Research Of Minocycline Protective Mechanisms In Glutamate Metabolism And Inflammatory Response After The Hypoxic-ischemic Brain Damage

Objective:Neonatal hypoxic-ischemic brain damage (HIBD) is a major cause of neurological impairment and cognitive deficits. Glutamate accumulation and inflammatory response in brain are believed to play a critical role in excitotoxicity-induced neuron damage. In this study, we investigated the dynamic changes of glutamate concentration, glutamate transporters (EAAT1 and EAAT2), marker of activated microglia (Ibal) and inflammatory mediators (IL-1β, TNF-a and TGF-β1) in periventricular zone and hippocampus in neonatal rats after systemic hypoxia exposure. In addition, we also detected the inflammatory cytokines (IL-1β and TNF-a) in peripheral serum after hypoxic exposure. This study investigated the effects of minocycline on glutamate’s metabolism (in periventricular and hippocampus zone), inflammatory reaction (in periphery and brain) and it’s putative protective mechanisms in neonatal rat hypoxia induced HIBD model.Methods:1 A model of HIBD was developed by putting postnatal 0 day rats in 5% O2 for 3.5h. Rats were intraperitoneal injected with normal saline (Hy group) or minocycline (Hy M group) after 2h hypoxia exposure respectively. Other littermates that subjected neither systemic hypoxia nor minocycline injection were used as normal control (NG group).2 Western blot analysis was applied to observe the expression of glutamate transporters (EAAT1 and EAAT2) dynamic changes in periventricular zone and hippocampus during a time range from postnatal Id to postnatal 15d. Glutamate transporters (EAAT1 and EAAT2), marker of activated microglia (Ibal) and inflammatory mediators (IL-1β, TNF-a and TGF-β1) expression in the indicated regions of each group were also determined by western blot assay after hypoxic exposure 4h, 1d,3d,7d and 14d.3 The glutamate concentration of hippocampus was measured by liquidchromatography coupled with tandem mass spectrometry assay after hypoxic exposure 4h, 1d and 3d.4 The inflammatory cytokines (IL-1β and TNF-a) of peripheral serum were measured using ELISA at 4h, Id and 3d after hypoxic exposure.Results:Part 1:The effect of minocycline on glutamate metabolism in periventricular zone of neonatal rats after hypoxia1 Western blot results revealed low expression levels of EAAT1 and EAAT2 at periventricular zone during the first postnatal week. However, a predominant elevation was found in the end of the second week after birth (EAAT1:***p<0.001 was postnatal 15d vs postnatal Id,2d,4d and 8d; oop<0.01 was postnatal 15d vs postnatal 4d; EAAT2: ***p<0.001 was postnatal 15d vs postnatal 1d,2d,4d and 8d).2 The expression of glutamate transporters in periventricular zone. EAAT1 level was increased at 1d (p<0.05 vs NG) and 7d (p<0.05 vs NG) after hypoxia exposure. The expression of EAAT2 was elevated after at 4h (p<0.05 vs NG), Id (p<0.05 vs NG),7d (p<0.05 vs NG) and 14d (p<0.05 vs NG) hypoxic exposure. However, a declined expression level of EAAT2 was found at 3d (p<0.05 vs NG) after hypoxic exposure. EAAT1 expression was up-regulated and EAAT2 expression was down-regulated at 1d (p<0.05 vs Hy) after hypoxic, and EAAT1 level was decreased at 7d (p<0.05 vs Hy) by minocycline treatment respectively.3 The expression of inflammatory mediators in periventricular zone. Ibal was increased significantly after hypoxic exposure at 1d (p<0.05 vs NG) and 7d (p<0.05 vs NG) time points. The expression of IL-1β maintained at high levels at 4h (p<0.05 vs NG), 1d (p<0.05 vs NG),3d (p<0.05 vs NG) and 7d (p<0.05 vs NG) after hypoxic exposure. In addition, the TNF-a expression was decreased at 3d (p<0.05 vs NG) after hypoxic exposure. While anti-inflammatory cytokines TGF-β1 was up-regulated at 1d (p<0.05 vs NG) and 7d (p<0.05 vs NG) after hypoxic exposure, it was down-regulated at 3d (p<0.05 vs NG) after hypoxia. Minocycline administration could facilitate IL-1β (p<0.0l vs Hy) and TNF-a (p<0.05 vs Hy) expression at 3d after hypoxic exposure.Part 2:The effect of minocycline on glutamate metabolism in hippocampus of neonatal rats after