P2X7 receptor activation ameliorates CA3 neuronal damage via a tumor necrosis factor-α-mediated pathway in the rat hippocampus following status epilepticus

Ji-Eun Kim 0 1 Hea Jin Ryu 0 Tae-Cheon Kang 0 0 Department of Anatomy & Neurobiology, Institute of Epilepsy Research, College of Medicine, Hallym University , Chunchon, Kangwon-Do 200-702 , South Korea 1 Ji-Eun Kim, Department of Neurology, UCSF, and Veterans Affairs Medical Center , San Francisco, California 94121 , USA Background: The release of tumor necrosis factor-a (TNF-a) appears depend on the P2X7 receptor, a purinergic receptor. In the present study, we addressed the question of whether P2X7 receptor-mediated TNF-a regulation is involved in pathogenesis and outcome of status epilepticus (SE). Methods: SE was induced by pilocarpine in rats that were intracerebroventricularly infused with saline-, 2',3'-O(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), adenosine 5'-triphosphate-2',3'-dialdehyde (OxATP), A-438079, or A-740003 prior to SE induction. Thereafter, we performed Fluoro-Jade B staining and immunohistochemical studies for TNF-a and NF- B subunit phosphorylations. Results: Following SE, P2X7 receptor agonist (BzATP) infusion increased TNF-a immunoreactivity in dentate granule cells as compared with that in saline-infused animals. In addition, TNF-a immunoreactivity was readily apparent in the mossy fibers, while TNF-a immunoreactivity in CA1-3 pyramidal cells was unaltered. However, P2X7 receptor antagonist (OxATP-, A-438079, and A-740003) infusion reduced SE-induced TNF-a expression in dentate granule cells. In the CA3 region, BzATP infusion attenuated SE-induced neuronal damage, accompanied by enhancement of p65-Ser276 and p65-Ser311 NF- B subunit phosphorylations. In contrast, OxATP-, A-438079, and A-740003 infusions increased SE-induced neuronal death. Soluble TNF p55 receptor (sTNFp55R), and cotreatment with BzATP and sTNFp55R infusion also increased SE-induced neuronal damage in CA3 region. However, OxATP-, sTNFp55R or BzATP+sTNFp55R infusions could not exacerbate SE-induced neuronal damages in the dentate gyrus and the CA1 region, as compared to BzATP infusion. Conclusions: These findings suggest that TNF-a induction by P2X7 receptor activation may ameliorate SE-induced CA3 neuronal damage via enhancing NF- B p65-Ser276 and p65-Ser311 phosphorylations. - Background Status epilepticus (SE) is a medical emergency with significant mortality [1]. SE has been defined as continuous seizure activity, which causes neuronal cell death [2,3], epileptogenesis [3] and learning impairment [4]. Cytokines are critical mediators of specific inflammatory responses and immune reactions in the brain [5]. Tumor necrosis factor-a (TNF-a) is a 17-kDa protein that is mainly produced by activated macrophages and T cells of the immune system. TNF-a is expressed at low levels in the normal brain and is rapidly upregulated in glia, neurons and endothelial cells in various pathophysiological conditions [6]. TNF-a shows various effects on brain function depending on its local tissue concentration, the type of target cells, and especially the specific receptor subtype: TNF receptor I, or p55 receptor (TNFp55R); and TNF receptor II, or p75 receptor (TNFp75R) [7,8]. Basically, TNF-related signal transduction pathways involve NF- B binding activity for TNFp55R contributing to cell death [9] and downstream signaling via TNFp75R involves activation of p38 mitogen-activated protein kinase to promote neuronal survival [10]. However, TNFp55R deficiency enhances KA-induced excitotoxic hippocampal injury in mice [11]. Furthermore, Marchetti et al. [12] has reported that TNFp75R-induced persistent NF- B activity is essential for neuronal survival against excitotoxic stress. Therefore, TNF-a clearly possesses the ability to simultaneously activate both cell death and cell survival pathways, and this balance ultimately determines whether TNF-a promotes neurodegeneration or neuroprotection. On the other hand, P2X7 receptor, a purinergic receptor, plays a role in intercellular signaling involving ATP and glutamate release. Furthermore, the release of TNFa appears to be dependent on the P2X7 receptor. Indeed, treatment of microglia in neuron-microglia cocultures with the P2X7 agonist 2-3-O-(benzoyl-benzoyl) ATP (BzATP) leads to significant reductions in glutamate-induced neuronal cell death, and either TNF-a converting enzyme inhibitor or anti-TNF-a IgG readily suppresses this protective effect [13]. In contrast, Choi et al. [14] have reported that the P2X7 receptor antagonist, oxidized ATP (OxATP), is effective in attenuating LPS-induced neuronal damage. These findings encouraged us to speculate that P2X7 receptor-mediated TNFa regulation is involved in outcomes of SE. In the present study, therefore, we address the question of whether the effects of P2X7 receptor on the TNF-a system represent general features of SE-induced neuronal death in the hippocampus following SE. Methods Experimental animals and chemicals This study utilized the progeny of Sprague-Dawley (SD) rats (male, 9-11 weeks old) obtained from Experimental Animal Center, Hallym University, Chunchon, South Korea. The animals were provided with a commercial diet and water ad libitum under controlled temperature, humidity and lighting conditions (22 2C 55 5% and a 12:12 light/dark cycle with lights). Procedures involving animals and their care were conducted in accord with our institutional guidelines that comply with NIH Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, 1996). In addition, we have made all efforts to minimize the number of animals used and their suffering. All reagents were obtained from Sigma-Aldrich (St. Louis, MO), except as noted. Intracerebroventricular drug infusion Rats were divided into eight groups, treated with either (1) saline, (2) vehicle (0.1% DMSO/saline, v/v), (3) BzATP (5 mM in saline), (4) OxATP (5 mM in saline), (5) A-438079 (10 M in saline; Tocris Bioscience, Ellis-ville, MO), (6) A740003 (10 M in 0.001% DMSO/saline, v/v; Tocris Bioscience, Ellis-ville, MO), (7) soluble TNFp55R (sTNFp55R 50 g/ml), or (8) BzATP (5 mM) + sTNFp55R (50 g/ml). The dosage of each compound was determined as the highest dose that did not affect seizure threshold in a preliminary study. Animals were anesthetized (Zolretil, 50 mg/kg, i.m.; Virbac Laboratories) and placed in a stereotaxic frame. For osmotic pump implantation, holes were drilled through the skull to introduce a brain infusion kit 1 (Alzet, Cupertino, CA) into the right lateral ventricle (1 mm posterior; 1.5 mm lateral;3.5 mm depth; flat skull position with bregma as reference), according to the atlas of Paxinos and Watson [15]. The infusion kit was sealed with dental cement and connected to an osmotic pump (1002, Alzet, Cupertino, CA). The pump was placed in a subcutaneous pocket in the dorsal region. Animals received 0.5 l/hr of vehicle or compound for 2 weeks [16-18]. Seizure induction Three days after the start of vehicle or compound infusion, rats were treated with pilocarpine (380 mg/kg, i.p.) 20 min af (...truncated)