2-(-2-benzofuranyl)-2-imidazoline induces Bcl-2 expression and provides neuroprotection against transient cerebral ischemia in rats

Abstract

Stroke is the third leading cause of death and disability in North America and is becoming the most frequent cause of death in the rapid developing China. Protecting neurons in order to minimize brain damage represents an effective approach towards stroke therapeutics. Our recent study demonstrated that 2-(-2-benzofuranyl)-2-imidazoline (2-BFI), a ligand for imidazoline I₂ receptors, is potently neuroprotective against stroke, possibly through transiently antagonizing NMDA receptor activities. In this study, we further investigated the characteristics and mechanisms of 2-BFI-mediated neuroprotection using a rat stroke model of transient occlusion of the middle cerebral artery. Here, we show that 2-BFI was most effective at the dose of 3 mg/kg in vivo, with significantly reduced brain infarct size and improved neurological deficits. Lower doses of 2-BFI at 1.5 mg/kg, or higher dose of 2-BFI at 6 mg/kg, were either not effective, or toxic to the brain, respectively. Treating stroke rats with 3 mg/kg 2-BFI significantly reduced the number of TUNEL positive cells and preserved the integrity of subcellular structures such as nuclear membranes and mitochondria as shown under the electron microscope, confirming neuroprotection. Most interestingly, 2-BFI-treated brains exhibited significant expression of Bcl-2, a gene with a known function in neuroprotection. Taken together, these studies not only demonstrated that 2-BFI at 3 mg/kg was effective in neuroprotection, but also, for the first time, showed that 2-BFI provided neuroprotection through up-regulating the neuroprotective gene Bcl-2. 2-BFI can be further developed as a therapeutic drug for stroke treatment.

Introduction

Stroke is becoming the most frequent cause of death in the rapid developing China (Liu et al., 2007, Truelsen & Bonita, 2008) and is the third leading cause of death in the developed North America (Hou et al., 2008, Hou & MacManus, 2002). Stroke survivors often carry long term disabilities which pose an enormous economical burden to the health care system. Despite worldwide efforts to develop ways to treat stroke and to protect brain functions, sadly, there are so far no clinical effective therapeutics against stroke, which represents one of the largest unmet medical needs.

Recent studies, including those of our own, showed that ligands to the type 2 immidazoline receptor (I₂R) are potently neuroprotective against hypoxia-ischemia damage both in vitro and in vivo (Han et al., 2009, Jiang et al., 2010). There are three types of imidazoline receptors based on imidazoline binding sites: I₁ site, labeled by clonidine and I₂ site labeled by idazoxan and other selective molecules such as 2-BFI. A putative I₃ site has also been described (Head and Mayorov, 2006). I₁R, found in the rostral ventrolateral medulla, mediates the sympathoinhibitory actions of imidazolines to lower blood pressure; I₂R represents an allosteric binding site of monoamine oxidase, while I₃R is known to regulate insulin secretion from pancreatic beta cells. So far only ligands to I₂R, such as 2-BFI and isazoxan, have been found to prevent ischemia-hypoxia induced brain damage both in vitro and in vivo (Dong et al., 2008, Jiang et al., 2010, Milhaud et al., 2000, Olmos et al., 1996). Possible mechanisms of neuroprotection by I₂R ligands are believed through ameliorating excitotoxicity by direct modulating NMDA receptor-mediated calcium influx in neurons (Jiang et al., 2010). I₂R ligands directly bind to NMDA receptors (Olmos et al., 1996) and block NMDA receptor-gated calcium channels (Dong et al., 2008, Milhaud et al., 2000, Milhaud et al., 2002).

Amongst I₂R ligands, 2-BFI was found to be the most effective in neuroprotection against glutamate toxicity in vitro (Jiang et al., 2010) and cerebral ischemia in vivo (Han et al., 2009). Our recent studies demonstrated that 2-BFI not only transiently and reversibly block calcium influx in cultured cortical neurons in response to glutamate-mediated excitotoxicity, but also provided a much longer lasting neuroprotection against glutamate toxicity in comparison with other I₂R ligands, such as idazoxan (Jiang et al., 2010). Therefore better understanding the dose response and mechanisms of 2-BFI-mediated neuroprotection is important in developing this compound further as a drug for stroke therapeutics.

Activation of components of the apoptotic machinery is a characteristic of cerebral ischemia-induced neuronal death. Depending on the energy level and its supply, neurons exhibit cell death features appearing on the continuum between typical necrosis and apoptosis. Expression of neuroprotective genes, such as Bcl-2, is known to play a key role in neuronal survival during cerebral ischemia. To gain further understanding of the role of 2-BFI in cerebral ischemia in vivo, in the present study, we firstly determined the optimal effective dose of 2-BFI in a rat model of transient focal cerebral ischemia and secondly examined the mode of neuronal death and expression of Bcl-2 following transient cerebral ischemia with 2-BFI treatment.