Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia

Abstract

Minocycline, a semisynthetic tetracycline derivative, protects brain against global and focal ischemia in rodents. We examined whether minocycline reduces excitotoxicity in primary neuronal cultures. Minocycline (0.02 microm) significantly increased neuronal survival in mixed spinal cord (SC) cultures treated with 500 microm glutamate or 100 microm kainate for 24 hr. Treatment with these excitotoxins induced a dose-dependent proliferation of microglia that was associated with increased release of interleukin-1beta (IL-1beta) and was followed by increased lactate dehydrogenase (LDH) release. The excitotoxicity was enhanced when microglial cells were cultured on top of SC cultures. Minocycline prevented excitotoxin-induced microglial proliferation and the increased release of nitric oxide (NO) metabolites and IL-1beta. Excitotoxins induced microglial proliferation and increased the release of NO metabolites and IL-1beta also in pure microglia cultures, and these responses were inhibited by minocycline. In both SC and pure microglia cultures, excitotoxins activated p38 mitogen-activated protein kinase (p38 MAPK) exclusively in microglia. Minocycline inhibited p38 MAPK activation in SC cultures, and treatment with SB203580, a p38 MAPK inhibitor, but not with PD98059, a p44/42 MAPK inhibitor, increased neuronal survival. In pure microglia cultures, glutamate induced transient activation of p38 MAPK, and this was inhibited by minocycline. These findings indicate that the proliferation and activation of microglia contributes to excitotoxicity, which is inhibited by minocycline, an antibiotic used in severe human infections.

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.

Fig. 2.

Minocycline (MC) inhibits excitotoxin-induced proliferation of microglial cells independently of neuronal cell death. a, In SC cultures, the number of OX-42-immunoreactive (OX-42-IR) microglial cells is significantly increased after a 24 hr exposure to 500 μmglutamate (Glu) and 100 μm kainate (KA). Minocycline (0.02 μm) treatment completely prevents the microglia proliferation and decreases the microglia cell number even in unexposed control cultures (0-ctrl). Data are presented as the mean ± SD counted in a blind manner and pooled from two independent experiments (n = 8). **p < 0.01; single-factor ANOVA. b, Representative photomicrographs of the effect of glutamate and minocycline on OX-42-immunoreactive cells. Scale bar, 250 μm. c, A 24 hr exposure of neuron-free glial cultures to 500 μmglutamate and 500 μm kainate results in microglia proliferation, which is inhibited by 0.2 μm minocycline treatment. Minocycline also inhibits the spontaneous microglia proliferation. Data are presented as the mean ± SD counted in a blind manner (n = 3). **p < 0.05; single-factor ANOVA. d, Exposure of SC cultures to excitotoxins causes increased IL-1β release, which is reduced by minocycline treatment. The exposure and treatment of these cultures were done as described in Figure 1a. Data are presented as the mean ± SD pooled from two independent experiments (n = 6). **p < 0.01; single-factor ANOVA. e, Increased number of microglia in SC cell cultures enhances the excitotoxicity, which was inhibited by minocycline. Adding microglia onto SC cell cultures (+MG), excitotoxicity of 500 μm glutamate and 100 μm kainate is increased significantly (*p < 0.05; single-factor ANOVA) when compared with the normal mixed SC culture (−MG). The basal LDH release is also enhanced significantly (*p < 0.05; single-factor ANOVA) in microglia-rich cultures. Minocycline (0.02 μm) treatment 30 min before excitotoxin exposures was able to provide significant (§p < 0.05; single-factor ANOVA) neuroprotection. Data were presented as the mean ± SD pooled from two independent experiments (n = 6).

Fig. 3.

Minocycline (MC) treatment inhibits activation of p38 MAPK in microglia at neuroprotective doses, and inhibition of p38 MAPK is neuroprotective in SC cell cultures.a, Quantitation of phospho-p38 MAPK-immunoreactive microglia in SC cell cultures after 10 min stimulation with 500 μm glutamate (Glu) and 100 μm kainate (KA) with and without 0.02 μm minocycline treatment. Both glutamate and kainate increased the number of immunoreactive cells within 10 min. Minocycline decreased the induced p38 MAPK activity in microglial cells. Data are presented as the mean ± SD counted in a blind manner from three independent experiments (n = 9). **p < 0.01; single-factor ANOVA.b–e, Representative photomicrographs of the effect of glutamate and minocycline on phospho-p38-immunoreactive (p38-IR) cells of SC cell cultures. Double-staining with phospho-p38 (b) and OX-42 (c) antibodies indicates that p38 MAPK activity is increased by glutamate stimulation only in microglia. Scale bar, 200 μm. f, LDH release induced by 500 μm glutamate and 100 μm kainate (24 hr) was reduced by 10 μmSB203580 (SB), a specific p38 MAPK inhibitor, but not by 10 μm PD98059 (PD), a specific p44/42 MAPK inhibitor. Data are presented as the mean ± SD from three independent experiments (n = 10–13). **p < 0.01; single-factor ANOVA.0-ctrl, Unexposed 0 control.

Fig. 4.

Stimulation of glutamate receptors causes microglial proliferation and activation, which are decreased by minocycline (MC). a, Immunoblot analysis of AMPA/kainate receptor GluR4 in pure microglia cultures. A specific immunoreactive band at appropriate size is detected. b, Quantitation of BrdU-positive cells in pure microglial culture 24 hr after stimulation with 500 μm glutamate (Glu) and 100 μm kainate (KA). Both 0.2 μm minocycline and 10 μm SB203580 (SB), a specific p38 MAPK inhibitor, treatments reduce the excitotoxin-induced microglial proliferation. Data are present as the mean ± SD pooled from two independent experiments (n = 9–11). **p < 0,01; single-factor ANOVA. A 24 hr stimulation with excitotoxins also causes increased NO (c) and IL-1β (d) release by a mechanism, which is reduced by 0.02 μm minocycline treatment. Data are presented as the mean ± SD pooled from two independent experiments (n = 8). *p < 0.05; **p < 0.01; single-factor ANOVA. 0-ctrl, Unexposed 0 control.

Fig. 5.

Glutamate-stimulated pure microglial cultures show an increased amount of phospho-p38 MAPK, which is reduced by minocycline. Quantitative immunoblot analysis of phospho-p38 (a) and p44/42 MAPK (b) after 5 min stimulation with 500 μm glutamate alone (Glu 5min), 5 min stimulation with 500 μmglutamate with minocycline administered 30 min before glutamate (Glu+MC 5min), 0.2 μm minocycline alone (MC 30min), and untreated cultures (0-ctrl). Below the graphs, representative immunoblots are shown. The experiments were repeated twice with similar results (*p < 0.05).