Anat Cell Biol.  2010 Dec;43(4):325-331. 10.5115/acb.2010.43.4.325.

Direct protection of cultured neurons from ischemia-like injury by minocycline

• Huang WC
• Qiao Y
• Xu L
• Kacimi R
• Sun X
• Giffard RG
• Yenari MA

• KMID: 1455347
• DOI: http://doi.org/10.5115/acb.2010.43.4.325

Abstract

Minocycline, a tetracycline antibiotic, is now known to protect cells via an anti-inflammatory mechanism. We further explored this effect using an in vitro model of ischemia-like injury to neurons. Coculturing neurons with microglia, the brain's resident immune cell, modestly increased cell death due to oxygen and glucose deprivation (OGD), compared to neurons alone. Treatment of cocultures with minocycline decreased cell death to a level significantly lower than that of neurons alone. Treatment of cocultures with minocycline or inhibitors of various immune mediators, also led to decreased cell death. Importantly, treatment of neuron cultures without added microglia with these same inhibitors of tissue plasminogen activator, matrix metalloproteinases, TNF-alpha and inducible nitric oxide synthase as well as minocycline also led to decreased cell death. Thus, anti-inflammatory treatments appear to be directly protective of neurons from in vitro ischemia.

Keyword

Minocycline; Microglia; Ischemia; Neurons

MeSH Terms

Cell Death
Coculture Techniques
Glucose
Ischemia
Matrix Metalloproteinases
Microglia
Minocycline
Neurons
Nitric Oxide Synthase Type II
Oxygen
Tetracycline
Tissue Plasminogen Activator
Tumor Necrosis Factor-alpha
Glucose
Matrix Metalloproteinases
Minocycline
Nitric Oxide Synthase Type II
Oxygen
Tetracycline
Tissue Plasminogen Activator
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Representative neuron-microglia (NM) coculture. Neurons were plated at a density of 106 cells/ml, microglia were plated on top of neurons at a density of 2×105 cells/ml. Fluoroscent immunostains identify neurons (MAP-2, red, arrowheads) and microglia (IB4, green, arrows). Scale bar = 25 µm.

• 

  Fig. 2 (A) The addition of microglia to neuron cultures (NM-BSS0) somewhat increased the extent of cell death due to OGD compared to neurons alone (N-BSS0). However, neuroprotection was markedly enhanced by treatment of cocultures with minocycline (NM-mino) as well as inhibitors of iNOS (aminoguanidine, NM-AG), MMPs (GM6001, NM-GM), and tPA (NM-PAI-1). (*P<0.05 vs. NM, †P<0.05 vs. N) (B) Treatment of neurons cultured alone markedly reduced OGD-induced injury (*P<0.05 vs. BSS0). BSS0 = balanced salt solution lacking glucose, used as a control, and also used in all washes with treatment, N = neurons, M = microglia (C). TNF-α inhibition protects neurons from OGD. Treatment of neuron cultures with a blocking antibody against TNF-α reduced neuron cell death as assessed by the LDH release assay. The extent of cell death reduction by the blocking antibody was comparable to that seen by minocycline treatment. (*P<0.01 vs. no injury; †P<0.01 vs. OGD; No inj = no injury control, OGD = oxygen glucose deprivation, TNF Ab = TNF-α blocking antibody, mino = minocycline).

• 

  Fig. 3 Minocycline prevented generation of TNF-α in primary microglia stimulated by LPS (A) or subjected to OGD (B). Microglia were stimulated by 10 µg/ml LPS for 24 h or subjected to 24 h OGD followed by 2 h reperfusion (a paradigm previously shown to cause some microglial death and activation (Yenari & Giffard, 2001)). TNF-α levels were measured in culture supernatants collected at the end of the experiments. In contrast, minocycline did not suppress NO generation, as estimated by the amount of nitrates and nitrated proteins in culture supernatants, in the BV2 microglial cell line (C) or primary microglia (D). Only LPS led to increased NO production, but this was not decreased by minocycline in a concentration found to be neuroprotective. (*P<0.001).

• 

  Fig. 4 Neurons increase NO generation after OGD, but not TNF-α. Neuron cultures were exposed to 90 min OGD followed by 24 h reperfusion. (A) Neurons secreted low levels of TNF-α, but this was not changed by OGD or minocycline treatment. (B) Low levels of nitrates and nitrated protein indicating NO generation were detected in culture supernatants. OGD caused NO increases which were decreased by minocycline treatment (*P<0.05, mino = 2 µM minocycline).

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