Effect of Enhanced Prostacyclin Synthesis by Adenovirus‐Mediated Transfer on Lipopolysaccharide Stimulation in Neuron‐Glia Cultures

Abstract

Prostacyclin (PGI2) is known as a short-lived, potent vasodilator and platelet anti-aggregatory eicosanoid. This work attempts to selectively augment PGI2 synthesis in neuron-glia cultures by adenoviral (Ad) gene transfer of PGI synthase (PGIS) or bicistronic cyclooxygenase 1 (COX-1)/PGIS and examines whether PGI2 confers protection against lipopolysaccharide (LPS) stimulation. Cultures released low levels of eicosanoids. Upon Ad-PGIS or Ad-COX-1/PGIS infection, cultures selectively increased prostacyclin release. Both PGIS- and COX-1/PGIS-overexpressed cultures contained fewer microglial numbers. Further, they significantly attenuated LPS-induced iNOS expression and lactate, nitric oxide, and TNF-alpha production. Taken together, enhanced prostacyclin synthesis in neuron-glial cultures reduced microglia number and suppressed LPS stimulation.

Enhanced Prostacyclin Synthesis by Adenoviral Gene Transfer Reduced Glial Activation and Ameliorated Dopaminergic Dysfunction in Hemiparkinsonian Rats

“…Adenovirus encoding GFP (expressing the green fluorescence protein of jelly fish), PGI 2 synthase (PGIS), or bicistronic COX-1/PGIS used phosphoglycerate kinase (PGK) as a driving promoter. The preparation, ex vivo expansion, and purification of these Ads followed methods described previously [18, 20, 21]. The viral titers of the purified Ads were determined by a plaque-forming assay and were in the range of ~10 10  pfu/mL.…”

“…Briefly, cultured cells were incubated in DMEM (serum-free) containing 10  μ M [1- 14 C] AA at 37°C for 10 min. The cells were saved for western blot analysis, while the released fractions, containing radioactive eicosanoids, were extracted by a Sep-Pak C 18 cartridge (Waters Associates, Milford, MA) as described [20, 21]. The resulted extracts of 14 C-labeled AA metabolites (eicosanoids) were analyzed by reverse phase high performance liquid chromatography (HPLC; Waters model 2690) equipped with an online radioisotope detector (Packard 150-TP) as previously described [18].…”

“…Using adenovirus-mediated transfer of COX1 or COX1/PGIS, Lin et al, [19] and our coworkers [20] have demonstrated that enhanced PGI 2 synthesis in neuronal cultures or in ischemic brain was neuroprotective and had prominent influence on microglia. This work attempts to selectively augment PGI 2 synthesis in mixed glial culture and in hemiparkinsonian rats by direct adenoviral gene transfer of PGIS or bicistronic COX-1/PGIS and examines whether it confers protection or induces cell damage in Parkinson's disease (PD).…”

Acid Fibroblast Growth Factor and Peripheral Nerve Grafts Regulate Th2 Cytokine Expression, Macrophage Activation, Polyamine Synthesis, and Neurotrophin Expression in Transected Rat Spinal Cords

“…Mixed neuron-glial cultures were prepared from the cerebrocortical regions of embryonic SD rat fetuses at ϳ15-17 d gestation, as described previously (Tsai et al, 2005(Tsai et al, , 2010b. Briefly, fetal cortexes were dissociated with mixtures of papain/protease/ deoxyribonuclease I (0.1/0.1/0.03%) and plated onto poly-lysine-coated multiwell plates or insert wells (for cocultures).…”

“…An SCI repair strategy using peripheral nerve grafts and acidic fibroblast growth factor (aFGF, FGF-1) improves hindlimb locomotor function in spinal cord-transected rats (Cheng et al, 1996;Lee et al, 2002Lee et al, , 2004Tsai et al, 2005). Repaired spinal cords induce the expression of the M2 macrophage marker arginase I (Arg I) 6 -14 d after repair and recruited large numbers of M2 macrophages to the graft area 10 d after repair .…”