Shock/Sepsis/Trauma/Critical Care
Rapamycin protects against apoptotic neuronal death and improves neurologic function after traumatic brain injury in mice via modulation of the mTOR-p53-Bax axis

https://doi.org/10.1016/j.jss.2014.09.026Get rights and content

Abstract

Background

Rapamycin has proven to be a neuroprotective agent in traumatic brain injury (TBI). However, there is a lack of data regarding the effect of rapamycin on apoptotic neuronal death after TBI. Thus, the present study was designed to detect the modulatory role of rapamycin on apoptosis and explore the potential involvement of the mammalian target of rapamycin (mTOR)-p53-Bax axis after TBI.

Material and methods

Neurologic severity score tests were performed to measure behavioral outcomes. The effect of rapamycin treatment on neuronal death was analyzed using immunofluorescence analysis of NeuN. Terminal deoxynucleotidyl transferase-mediated dUTP nick 3′-end labeling was performed to detect apoptotic cells. The expression of Bax and phosphorylated protein of p53 was detected using Western blotting analyses and immunofluorescence staining. Phosphorylated protein of the mTOR in the ipsilateral cortex was detected using Western blotting analyses.

Results

Rapamycin administration after TBI was associated with an increased number of neurons, decreased apoptosis index, and improved neurobehavioral function, which was potentially mediated by inactivation of the mTOR-p53-Bax axis.

Conclusions

Rapamycin can protect neurons from apoptotic neuronal death after TBI. This study presents a new insight into the antiapoptosis mechanisms, which are responsible for the neuroprotection of rapamycin, with the potential involvement of the mTOR-p53-Bax axis.

Introduction

Traumatic brain injury (TBI) is a serious public health problem affecting millions of people worldwide. Each year approximately 10 million people are hospitalized for TBI worldwide [1]. TBI causes primary mechanical injury of cerebral cells and also initiates secondary damage, which occurs immediately after the primary damage. The secondary nonmechanical injury is progressive and lasts from hours, days to months [2]. There are several pathologic processes that have been reported to be responsible for the neuronal death observed in the secondary damage of TBI, such as inflammation, disruption of the blood–brain barrier, apoptosis, and oxidative stress [3], [4], [5], [6], [7], [8], [9], [10]. Among these processes, apoptosis plays a key role in secondary injury [6], [7]. Apoptosis, which is also known as programmed cell death, has been observed in the injured brain in an experimental model [11], [12] and humans [13], [14] after TBI. Its phenotypic features include DNA fragmentation and chromatin condensation, cell shrinkage, and formation of apoptotic bodies, which are cleared by phagocytosis without initiating a systemic inflammatory response [15]. It has been reported that approximately two-third of cell death might be attributable to apoptosis and one-third to necrosis in experimental models of TBI [15]. Apoptotic neuronal death presents within a time window that may be responsive to targeted therapies, and an increasing number of researchers have focused on it to retard the pathologic process related to apoptosis after TBI [6], [7], [16], [17], [18].

Rapamycin is a macrolide antibiotic obtained from Streptomyces hygroscopicus that can specifically inhibit the activity of mammalian target of rapamycin (mTOR). The function of rapamycin has been intensively investigated in cancer research, development, metabolism, and central nervous system diseases [19]. Rapamycin has also been investigated for its neuroprotective effects in closed head injury models. Injection of rapamycin increased the survival of neurons and significantly improved brain functional recovery [20]. However, the effect of rapamycin on apoptosis after TBI has not been extensively investigated. Thus, we present the hypothesis that rapamycin can inhibit the mTOR-p53-Bax axis, thereby decreasing the number of apoptotic neuronal death after TBI.

Section snippets

Animals

Male imprinting control region mice (Experiment Animal Centre of Nanjing Medical University, Jiangsu, China) aged 6–8 wk and weighing 28–32 g were used in this study. The experimental 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. The mice were housed on a 12 h light–dark cycle with free access to food and water.

Model of TBI

The model of TBI used in the present

Effects of rapamycin on neurobehavioral scores after TBI

As shown in Figure 1, mice in the sham group presented no neurologic dysfunction. The NSS scores of the other three groups were gradually improved from 1 h to 7 d post-TBI. At 24 h after TBI, the NSS score of the rapamycin-treated mice was significantly lower compared with vehicle-treated mice (P < 0.01). At 72 h, a significant difference was still detectable (P < 0.05). At 1 h and 7 d, no significant difference was observed between the TBI + DMSO group and TBI + rapamycin group (P > 0.05).

Effects of rapamycin on the neuronal death in the pericontusive cortex after TBI

Discussion

In this study, we examined the protective effect of rapamycin against apoptotic neuronal death and neurologic deficits in an experimental mice model of TBI. Our data showed that 24 h after TBI, (1) the mTOR-p53-Bax axis was activated, (2) rapamycin improved neurobehavioral deficits and increased the number of neurons in the pericontusive cortex, (3) rapamycin decreased neuronal apoptosis, and (4) rapamycin inactivated the mTOR-p53-Bax axis induced by TBI. These findings suggested that early

Conclusions

In summary, to the best of our knowledge, this is the first study to suggest the potential involvement of rapamycin in protecting neurons from apoptotic neuronal death via modulation of the mTOR-p53-Bax axis after TBI. These findings extend our current understanding of the mechanisms underlying the neuroprotective effect of rapamycin in TBI. We demonstrated that treatment with rapamycin resulted in a significant decrease in apoptotic neuronal death after TBI. This neuroprotective effect was

Acknowledgment

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

The authors declare that they have no competing interests.

Authors' contributions: K.D. and L.Z. performed most of the experiments. J.X. performed the Western blotting experiments. T.L. and Y.D. performed analysis on the IHC data and statistical analyses and provided the discussion of the results. L.Z. and J.H. performed the IHC

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