Neuroglobin protects the brain from experimental stroke in vivo

Abstract

Neuroglobin (Ngb) is an O(2)-binding protein localized to cerebral neurons of vertebrates, including humans. Its physiological role is unknown but, like hemoglobin, myoglobin, and cytoglobin/histoglobin, it may transport O(2), detoxify reactive oxygen species, or serve as a hypoxia sensor. We reported recently that hypoxia stimulates transcriptional activation of Ngb in cultured cortical neurons and that antisense inhibition of Ngb expression increases hypoxic neuronal injury, whereas overexpression of Ngb confers resistance to hypoxia. These findings are consistent with a role for Ngb in promoting neuronal survival after hypoxic insults in vitro. Here we report that in rats, intracerebroventricular administration of an Ngb antisense, but not sense, oligodeoxynucleotide increases infarct volume and worsens functional neurological outcome, whereas intracerebral administration of a Ngb-expressing adeno-associated virus vector reduces infarct size and improves functional outcome, after focal cerebral ischemia induced by occlusion of the middle cerebral artery. We conclude that Ngb acts as an endogenous neuroprotective factor in focal cerebral ischemia and may therefore represent a target for the development of new treatments for stroke.

Figure 1

Ngb antisense ODN, given by the intracerebroventricular route, reduces Ngb expression in rat brain. (a) Western blot of Ngb expression in cerebral cortex from control, antisense-treated, and sense ODN-treated rats. (b) Immunohistochemical localization of fluorescein-labeled ODN (green) within cerebral cortical neurons.

Figure 2

Ngb antisense ODN exacerbates focal cerebral ischemia in the rat. (a) Antisense-treated (Left) and sense ODN-treated (Right) brains after MCA occlusion and TTC staining, showing an increase in infarct area (white region at left; note greater superior and inferior extent of cortical infarction indicated by asterisks) in antisense-treated rats. (b) Infarct volume (mm³) increased by antisense ODN. (c) Neurological deficit worsened by antisense ODN. *, P < 0.05 compared with aCSF- (Con) and sense ODN-treated rats (n = 5–6; ANOVA and post hoc t test).

Figure 3

AAV-Ngb vector increases Ngb expression in vitro and in rat brain in vivo. (a) Western blot of Ngb expression in AAV-gfp control vector- (Left) and AAV-Ngb-transfected (Right) 293 cells. (b) AAV-Ngb partially protects cultured cortical neurons from hypoxic death, measured with MTT. (Dead, freeze-thawed cells). *, P < 0.05 (n = 4; ANOVA and post hoc t test). (c) Intracerebral injection of AAV-Ngb increases Ngb expression (top left) over basal levels (bottom right), as shown by immunohistochemistry with an anti-Ngb Ab (brown). Arrow, example of an uninfected cell with basal expression of Ngb. (d) Intracerebral administration of AAV-Ngb is associated with expression of Ngb in cortical neurons (Upper) but not astroglia (Lower). NeuN, neuronal nuclear antigen; GFAP, glial fibrillary acidic protein; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 4

Overexpression of Ngb reduces ischemic cerebral injury in the rat. (a) Intracerebral AAV-Ngb reduces infarct size, shown as outlined areas in hematoxylin-stained sections (Left, AAV-gfp control vector-treated; Right, AAV-Ngb-treated). (b) Infarct volume (mm³) reduced by AAV-Ngb. (c) Neurological deficit improved in AAV-Ngb-treated animals. *, P < 0.05 compared with aCSF control and AAV-gfp control vector-treated rats (n = 3–8; ANOVA and post hoc t test).