Methylene Blue Reduces Neuronal Apoptosis and Improves Blood-Brain Barrier Integrity After Traumatic Brain Injury

WHOM will your doctor contact to get this tested for stroke in humans?  No contact then you need to have that doctor fired for incompetency and dereliction of duty. We need to start clearing out a lot of dead wood in stroke, probably starting with your stroke hospital board of directors.

Methylene Blue Reduces Neuronal Apoptosis and Improves Blood-Brain Barrier Integrity After Traumatic Brain Injury

Jun Shen^1,2,3,4†, Wenqiang Xin^2,3,4, Qifeng Li^2,3,4†, Yalong Gao^2,3,4†, Lili Yuan^5* and Jianning Zhang^2,3,4*

• ¹Department of Neurosurgery, Yijishan Hospital of Wannan Medical College, Wuhu, China
• ²Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin, China
• ³Tianjin Neurological Institute, Tianjin, China
• ⁴Key Laboratory of Post-trauma Neuro-repair and Regeneration in Central Nervous System, Ministry of Education, Tianjin, China
• ⁵Department of Neurology, Yijishan Hospital of Wannan Medical College, Wuhu, China

Objective: To investigate whether methylene blue (MB) treatment can reverse neuronal mitochondrial dysfunction caused by oxygen glucose deprivation/reoxygenation (OGD) injury and then investigate whether MB treatment can reduce neuronal apoptosis and improve blood-brain barrier (BBB) integrity in traumatic brain injury (TBI) animals.

Methods: Reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and adenosine triphosphate (ATP) were used to evaluate mitochondrial function. The terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay was used to assess neuronal apoptosis in vitro. TUNEL and immunofluorescence staining for neuronal nuclei (NeuN) were combined to assess neuronal apoptosis in vivo. An Evans blue (EB) permeability assay and brain water content (BWC) were used to measure BBB permeability in vivo. The Morris water maze (MWM), rotarod test, and modified Neurological Severity Score (mNSS) test were employed to assess the prognosis of TBI mice.

Results: MB treatment significantly reversed neuronal mitochondrial dysfunction caused by OGD injury. Both in vitro and in vivo, MB treatment reduced neuronal apoptosis and improved BBB integrity. In TBI animals, treatment with MB not only improved cognitive and motor function caused by TBI but also significantly improved overall neurological function.

Conclusions: Our findings suggest that MB is a potential candidate for the treatment of TBI. Future research should focus on other therapeutic effects and mechanisms of MB in secondary brain injury.

Introduction

Traumatic brain injury (TBI) is the most common cause of mortality and disability among working-age adults and young individuals worldwide (1). In the United States, ~2 million people suffer a TBI each year, and TBI accounts for nearly one-third of all trauma-related mortality (1, 2). TBI damages brain tissue through two pathological processes, primary and secondary injury. Primary injury is characterized by immediate bleeding and loss of brain tissue when a blunt or sharp object impacts the head. Secondary injury involves complicated cellular and biochemical cascade reactions, including oxidative stress, excitotoxicity, neuroinflammation, free radical-induced injury, and calcium-mediated damage, which lead to blood-brain barrier (BBB) damage, elevated intracranial pressure, cerebral hypoxia, brain edema, and neuronal apoptosis (3–8). Mitochondrial dysfunction has been demonstrated to be a key participant in the pathological processes of secondary brain injury (9, 10).

Methylene blue (MB) is an FDA-approved drug used to treat cyanide poisoning, carbon monoxide poisoning, and methemoglobinemia (11). Previous studies have demonstrated that MB can improve mitochondrial function (12). Under pathological conditions, MB acts as an alternative electron carrier that bypasses complex I/III blockage and efficiently transfers electrons from NADH to cytochrome c (cyt c). This process reduces electron leakage, enhances adenosine triphosphate (ATP) production, and decreases the overproduction of reactive oxygen species (ROS) (13). In recent years, MB has been shown to attenuate pathological and neurobehavioral impairments in animal models of Alzheimer's disease (AD) (14, 15), Parkinson's disease (PD) (16), ischemic stroke (17, 18), and TBI (19–21). After TBI, MB treatment can attenuate neuroinflammation, reduce lesion volume, and improve neurological damage (19–21).

Since MB treatment can reduce the release of ROS and increase the production of ATP, it may have the potential to reduce neuronal apoptosis and improve BBB integrity. However, these effects of MB on TBI have not been investigated. In the present study, we first investigated whether MB treatment can reverse neuronal mitochondrial dysfunction and then investigated whether MB treatment can reduce neuronal apoptosis and improve BBB integrity after TBI.