Neuroprotective effect of fucoidin on lipopolysaccharide accelerated cerebral ischemic injury through inhibition of cytokine expression and neutrophil infiltration
Introduction
Ischemic stroke, which is induced by the loss of blood flow, causes a cascaded damage in tissues: excitotoxicity and oxidative damages, dysfunction of blood–brain barrier (BBB) and post-ischemic inflammation [1], [2], [3], [4]. The traffic of leukocytes from blood vessels into inflammatory sites is a co-operative multistep process involving an initial leukocyte rolling on endothelium mediated via the selectin family of adhesion molecules [5]. Leukocyte infiltration in cerebral ischemia has been studied in vivo [6], [7], [8], and inflammatory cell recruitment in an ischemic brain during reperfusion increases tissue injury [9], [10]. Cerebral damage is regulated by soluble pro-inflammatory mediators, such as cytokine, chemokine and adhesion molecules [3], [11].
Lipopolysaccharide (LPS) represents characteristic components of the cell envelope of Gram-negative bacteria. LPS is considered to be a typical inducer of inflammation and induces the release of pro-inflammatory cytokines such as TNF-α and IL-8 through the binding of its receptors on the cell membrane [12], [13]. Released cytokines or chemokines are thought to contribute to developing the infiltration of peripheral inflammatory cells such as neutrophils [14], [15]. Therefore, the inhibition of leukocyte recruitment can reduce brain injury [16], [17].
Inflammation plays a central role in the pathogenesis of cerebral ischemia and secondary damage [18]. It has been reported that, in the parenchyma of the brain, LPS induced activated microglia can rapidly release cytokines that activate the endothelium to recruit leukocytes, which lead to neuronal injury [19]. In addition, LPS induced activated microglia can contribute to amplified local inflammation and exacerbated brain injury [20], [21]. We have reported that, in cerebral ischemia, activated microglia induced by LPS microinjection into the corpus callosum accelerated cerebral ischemic injury in the rat [22].
Fucoidin is a sulfated polysaccharide which is one of the main constituents of brown seaweeds. As a potent inhibitor of leukocyte rolling, the physiological and biochemical effects of fucoidin have been examined in several animal studies. For instance, fucoidin reduces the recruitment of leukocytes, and levels of TNF-alpha and IL-1 in a rabbit model of meningitis [23], and the migration of neutrophils and eosinophils to sites of inflammation are also inhibited by fucoidin [24], [25]. In addition, fucoidin can reduce neutrophil infiltration and protect heart from myocardial injury after ischemia-reperfusion [26]. However, few studies have focused on effects of fucoidin on cerebral ischemia. Therefore, in the present study, we investigated the effect of fucoidin administration on neuronal injury and changes in cytokines in LPS accelerated cerebral ischemic injury.
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Animals
Male Sprague–Dawley rats weighing between 260 and 270 g were purchased from Charles River Laboratories (Seoul, Korea) and kept on a 12 h light/dark cycle with ad libitum access to food and water. The rats were acclimated to their environment for 5 days before use for experiments.
Microinjection of LPS into the corpus callosum
The rats were anesthetized with chloral hydrate (300 mg/kg) and positioned in a small-animal stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) to conform to the brain atlas (Paxinos and Watson, 1982). LPS was
Physiological parameters
Physiological variables are summarized in Table 2. The physiological parameters, including mean arterial blood pressure, blood gases, blood pH and serum glucose, were not significantly different in all the groups at 30 min both before MCAO and after reperfusion.
The levels of IL-8, CXCR1 and CXCR2 mRNA
In the LPS-group, IL-8 level was increased 6 h after LPS injection, and it was at its highest 9 h after LPS injection, thereafter it was gradually decreased until 24 h after LPS injection (Fig. 1A and B). In the LPS/fucoidin-group, IL-8
Discussion
It is well known that inflammation plays a central role in the pathogenesis of cerebral ischemia and in its secondary damage [18], [28], [29]. It is characterized by peripheral leukocyte infiltration and neurotoxicity to the cerebral parenchyma [30]. Leukocytic infiltration, particularly the infiltration of polymorphonuclear neutrophils (PMNs) is critical for the deleterious aspects of inflammation in stroke [31], [32]. Activated PMNs may contribute to ischemic brain injury by production of a
Conflicts of interest
The authors have no financial conflicts of interest.
Acknowledgments
This work was supported by a grant (2010K000823) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology, the Republic of Korea, and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0007307).
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