Neuroprotective and Functional Improvement Effects of Methylene Blue in Global Cerebral Ischemia

Abstract

Transient global cerebral ischemia (GCI) causes delayed neuronal cell death in the vulnerable hippocampus CA1 subfield, as well as behavioral deficits. Ischemia reperfusion (I/R) produces excessive reactive oxygen species and plays a key role in brain injury. The mitochondrial electron respiratory chain is the main cellular source of free radical generation, and dysfunction of mitochondria has a significant impact on the neuronal cell death in ischemic brain. The aim of the present study is to investigate the potential beneficial effects of methylene blue (MB) in a 4-vessel occlusion (4VO) GCI model on adult male rats. MB was delivered at a dose of 0.5 mg/kg/day for 7 days, through a mini-pump implanted subcutaneously after GCI. We first found that MB significantly improved ischemic neuronal survival in hippocampal CA1 region as measured by cresyl violet staining as well as NeuN staining. We also found that MB has the ability to rescue ischemia-induced decreases of cytochrome c oxidase activity and ATP generation in CA1 region following I/R. Further analysis with labeling of MitoTracker® Red revealed that the depolarization of mitochondrial membrane potential (MMP) was markedly attenuated following MB treatment. In addition, the induction of caspase-3, -8 and -9 activities, and the increased numbers of TUNEL positive cells of CA1 region were significantly reduced by MB application. Correspondingly, Barnes maze tests showed that the deterioration of spatial learning and memory performance following GCI were significantly improved in MB-treatment group compared to ischemic control group. In summary, our study suggests that MB may be a promising therapeutic agent targeting neuronal cell death and cognitive deficits following transient global cerebral ischemia.

Low-Level Laser Irradiation Improves Depression-Like Behaviors in Mice

“…As previously described [25], ATP concentration was determined using a mouse ATP Elisa kit (68CV-ATP-S1000, Rapidbio; America) following vendor’s instructions. Briefly, brain regions of PFC, Hip, and Hy were dissected and homogenized.…”

Methylene Blue promotes cortical neurogenesis and ameliorates behavioral deficit after photothrombotic stroke in rats

“…The level of ATP was quantified in the total protein sample using a kit ENLITEN ® rLuciferase/Luciferin reagent (FF2021, Promega, Madison, WI), as previously described by our lab (Lu et al, 2015). In brief, 30 μg of protein samples were diluted in 100 μL of reconstituted rL/L reagent buffer containing luciferase, D-luciferin, Tris-acetate buffer (pH 7.75), ethylene diamine tetra acetic acid (EDTA), magnesium acetate, bovine serum albumin (BSA), and dithiothreitol (DTT).…”

“…MB receives an electron from NADH and transfers it to cytochrome c, bypassing complex I/III. This facilitates enhanced ATP production as well as decreased reactive oxygen species (ROS) output from the mitochondrial electron transport chain (Chen et al, 2003; Lu et al, 2015; Shen et al, 2013). This has made MB a promising candidate for many neurodegenerative conditions.…”

“…This has made MB a promising candidate for many neurodegenerative conditions. It has been reported that low dose MB treatment attenuates behavioral, neurochemical and pathological impairments in animal models of Parkinson's disease (PD), Alzheimer's disease (AD), global cerebral ischemia (GCI) and stroke (Oz et al, 2009; Medina et al, 2011; Lu et al, 2015; Wen et al, 2011). The neurogenic property of MB in ischemic stroke, however, remains poorly understood.…”

Beneficial Effects of a CaMKIIα Inhibitor TatCN21 Peptide in Global Cerebral Ischemia

“…The experimental design was shown in Figure 1. Four vessel occlusion (4VO) GCI model was developed as previously described (Zhou et al , 2011; Lu et al , 2015). Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg ip), then both vertebral arteries were electro-cauterized and the bilateral common carotid arteries (CCAs) were exposed and a silastic ligature was loosely secured around each artery.…”