Nix Plays a Neuroprotective Role in Early Brain Injury After Experimental Subarachnoid Hemorrhage in Rats

So we need followup human testing. WHOM IS YOUR STROKE HOSPITAL CONTACTING TO GET THAT DONE?  NOTHING? Then I sugget firing the whole hospital starting with the board of directors.  This would be better said, 'This stops one of the causes of the neuronal cascade of death in the first week.'  Neuroprotection doesn't specify urgency, CASCADE OF DEATH does

Nix Plays a Neuroprotective Role in Early Brain Injury After Experimental Subarachnoid Hemorrhage in Rats

Juyi Zhang^†, Guiqiang Yuan^†, Tianyu Liang, Pengjie Pan, Xiang Li, Haiying Li, Haitao Shen^*, Zhong Wang^* and Gang Chen

• Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, China

Nix is located in the outer membrane of mitochondria, mediates mitochondrial fission and implicated in many neurological diseases. However, the association between Nix and subarachnoid hemorrhage (SAH) has not previously been reported. Therefore, the present study was designed to evaluate the expression of Nix and its role in early brain injury (EBI) after SAH. Adult male Sprague-Dawley (SD) rats were randomly assigned to various time points for investigation after SAH. A rat model of SAH was induced by injecting 0.3 ml of autologous non-heparinized arterial blood into the prechiasmatic cistern. The expression of Nix was investigated by Western blot and immunohistochemistry. Next, Nix-specific overexpression plasmids and small interfering RNAs (siRNAs) were separately administered. Western blot, neurological scoring, Morris water maze, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and fluoro-jade B (FJB) staining were performed to evaluate the role of Nix in EBI following SAH. We found that Nix was expressed in neurons and its expression level in the SAH groups was higher than that in the Sham group, which peaked at 24 h after SAH. Overexpression of Nix following SAH significantly decreased the expression of translocase of outer mitochondrial membrane 20 (TOMM20, a marker of mitochondria), ameliorated neurological/cognitive deficits induced by SAH, and reduced the total number of apoptotic/neurodegenerative cells, whereas siRNA knockdown of Nix yielded opposite effects. Taken together, our findings demonstrated that the expression of Nix is increased in neurons after experimental SAH in rats, and may play a neuroprotective role in EBI following SAH.

Introduction

Spontaneous subarachnoid hemorrhage (SAH) is a cerebrovascular disease with high disability and mortality rates. Rupture aneurysm is the main cause of SAH, accounting for about 85% of all spontaneous SAH (Ji and Chen, 2016). Although great progress has been made in current treatment of intracranial aneurysms, including microsurgery and endovascular embolization, it is reported that the disability and mortality rates of SAH have still not significantly decreased. The mortality rate of SAH is between 25 and 35% in high-income countries, and is as high as 48% in low-income countries (Pluta et al., 2009). Early brain injury (EBI) is an important factor leading to the deterioration of SAH patients. The occurrence of EBI after SAH is a complex pathophysiological process, and its mechanisms may be related to autophagy, apoptosis, inflammation, destruction of the blood-brain barrier (BBB), cytotoxic brain edema and oxidative stress (Fujii et al., 2013). Therefore, there is an urgent need for continued scientific investigations of novel approaches for preventing and treating EBI after SAH.

Autophagy involves the identification of cellular proteins and organelles for degradation that are then wrapped by double layered membranous structures that are detached from the ribosome-free attachment region of the rough-surface endoplasmic reticulum (ER) to form autophagosomes; subsequently, autophagosomes are further degraded after being fused with lysosomes, to meet the metabolic demands of cells and to regenerate organelles as needed (Nah et al., 2015). Studies have shown that mitochondrial dysfunction affects a series of intracellular biological processes that are involved in the process of EBI after SAH, including oxidative damage, calcium homeostasis disorder, and the collapse of ATP synthesis (Li et al., 2018). Mitophagy is the process in which injured or unwanted mitochondria are selectively cleared via autophagy, thereby maintaining the homeostasis of cells (Liu et al., 2014). Taken together, mitophagy may play an important role during EBI after SAH.

Nix, also known as B-cell lymphoma 2 (Bcl-2)-interacting protein 3 like (Bnip3L), was originally thought to belong to the Bcl-2 family and BH3-only pro-apoptotic proteins, but its ability to induce apoptosis was found to be weak (Sandoval et al., 2008; Zhang and Ney, 2009). With the development of further research, it has been found that Nix is different from other typical BH-3-only proteins and that pro-apoptosis may not be its main function (Mellor and Harris, 2007; Ding et al., 2010). Nix was first described as a mitophagic receptor when it was discovered to be involved in the procedural clearance of mitochondria during reticulocyte maturation (Ney, 2015). In recent years, it has been considered that the mechanisms of EBI after SAH are complex, which are the result of a combination of various factors. Although recent studies have suggested that autophagy is activated after SAH and can ameliorate EBI, the specific mechanism underlying this process remains unknown (Li et al., 2014). As mitochondria are the most important organelles for sources of intracellular energy, mitochondrial dysfunction plays a significant role in EBI after SAH (Wang et al., 2017; Zhou et al., 2017). Mitophagy clears impaired mitochondria in cells. Hence, discovery of the key factors that regulate the occurrence of mitophagy after SAH may reverse brain injury caused by mitochondrial dysfunction (Chen et al., 2014). Nix is located on the outer mitochondrial membrane and has been demonstrated to be a vital protein for regulating mitophagy. Moreover, Nix has been shown to be involved in the pathophysiological processes of various central nervous system (CNS) diseases, including cerebral ischemia-reperfusion (I/R) injury, intracranial hemorrhage, spinal cord injury and neurodegenerative diseases; however, the specific mechanisms of Nix in the pathophysiology of these CNS diseases remain unclear (Rui et al., 2013; Yu et al., 2013; Park et al., 2017; Yuan et al., 2017).

In the present study, we determined the expression of Nix after SAH, and further investigated the role of Nix in EBI after SAH. Our findings elucidating the role of Nix in EBI after SAH may provide a novel approach of thought for the treatment of SAH patients.