Effects of neuroglobin overexpression on acute brain injury and long-term outcomes after focal cerebral ischemia

Abstract

Background and purpose: Emerging data suggest that neuroglobin (Ngb) may protect against hypoxic/ischemic neuronal insults. However, the underlying mechanisms in vivo and implications for long-term outcomes are still not well understood.

Methods: Using our newly created Ngb overexpressing transgenic (Ngb-Tg) mice, we measured brain infarction on day 1 and day 14 after transient focal cerebral ischemia and performed neurobehavioral assessments in sensorimotor deficits on days 1, 3, 7, and 14. To test the hypothesis that Ngb may play a role in reducing oxidative stress after stroke, intracellular malondialdehyde levels were measured and compared in Ngb-Tg and wild-type mice.

Results: Increased Ngb mRNA and protein levels were identified in Ngb-Tg brains. Malondialdehyde levels in ischemic hemispheres of Ngb-Tg were significantly reduced compared with wild-type controls at 8 hours and 22 hours after transient focal cerebral ischemia. Compared with wild-type controls, brain infarction volumes 1 day and 14 days after transient focal cerebral ischemia were significantly reduced in Ngb-Tg mice. However, there were no significant improvements in sensorimotor deficits for up to 14 days after stroke in Ngb-Tg mice compared with wild-type controls.

Conclusions: Ngb reduces tissue infarction and markers of oxidative stress after stroke. Tissue protection by overexpressing Ngb can be sustained for up to 2 weeks.

Figure 1

Production and characterization of Ngb-overexpressing transgenic mice. A, Tail DNA was used for PCR analysis with primers flanked CMV and Ngb sequence. The detected 940-bp band of PCR production indicates the presence of mouse Ngb transgene. DNA markers (Marker) appeared on the first lane; pCMV-Ngb plasmid served as a positive control (P). B, Brain RNA was extracted for measuring Ngb mRNA levels by quantitative real-time PCR. There was an approximately 2.6-fold increases of Ngb mRNA levels in Ngb-Tg mouse brains compared with WT and WT littermates (WT-L); similar Ngb mRNA levels were detected in WT (C57BL/6) and WT-L mouse brains. All animals used were 22 to 26 males between 10 and 12 weeks of age. Mean±SEM, n=6 per group, *P<0.05. C, Top panel is a representative Western blot showing the increase of Ngb protein in Ngb-Tg mouse brains (Tg) compared with WT controls. Actin served as equal loading controls. Lower panel shows relative Ngb protein expression levels quantified by optical density of Ngb protein bands. Mean±SEM, n=4 per group, *P<0.05. D, In cerebral cortex, immunohistochemistry showed Ngb immunoreactivity was mainly colocalized with neuron marker neuronal nuclei (D, A–C), not colocalized with astrocyte marker glial fibrillary acidic protein (D, D–F) in WT mice. By comparison, Ngb immunoreactivity was enhanced in neurons (D, G–I), some colocalized with astrocyte marker glial fibrillary acidic protein as pointed out by arrows (D, J–L) in Ngb-Tg mice. Bar: 50 μmol/L.

Figure 2

A, Reduction of acute ischemic infarction in Ngb transgenic mice after focal cerebral ischemia. After 2 hours transient focal cerebral ischemia followed by 22 hours reperfusion, 2,3,5-triphenyltetrazolium hydrochloride infarction volume in the whole hemisphere and cortical regions was significantly reduced in Ngb-Tg mice compared with matching WT littermates. Mean±SEM, n=8 per group, *P<0.05. B, Reduction of late ischemic infarction in Ngb-Tg mouse brains. At 14 days after transient 1-hour focal cerebral ischemia, brain infarction was measured by hematoxylin & eosin staining and image analysis. The infarction was only located in subcortical regions, and the volume was significantly reduced in Ngb-Tg mice compared with WT mice. Mean±SEM, n=6 for Tg and 7 for WT per group, *P<0.05. All mice were 22 to 27 g males between 8 and 9 weeks old.

Figure 3

Reduction of MDA production in Ngb-Tg mouse brains after acute focal stroke. In nonischemic control hemispheres (controls), MDA levels were not significantly different between WT and Tg mice, whereas MDA production was significantly decreased in Ngb-Tg mice compared with WT controls at 8 hours (n=10 per group) and 22 hours (n=4 per group) after 2-hour transient focal cerebral ischemia. Mean±SEM, *P<0.05.