hypoxia1 The dynamic changes of glutamate transporters expression during development period in hippocampus. EAAT1 expression had a incessantly ascending at early development stages (***p<0.001 is postnatal 15d vs postnatal Id,2d and 4d; °p<0.05 is postnatal 15d vs postnatal 8d; ###p<0.001 is postnatal 8d vs postnatal 1d;☆☆p<0.01 is postnatal 8d vs postnatal 2d and 4d; △△p<0.01 is postnatal 4d vs postnatal 1d; □p<0.05 is postnatal 2d vs postnatal 1d). A rapid increase of EAA2 level we also found at postnatal 4d (***p<0.001 is postnatal 15d vs postnatal Id,2d and 8d; oop<0.01 is postnatal 15d vs postnatal 4d;***p<0.001 is postnatal 8d vs postnatal 2d;☆p<0.01 is postnatal 8d vs postnatal 1d and 4d).2 In hippocampus, the glutamate level was increased at 4h (p<0.05 vs NG) but was decreased at 3d (p<0.05 vs NG) after systemic hypoxia. However, minocycline treatment had no effects on the glutamate levels in hippocampus (p>0.05 vs NG).3 The dynamic changes of glutamate transporters in hippocampus after hypoxic exposure. EAAT1 expression level appeared declined at both 4h (p<0.05 vs NG) and 1d (p<0.05 vs NG) after hypoxic exposure. However, the expression of EAAT2 was elevated at 7d (p<0.05 vs NG) and 14d (p<0.05 vs NG) after hypoxic. Minocycline administration could down-regulate the expression of EAAT2 at 7d (p<0.05 vs Hy) after hypoxic exposure, but it failed to regulate the EAAT1 expression (p>0.05 vs Hy).4 The expression of inflammatory mediators in hippocampus. Ibal was up-regulated at 1d (p<0.015 vs NG) and 7d (p<0.05 vs NG) after hypoxic, but a predominant decline was found at 3d (p<0.05 vs NG) after hypoxic exposure. IL-1β expression was decreased at 3d (p<0.05 vs NG) after hypoxic exposure and was increased at 7d (p<0.05 vs NG) after hypoxic exposure. In addition, the expression of TNF-a was up-regulated after hypoxic exposure at both 3d (p<0.05 vs NG) and 7d (p<0.05 vs NG) time points. The TGF-β1 level was down-regulated at 4h (p<0.05 vs NG) and 14d (p<0.05 vs NG) after hypoxic exposure. Treatment with minocycline inhibited IL-1β and TNF-α expression at both 3d (p<0.05 vs Hy) and 7d (p<0.05 vs Hy) after hypoxic exposure. Ibal expression was inhibited by minocycline at 7d (p<0.05 vs Hy) after hypoxic exposure, but with a significant increase of TGF-β1 expression by minocycline at 4h (p<0.05 vs Hy) after hypoxic exposure.Part 3:The effect of minocycline on periphery serum inflammatory responses of neonatal rats after hypoxia1 The results of ELISA shown that serum IL-1β concentration was increased at 4h (p<0.05 vs NG) and 1d (p<0.01 vs NG) after hypoxic exposure, while it was decreased at 3d (p<0.01 vs NG) after hypoxic exposure. In addition, the level of serum TNF-a was also elevated at both 4h (p<0.01 vs NG) and 3d (p<0.05 vs NG) after hypoxia. Treatment of minocycline could up-regulate the concentration of IL-1β at Id (p<0.05 vs Hy) after hypoxic exposure, but it down-regulated TNF-a level at 4h (p<0.05 vs Hy) and 3d (p<0.05 vs Hy) after hypoxic exposure.Conclusions:1 During the early developmental stages, expression of EAAT1 and EAAT2 gradually increased, which begins from the second week after birth in periventricular zone. The high expression level of EAAT1 occurs earlier than that of EAAT2 in hippocampus.2 Minocycline inhibits the increase of the glutamate level after hypoxia in periventricular region of neonatal rats, which might relate with regulation of glutamate transporters selectively, rather than suppressing neuroinflammation in periventricular zone.3 In hippocampus, minocycline had no effect on hypoxia-induced glutamate level rise in this region of neonatal rats. Therefore, the protective effects of it might not involve glutamate transporters in hippocampus.4 The protective role of minocycline in HIBD might through its effects on inhibiting the neuroinflammation and dysfunctional glutamate transporters in hippocampus at 7d after hypoxic exposure.5 The periphery inflammatory response of neonatal rats happened after systemic hypoxia. Minocycline inhibits the periphery serum inflammatory response and therefore ameliorates the hypoxic-ischemic brain injury.