Shock/sepsis/trauma/critical careThe Effects of Riluzole on Neurological, Brain Biochemical, and Histological Changes in Early and Late Term of Sepsis in Rats
Introduction
Sepsis is a generalized inflammatory response, which involves organ systems remote from the locus of the initial infectious insult [1]. One of the underlying mechanisms of sepsis is thought to be the oxidative damage due to the generation of free radicals. Recent studies have shown that sepsis is associated with enhanced generation of reactive oxygen species (ROS), which lead to multiple organ dysfunctions [2]. One of the complications of sepsis is central nervous system dysfunction that leads to septic encephalopathy, a situation of mental disturbances, and has a negative influence on survival [3]. Septic encephalopathy is the most common form of encephalopathy among patients in intensive care units. Septic encephalopathy possibly arises from the action of inflammatory mediators in the brain or a cytotoxic response of brain cells to these mediators [4].
The release of endotoxin (lipopolysaccharide, LPS) from bacteria is generally believed to be the initial event in the development of sepsis. Activation of macrophages and cytokines by endotoxin and the subsequent formation of reactive oxygen and nitrogen species have central pathogenic importance in various inflammatory diseases including sepsis. LPS activates inflammatory cells of the myeloid lineage that subsequently amplify the inflammatory response by releasing various cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1β. This systemic inflammatory cascade results in polymorphonuclear leukocytes (PMNs) sequestration in the various organs. Subsequent PMN extravasation can lead to vascular endothelium dysfunction as well as axonal damage [5].
Endothelium is known to regulate transvascular flux of fluid, nutrients, and mediators [6, 7]. The endothelium is the major target of sepsis-induced events and endothelial dysfunction is one of the underlying mechanisms of the pathology of sepsis. During septic shock, the breakdown of endothelial barrier function contributes to the loss of fluid into the extravascular space leading to life-threatening edema in the lung, kidney, and brain of the septic patient [8]. The sepsis associated hypoxia and inflammation are linked to an alteration in the production of ROS and nitrogen species. ROS-induced oxidative stress plays a significant role in endothelial damage that leads to tissue injury. The initiation of lipid peroxidation, direct inhibition of mitochondrial respiratory chain enzymes, and other oxidative protein modifications contribute to the cytotoxic effect of ROS [8].
Glutamate, the major excitatory amino acid in the brain, is known to play an important role in blood brain barrier (BBB) permeability, brain edema, and oxidative damage in pathological conditions. Glutamate-induced neuronal death can be mediated by (1) activation of NMDA subtype of glutamate receptor, resulting in Ca2+ and/or Na+ overload of the neuron; (2) activation of AMPA receptors, resulting in Ca2+ and/or Na+ overload of the neuron; and (3) glutamate inhibition of cysteine uptake, resulting in oxidative stress [9]. Also a possible mediating event is mitochondrial dysfunction. Inhibition of the respiratory chain results in neuronal adenosine triphosphate depletion, decrease in plasma membrane Na+ gradient and membrane potential, causing release of glutamate and NO, which together increase the oxidative stress. Recently, Gilgun-Sherki et al. [10]) have emphasized the importance of antioxidant therapy for neuroprotection and suggested that there was a need for antioxidant drugs that penetrate BBB.
It has previously been shown that glutamate antagonists have beneficial effects in sepsis, ischemia ,and trauma models. Riluzole, a glutamate release inhibitor, has been shown to have psychotropic, anticonvulsant, hypnotic, anesthetic, and neuroprotective effects, and is used against neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) [11, 12, 13]. Riluzole is the first FDA approved drug for the treatment of ALS. It is well-absorbed (approximately 90%), with average absolute oral bioavailability of about 60%. Pharmacokinetics are linear over a dose range of 2 to 100 mg given every 12 h. The mean elimination half-life of riluzole is 12 h after repeated doses. Riluzole is highly bound (96%) to plasma proteins, binding mainly to serum albumin and to lipoproteins. Riluzole is extensively metabolized to 6 major and a number of minor metabolites, not all of which have been identified. Some metabolites appear pharmacologically active in in vitro assays. The metabolism of riluzole is mostly hepatic and consists of cytochrome P450-dependent hydroxylation and glucuronidation. With multiple-dose administration, riluzole accumulates in plasma by about 2-fold and steady-state is reached in less than 5 d [11, 14].
It has been suggested that riluzole exerts its effects via the inhibition of glutamate release and GABA uptake in the striatal synaptosomes [15, 16] and blockade of voltage-dependent sodium channels [17, 18]. Riluzole has also additional antioxidant effects [19, 20, 21].
In the light of this knowledge, the aim of our study was to investigate the time course of putative protective effect of riluzole against sepsis-induced injury in cerebral cortex and cerebellum.
Oxidative stress was assessed by the parameters of malondialdehyde (MDA), an index for lipid peroxidation, and glutathione (GSH), which is an important constituent of intracellular protective mechanisms against various noxious stimuli, including oxidative stress.
Section snippets
Animals and Protocol for the Induction of Sepsis
Wistar albino rats of both sexes, weighing 180 to 220 g, were fasted for 12 h, but allowed free access to water before the experiments. The animals were kept in individual wire-bottom cages, in a room at a constant temperature (22 ± 2°C) with 12-h light and dark cycles, and fed standard rat chow. The study was approved by the Marmara University School of Medicine Animal Care and Use Committee (2004-76.2003.MAR).
The rats were divided into the following 4 groups of 12 rats: 1, vehicle-treated
Results
Sepsis significantly affected survival, with survival rates of 89%, 50%, and 28% at 6 h, 24 h, and 48 h, respectively. Survival was improved in riluzole-treated septic rats to 94%, 72%, and 50% at the same time points (Table 2). After the CLP operation, rats were followed for 48 h to evaluate weight loss, rectal body temperature, leukocyte counts, plasma TNF-α levels, and neurological examination scores; 24 h after the operation there was an insignificant loss of weight in all animals, but at
Discussion
Sepsis caused biochemical and histological changes in the brain that could be observed at 6 h. These changes were most severe in the 48th h resulting in poor prognosis and survival. Riluzole treatment significantly improved survival rate and prognosis of the disease as assessed by histological, biochemical, and neurological parameters.
Sepsis caused an increase in the leukocyte counts in 48 h, and riluzole treatment did not seem to have an effect on it. The standard error of sepsis group was
Acknowledgments
The authors gratefully acknowledge the support given for this work by the Marmara University Research Fund (project no: SAG-DKR-120905-0184).
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