Hyperbaric Oxygen Reduces Infarction Volume and Hemorrhagic Transformation Through ATP/NAD+/Sirt1 Pathway in Hyperglycemic Middle Cerebral Artery Occlusion Rats

Abstract

Background and purpose: Energy depletion is a critical factor leading to cell death and brain dysfunction after ischemic stroke. In this study, we investigated whether energy depletion is involved in hyperglycemia-induced hemorrhagic transformation after ischemic stroke and determined the pathway underlying the beneficial effects of hyperbaric oxygen (HBO).

Methods: After 2-hour middle cerebral artery occlusion, hyperglycemia was induced by injecting 50% dextrose (6 mL/kg) intraperitoneally at the onset of reperfusion. Immediately after it, rats were exposed to HBO at 2 atmospheres absolutes for 1 hour. ATP synthase inhibitor oligomycin A, nicotinamide phosphoribosyl transferase inhibitor FK866, or silent mating type information regulation 2 homolog 1 siRNA was administrated for interventions. Infarct volume, hemorrhagic volume, and neurobehavioral deficits were recorded; the level of blood glucose, ATP, and nicotinamide adenine dinucleotide and the activity of nicotinamide phosphoribosyl transferase were monitored; the expression of silent mating type information regulation 2 homolog 1, acetylated p53, acetylated nuclear factor-κB, and cleaved caspase 3 were detected by Western blots; and the activity of matrix metalloproteinase-9 was assayed by zymography.

Results: Hyperglycemia deteriorated energy metabolism and reduced the level of ATP and nicotinamide adenine dinucleotide and exaggerated hemorrhagic transformation, blood-brain barrier disruption, and neurological deficits after middle cerebral artery occlusion. HBO treatment increased the levels of the ATP and nicotinamide adenine dinucleotide and consequently increased silent mating type information regulation 2 homolog 1, resulting in attenuation of hemorrhagic transformation, brain infarction, as well as improvement of neurological function in hyperglycemic middle cerebral artery occlusion rats.

Conclusions: HBO induced activation of ATP/nicotinamide adenine dinucleotide/silent mating type information regulation 2 homolog 1 pathway and protected blood-brain barrier in hyperglycemic middle cerebral artery occlusion rats. HBO might be promising approach for treatment of acute ischemic stroke patients, especially patients with diabetes mellitus or treated with r-tPA (recombinant tissue-type plasminogen activator).

Keywords: NAD; NAMPT; Sirt1 and hemorrhagic transformation; hyperbaric oxygenation; stroke.

Figure 1

HBO attenuated energy depletion, resulting in improved neurological functions in hyperglycemic MCAO rats. Hyperglycemia/MCAO generated a significant depletion of energy evaluated as level of ATP (Figure 2A) and NAD⁺ (Figure 2B) 6 hours after MCAO, and presented with severe neurological dysfunction assessed by neurological scores (Figure 2C) and Foot-fault test (Figure 2D) 24 hours after MCAO. *p < 0.05 vs. Sham; #p < 0.05 vs. MCAO+DX.

Figure 2

HBO decreased infarction and attenuated HT 24 hours after MCAO in hyperglycemic rats. Hyperglycemia/MCAO caused significant brain infarction (Figure 1A and 1B), hemorrhagic transformation (Figure 1A and 1C) and brain edema (Figure 1D). *p < 0.05 vs. Sham; #p < 0.05 vs. MCAO+DX.

Figure 3

Hyperglycemia/MCAO increased production of neuronal and astrocytic NAMPT but decreased the activity of NAMPT. NAMPT expression was mostly observed in neurons (NeuN/NAMPT positive cells) and astocytes (GFAP/NAMPT positive cells) of penumbra (Figure 3A). Hyperglycemia/MCAO increased production of NAMPT (Figure 3B) but decreased its activity (Figure 3C). *p < 0.05 vs. Sham.

Figure 4

Beneficial effects of HBO were dependent on ATP and NAD⁺ in hyperglycemic MCAO rats. NAD⁺ administration mimicked the effects of HBO to reduce infarction volume (Figures 4A) and hemorrhagic volume (Figures 4B), and improved neurological functions in neurological scores (Figures 4C) and food-fault test (Figures 4D) 24 hours after MCAO. NAMPT or ATP synthase inhibition attenuated the beneficial effects of HBO treatment (Figures 4A–D). #p < 0.05 vs. MCAO+DX; &p < 0.05 vs. MCAO+DX+HBO.

Figure 5

HBO ability to restore NAD⁺ and Sirt1 was dependent on NAMPT and ATP. Hyperglycemia/MCAO significantly decreased activity of NAMPT at 6 hours (Figure 5A) and diminished NAD⁺ production at the same time point (Figure 5B). HBO or NAD⁺ administration restored NAD⁺ and activated NAMPT (Figure 5A and 5B). Co-expression of Sirt1/NAMPT in cells of the penumbra area was observed (Figure 5C). HBO increased the expression of Sirt1 and NAD⁺ mimicked the effects of HBO, and ATP synthase or NAMPT inhibition reduced the protective effects of HBO (Figure 5D). *p < 0.05 vs. Sham; #p < 0.05 vs. MCAO+DX.

Figure 6

HBO induced activation of ATP/NAD⁺/Sirt1 pathway, decreased apoptosis and inhibited MMP-9. Protective effects of HBO were accompanied with decreased expression of p53 (Figure 6A), CC-3 (Figure 6B) and NF-κB (Figure 6C), and decreased activity of MMP-9 (Figure 6D). ATP synthase inhibitor and NAMPT inhibitor as well as Sirt1 siRNA reduced the protective effects of HBO (Figure 6A-D). #p < 0.05 vs. MCAO+DX; &p < 0.05 vs. MCAO+DX+HBO.