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Abstract. Gap junctions are conductive channels formed by membrane proteins termed connexins, which permit the intercellular exchange of metabolites, ions and small molecules. Previous data demonstrated that traumatic brain injury (TBI) activates autophagy and increases microtubule-associated protein 1 light chain 3 (LC3) immunostaining predominantly in neurons. Although previous studies have identified several extracellular factors that modulate LC3 expression, knowledge of the regulatory network controlling LC3 in health and disease remains incomplete. The aim of the present study was to assess whether gap junctions control the in vivo expression of LC3 in TBI. Using a modified weight-drop device, adult male Sprague-Dawley rats (weight, 350-375 g) were subjected to TBI. Phosphorylated gap junction protein levels and LC3-Ⅱ levels were quantified using western blot analysis. The spatial distribution of immunoreactivity for phosphorylated connexin 43 (p-CX43) and LC3-Ⅱ was analyzed by immunofluorescence. The results showed that p-CX43 expression in the hippocampus reached a maximum level 6 h following injury. In addition, the immunoreactivity of p-CX43 was localized in the astrocytes surrounding pyramidal neurons. The LC3-Ⅱ protein content remained at high levels 24 h following injury. Double immunolabeling demonstrated that LC3-II dots colocalized with the hippocampus pyramidal neurons. Furthermore, inhibition of p-CX43 reduced TBI-induced autophagy, according to western blot analysis. As astrocytic gap junction coupling is affected in various forms of brain injury, the results suggest that point gap junctions/connexins are important regulators of autophagy in the hippocampal neurons following TBI.

Traumatic brain injury (TBI) can initiate a series of complicated pathological events, and induce various types of neuronal cell death including autophagy and apoptosis. Currently, the treatment of TBI is one of the main challenges in neurobiology. In this regard, the administration of bone marrow stromal cells (BMSCs) represents a novel treatment modality for TBI. However, the protective mechanism of BMSCs was unknown in the TBI. The aim of the present study was to assess the effects of BMSCs on connexin 43(CX43) and autophagy in the hippocampus following TBI in rats. A rat model of TBI was created using a modified weight-drop device. Double-membrane structures in the process of autophagy formation were frequently observed in injured brain by electron microscopy. The levels of autophagic pathway associated proteins and CX43 were also detected by western blot analysis. Specifically, immunoblotting results showed that BMSCs treatment after TBI could down-regulate light chain 3 (LC3), Beclin-1 and CX43 expression in the hippocampus. Taken together, our results demonstrated that BMSCs were able to significantly suppress TBI-induced autophagy activity, and the potential mechanism by regulating CX43 levels.

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