Research ReportBlood micromolar concentrations of kaempferol afford protection against ischemia/reperfusion-induced damage in rat brain
Introduction
Neurodegeneration induced by ischemic brain insults has been shown to follow characteristic spatio-temporal patterns, and this has lead to the identification of areas displaying rapid necrotic death surrounded by areas undergoing slowly developing cell death (Hossmann, 1994, Lehrmann et al., 1997, Iadecola, 1997). Extracellular matrix degradation upon activation of matrix metalloproteinases (MMP) has been shown to take place in transient focal cerebral ischemia, which likely plays a major role in the spatial spreading of neurodegeneration in brain, largely due to the activation of MMP-2 and MMP-9 (Romanic et al., 1998, Heo et al., 1999, Asahi et al., 2000, Gu et al., 2002, Gu et al., 2005, Chang et al., 2003). These experimental observations are consistent with the occurrence of different types of neuronal death after brain ischemia/reperfusion insults developing with very different time windows, like glutamate-mediated excitotoxicity (Szatkowski and Atwell, 1994) or apoptosis (Saito et al., 2004, Copin et al., 2005, Gu et al., 2005). This is particularly relevant to protect against brain degeneration after ischemia/reperfusion, because the slow time course of neurodegeneration opened a realistic time window for the application of protective therapies. Basal ganglia, particularly the striatum and the hippocampus, and cortical neurons have been shown to be the brain areas most sensitive to ischemia/reperfusion (Lehrmann et al., 1997, Gu et al., 2005). Caspases involved in apoptotic cell death in experimental models of focal and global brain ischemia have been reported to follow different temporal profiles of activation in rat brain (Cao et al., 2002, Cho et al., 2003). For instance, while caspase-3 peaked in the penumbral cortex at 6–12 h following ischemia, significant induction and levels of proteolytically activated caspase-9 can be better seen after 24 h post-ischemia (Cho et al., 2003). Moreover, administration of the caspase-9 inhibitor Z-Leu-Glu(Ome)-His-Asp(Ome)-FMK after focal cerebral ischemia has been reported to reduce the infarct volume by 49% and to improve neurological outcome (Mouw et al., 2002). This experimental observation is consistent with the proposal of caspase-9 as a mediator of oxidative stress-induced apoptosis after focal cerebral ischemia (Saito et al., 2004).
On the other hand, it has been shown that peroxynitrite and other reactive oxygen species play a major role in brain damage associated with ischemia/reperfusion (Chan, 1996, Iadecola, 1997, Gu et al., 2002, Saito et al., 2004). Plant flavonoids are peroxynitrite scavengers of low toxicity for mammals found in many vegetables commonly used in human nutrition (Manach et al., 1996, Hollman and Katan, 1997). Several studies have shown protective effects of different flavonoids against ischemia/reperfusion brain damage after transient focal cerebral ischemia or global cerebral ischemia in model experimental animals, e.g. Crataegus flavonoids in gerbils (Zhang et al., 2004), epigallocatechin gallate in rats (Choi et al., 2004) and gerbils (Lee et al., 2004), ginsenosides in rats (Tian et al., 2005, Zhou et al., 2006), isoliquiritigenin in rats (Zhan and Yang, 2006), Scutellaria baicalensis Georgi flavonoids in rats (Shang et al., 2006) and resveratrol in rats (Tsai et al., 2007). In a previous study, we showed that micromolar concentrations of kaempferol afforded full protection against the apoptosis of rat cerebellar granule neurons in culture induced by K+-deprivation in the extracellular medium (Samhan-Arias et al., 2004), a well established model for neuronal apoptosis (D'Mello et al., 1993, Nardi et al., 1997, Martin-Romero et al., 2000, Martin-Romero et al., 2002). Other flavonoids, such as apigenin and quercetin, were found to be much less effective than kaempferol (Samhan-Arias et al., 2004). In contrast, kaempferol protection against glutamate-induced excitotoxicity to neurons in culture is relatively low at micromolar concentrations of this flavonoid (Ishige et al., 2001, Kim et al., 2001).
Transient focal cerebral ischemia in rats can be readily attained by middle cerebral artery occlusion (Longa et al., 1989, Belayev et al., 1996). We have recently developed a new surgical approach, which we called “transfemoral selective ‘intraluminal wiring’ technique” (Sun et al., 2005), by selective endovascular placement of a guidewire into a target vessel under fluoroscopic guidance. Transfemoral selective ‘intraluminal wiring’ is a minimum invasive technique that minimized subarachnoid hemorrhage (Sun et al., 2005), which has been reported to occur with the intraluminal thread model of middle cerebral artery occlusion in rats (Longa et al., 1989, Schmid-Elsaesser et al., 1998).
The major aim of this work is to evaluate the efficiency of acute administration of micromolar concentrations of kaempferol in blood as neuroprotector agent against brain degeneration after transient focal ischemia/reperfusion elicited by middle cerebral artery occlusion in rats.
Section snippets
Protection by micromolar kaempferol in blood against brain damage after ischemia/reperfusion
The low toxicity of kaempferol to Wistar rats was ascertained before running experiments with transient focal cerebral ischemia. None of the adult Wistar rats (n = 6) treated during 3 days with intraperitoneal injections of 11 mg kaempferol per day died within a 2 week observation period, and this was consistent with the lack of toxicity of this flavonoid noticed previously (Manach et al., 1996, Hollman and Katan, 1997). Taking into account that kaempferol affords nearly complete protection of
Discussion
The regional brain damage after transient focal ischemia/reperfusion in our rat model shows that basal ganglia, mostly the striatum, the hippocampus and close areas of the neocortex are by far the most sensitive areas to this insult, in excellent agreement with earlier works (Lehrmann et al., 1997, Gu et al., 2005). In addition, damage at the corpus callosus was also noticed, although in this case it is likely that there is a large contribution coming from the slice processing, as revealed by
Experimental procedures
Adult male Wistar rats weighting 350–400 g were used in this study. Animal care and all experimental procedures were carried out in accordance with guidelines of the European Communities Council Directive (86/609/EEC). Protocols were approved by the Ethics Committee for Animal Research of the local government.
Acknowledgments
Professor J. Usón, Scientific Director of the CCMI (Cáceres, Spain) for helpful discussions. Supported by grants no. SAF2003-08275 from the Spanish MEC and SCSS0633 and 3PR05A078 from Junta de Extremadura. AKSA holds a predoctoral fellowship of Junta de Extremadura.
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