C: None of the purified cells showed a positive reaction for GFAP. Minocycline and sulforaphane inhibited lipopolysaccharide-induced microglial activation After LPS stimulation for 24 h, the purified microglial cells became bigger and rounder and developed the characteristic ameboid shape of activated microglia (Physique 1C and Physique 2B). and the p65 subunit of NF-B were also upregulated. However, the protein expression of p-p44/42 was not α-Estradiol significantly changed. Pretreatment with minocycline or sulforaphane for 1 h before LPS administration inhibited LPS-induced microglial morphological change and inhibited LPS-induced upregulation of p-p38, but had no effect on the expression of Rabbit Polyclonal to mGluR2/3 p-p44/42, p-JNK, and the p65 subunit of NF-B. Conclusions Minocycline and sulforaphane inhibited LPS-induced retinal microglial activation, Western blot and immunofluorescent studies showed decreased p-p38 MAPK expression. We suggested that this inhibitory effect of minocycline and sulforaphane was partly through a p38 MAPK-dependent mechanism. Introduction Microglia, major glia of the central nervous system (CNS), play a critical role as resident immunocompetent and phagocytic cells in the CNS [1]. Presence of activated microglia was exhibited in pathological lesions in several neurological and retinal diseases including Alzheimer’s disease, Parkinson’s disease [2], multiple sclerosis [3] and retinal degeneration [4,5]. Although microglia α-Estradiol promoted neuronal cell viability and survival by producing growth factor and removing potentially toxic cellular debris [6], there was also evidence that activated microglia was deleterious to neurons through excessive production of inflammatory mediators [5,7]. Microglia and their secretions were major contributors to the enhanced death of neurons in neurodegenerative diseases [8]. Hence, understanding the secretion of microglia and the mechanisms regulating microglial activation is an important step in developing therapeutic strategies that ameliorate symptoms of these diseases. Studies exhibited that brain-derived microglial cells released immuno-regulatory and neuroprotective brokers in conversation with phosphatidyl serine-expressing apoptotic cells [9]. However retinal microglial cells promoted photoreceptor death in vitro [10,11]. Previous studies mainly reported the expression of cytokines/chemokines in brain microglia, which was α-Estradiol different from retinal microglia. In this study the cultured retinal microglia was used to study microglial activity. Lipopolysaccharide (LPS) is used as a tool to simulate a challenge by gram-negative bacteria and to study the microglial activation process. Minocycline, a semi-synthetic, long-acting tetracycline derivative with good penetration of the blood-brain barrier, has recently been shown to have amazing neuroprotective properties in models of neurodegeneration [12,13], brain ischemia [14], and multiple sclerosis [15]. Aside from its direct anti-apoptosis effect, this neuroprotective function was also associated with reduced activation of microglia and reduction of inducible nitric oxide synthase (iNOS) and interleukin-1 (IL-1)-converting enzyme (ICE) expression [4,14]. However, the mechanism regulating this inhibition was not clear. Sulforaphane, a naturally occurring malignancy chemopreventive agent found in broccoli [16], has been shown to suppress LPS-mediated expression of iNOS, Cox-2 and tumor necrosis factor- (TNF-) in Natural 264.7 macrophage cells [17]. In view of this observation, we hypothesized that sulforaphane may modulate the inflammatory response of activated retinal microglia. Because sulforaphane occurs naturally in the widely consumed vegetable broccoli, this might provide a convenient approach to militate retinal degenerative diseases. In the present study, we investigated (1) the expression of immunological signaling molecules in cultured retinal microglia with or without LPS treatment; (2) the cellular pathways regulating the LPS-mediated microglial activation processes; and (3) the inhibitory effect of minocycline and sulforaphane on LPS-mediated microglial activation and the mechanisms through which they exert their effects. Methods Primary retinal microglial culture A primary culture of murine retinal microglial cells was prepared from newborn Sprague-Dawley rat retinas according to the technique by Roque and Caldwell [18], with minor modifications. Briefly, the eyes were enucleated and dissected, the retinas were peeled out and incubated in Ca++/Mg++-free Hank’s balanced salt solution made up of 0.25 mg/ml trypsin and 0.4 mg/ml EDTA at 37 C for 20 min (Sigma-Aldrich, St. Louis, MO). Afterwards, enzyme-treated tissues were dissociated into single cells by gentle pipetting and centrifuged. The dissociated cells were resuspended in DMEM/F-12 (1:1; Invitrogen Corporation, Grand Island, NY, USA) made up of 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Sigma-Aldrich). Cells were seeded at a density of 106 cells/ml in T75 culture flasks (Corning Incorporation, Corning, NY) and α-Estradiol incubated at 37 C in a humidified atmosphere of 5% CO2 and 95% air. After 2-3 weeks in vitro, microglial enriched cultures were shaken at 200 rpm on an orbital shaker (Lab-Line Devices,.