Elsevier

Neurochemistry International

Volume 76, October 2014, Pages 23-31
Neurochemistry International

Melatonin reduced microglial activation and alleviated neuroinflammation induced neuron degeneration in experimental traumatic brain injury: Possible involvement of mTOR pathway

https://doi.org/10.1016/j.neuint.2014.06.015Get rights and content

Highlights

Abstract

This study was designed to detect the modulation manner of melatonin on microglial activation and explore herein possible involvement of mammalian target of rapamycin (mTOR) pathway following traumatic brain injury (TBI). ICR mice were divided into four groups: sham group, TBI group, TBI + sal group and TBI + Melatonin group. A weight-drop model was employed to cause TBI. Neurological severity score (NSS) tests were performed to measure behavioral outcomes. Nissl staining was conducted to observe the neuronal degeneration and wet-to-dry weight ratio indicated brain water content. Immunofluorescence was designed to investigate microglial activation. Enzyme-linked immunosorbent assay (ELISA) was employed to evaluate proinflammatory cytokine levels (interleukin-beta (IL-1β), tumor necrosis factor-alpha (TNF-α)). Western blotting was engaged to analyze the protein content of mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase (p70S6K) and S6 ribosomal protein (S6RP). Melatonin administration was associated with markedly restrained microglial activation, decreased release of proinflammatory cytokines and increased the number of surviving neurons at the site of peri-contusion. Meanwhile, melatonin administration resulted in dephosphorylated mTOR pathway. In conclusion, this study presents a new insight into the mechanisms responsible for the anti-neuroinflammation of melatonin, with possible involvement of mTOR pathway.

Introduction

Traumatic brain injury (TBI) is the leading cause of death under the age of 40 in developed countries, and approximately 10 million persons are in hospital for TBI annually worldwide (Langlois et al., 2006). TBI cause primary mechanical injury of cerebral cells and also initiates secondary damage occur immediately following the primary damage. The secondary nonmechanical injury is progressive and lasts from hours to days (Cernak, 2005).There are several pathological process reported to be responsible for the neuronal death in the secondary damage of TBI (Yatsiv et al., 2005, Xu et al., 2013, Cornelius et al., 2013), including inflammation (Ziebell and Morganti-Kossmann, 2010, Xu et al., 2013). The activation of microglia is wildly accepted as a prominent histological evidence of brain injury (Morganti-Kossmann et al., 2007, Venkatesan et al., 2010). It is also thought as the major source of proinflammatory cytokines (Cao et al., 2012, Kelley et al., 2007, Morganti-Kossmann et al., 2007, Nakajima and Kohsaka, 2004, Zhang et al., 2013) after TBI. Now more and more researchers targeted it to retard the pathological process related inflammation after TBI (Sanchez et al., 2001, Ng et al., 2012, Siopi et al., 2012, D’Avila et al., 2012).

Melatonin (N-acetyl 5-methoxytryptamine) has been proved to be a neuroprotective agent in TBI (Tsai et al., 2011, Dehghan et al., 2013, Cirak et al., 1999, Beni et al., 2004). The anti-inflammation characteristic of melatonin have been accepted as a major protective mechanism for melatonin to brain injury (Tsai et al., 2011, Beni et al., 2004, Samantaray et al., 2009). However, the modulation manners of melatonin to microglial activation have not been particular investigated in the research with respect to TBI.

Mammalian target of rapamycin (mTOR) is a rapamycin sensitive serine/threonine protein kinase which plays a major role in modulating protein synthesis initiation (Dazert and Hall, 2011). Growth factors, hormones and nutrients activate the phosphoinositide 3-kinase pathway, leading to the phosphorylation and activation of mTOR on Ser2448 (Reynolds et al., 2002, Chong et al., 2010). Activated mTOR exerts its functions of stimulation of translation through phosphorylation and activation of p70 ribosomal S6 kinase (p70S6K), which in turn phosphorylates S6 ribosomal protein (S6RP) (Park et al., 2012). S6RP phosphorylation is a critical effector of mTOR in the regulation of cell proliferation and protein synthesis. The up-regulated expression of phospho-S6RP has been found in microglia rather than neuron after TBI (Park et al., 2012) and mTOR play a crucial role in microglial viability (Dello et al., 2009). Phosphorylation of mTOR pathway is a considerable reason for activation of microglia after TBI. Therefore, mTOR is a reasonable therapeutic target for inflammatory response after TBI. Thus, in the present study we explored the possible involvement of melatonin in modulation the activation of mTOR pathway in a mice TBI model and revealed a marked beneficial effect of melatonin on neurologic outcome associated its suppression microglia activation following TBI.

Section snippets

Animals and experimental protocols

Male ICR mice (Experiment Animal Centre of Nanjing Medical University, Jiangsu, China) ages 6–8 weeks, weighing 28–32 g were used in this study. Experiment protocols were approved by the Animal Care and Use Committee of Nanjing University and conformed to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH). The mice were housed on a 12 h light/dark cycle circumstance with free access to food and water.

Male ICR mice were divided into four groups: sham

Melatonin improved neurobehavioral performance of mice after TBI

At 24 h after TBI, the NSS score of the melatonin injection mice was significantly better than that of the vehicle treated mice (P < 0.05). At 3 days, the scores of both groups had improved, and the difference was still significant (P < 0.05) (Fig. 1 A).

Melatonin alleviated cerebral oedema after TBI

To confirm the protective effect of melatonin at the macroscopic level, we measured the brain water content of mice in four groups (Fig. 1B). Compared with the sham group, mice in the TBI and TBI + sal groups had significantly greater brain water

Discussion

In agreement with previous studies in rodents, p-mTOR, p-p70S6K and p-S6RP were markedly increased in ipsilateral cortex 24 h post-TBI in mice, suggesting activation of mTOR pathway following TBI (Chen et al., 2007, Park et al., 2012, Erlich et al., 2007). Our observations also showed that injection of melatonin repressed phosphorylation of mTOR and its downstream substrates. Furthermore, melatonin restrained the activation of microglia and decreased protein expression of IL-1β and TNF-α,

Conclusions

These findings extend the current understanding of the mechanisms of melatonin’s neuroprotective effect in TBI. We demonstrated that inactivation mTOR pathway by melatonin plays a protective role in TBI. This neuroprotective effect was associated with the inactivation of microglia and the reduced release of proinflammatory cytokines (Fig. 7). However, further studies are needed to resolve outstanding issues regarding the mechanisms by which melatonin dephosphorylates mTOR in TBI.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

This work was supported by Grants from the Natural Science Fund of China (Nos. 81271377 and 81371357) and the Research Project of Jinling Hospital (No. 2014040).

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