Fig. 1.

Excitotoxin-induced neuronal death is inhibited by minocycline treatment. a, Dose-dependent neuroprotection of minocycline detected by measuring LDH release from SC cell culture medium after 24 hr exposure to 500 μm glutamate with and without 2 μm to 0.01 nm minocycline treatment. Ten nanomolar minocycline was the lowest dose to provide significant neuroprotection, but the most consistent (with the least variation) and efficient protection was seen at 20 nm to 2 μm concentrations. Data are presented as the mean ± SD pooled from two independent experiments (n = 6). *p < 0.05; **p < 0.01 versus glutamate; single-factor ANOVA. 100% LDH release refers to the LDH release observed 24 hr after adding 500 mm glutamate alone.b, LDH release into SC cell culture medium, measured after a 24 hr exposure to 500 μm glutamate and 100 μm kainate (KA), is significantly reduced by 0.02 μm minocycline treatment. Data are presented as the mean ± SD pooled from three independent experiments (n = 12). **p < 0.01; single-factor ANOVA. 100% LDH release refers to the LDH release observed 24 hr after adding 500 μm glutamate or 100 μm kainate alone. c, The neuronal cell loss in SC cell cultures caused by 24 hr exposure to 500 μm glutamate and 100 μm kainate was decreased by 0.02 μm minocycline treatment. NeuN-immunoreactive cells were counted in a blind manner. Data are presented as the mean ± SD pooled from two independent experiments (n = 6). **p < 0.01; single-factor ANOVA. d–f, Representative fluorescence micrographs of bis-benzimide chromatin staining in SC cell cultures exposed to 500 μm glutamate for 24 hr with and without 0.02 μm minocycline treatment. Healthy surviving neurons have a round and large nucleus, whereas in preapoptotic neurons the nuclei are condensed (arrows) and in apoptotic neurons chromatin has fragmented (arrowheads). Preapoptotic and apoptotic neurons can been seen in cultures exposed to glutamate (d). In minocycline-pretreated (e) and control (f) cultures, preapoptotic and apoptotic neurons are not observed. Scale bars: d–g, 200 μm; d′, 100 μm.g, The apoptotic neuronal death and its inhibition by minocycline was also studied by semiquantitative ligation-mediated PCR assay. The number of DNA fragments generated in 500 μmglutamate-exposed (Glu), unexposed (0-ctrl), and 0.02 μmminocycline-treated glutamate-exposed (Glu+MC) cultures was amplified by 23, 26, 29, 32, and 35 (the five lanesfrom right to left in the gel, representing each kind of sample) thermal cycles and compared with a positive control sample [(+)Ctrl, provided by the manufacturer of the kit], which was run in parallel with other samples. DNA ladder became clearly visible only in the samples from glutamate-treated cells. The experiment was repeated twice with similar results.