanent middle cerebral artery (MCA) occlusion in cats. MATERIALS AND METHODSAnimals Twenty-three male Ico: Fec Eur (Tif) cats (IFFA-CREDO, L'arbresle, France), weighing 2.7 to 3.3 kg, were used in this study. The animals were maintained on canned food (CK, Oriental Yeast, Tokyo, Japan), solid food (NK-C, Nihon Nosan, Yokohama, Japan) and tap water, which were given once a day under a constant 12-h dark-light cycle. All experimental procedures were performed in accordance with the in-house guidelines of the Institutional Animal Care and Use Committee ...