Upregulation of neuroglobin expression and changes in serum redox indices in a rat model of middle cerebral artery occlusion

Abstract

Neuroglobin (NGB) is a recently identified protein, which is localized in the neurons and retinal cells of the central and peripheral nervous systems in vertebrates. It is hypothesized to function as a scavenger for reactive oxygen species, or as a stress-responsive sensor for signal transduction in hypoxic-ischemic brain insults. However, the mechanism underlying the physiological function of this protein remains to be elucidated. In the present study, the profiling of changes in the serum redox index of morphological features of the hippocampus and cortex, and of the expression of NGB and hypoxia-inducible factor-1α (HIF-1α), are described in a rat middle cerebral artery occlusion (MCAO) model. The necrotic zone of the rat neural tissues increased in size with increasing reperfusion time, and different brain slices exhibited necrosis in different regions. The number of NGB-positive hippocampal and cortical cells, as well as NGB and HIF-1α transcript and protein levels in the ischemic cortex, increased with increasing reperfusion time. NGB and HIF-1α mRNA and protein levels peaked in the group that received reperfusion at 32 h after MCAO. These findings indicated that HIF-1α may be involved in ischemic pathology in an MCAO model and that NGB expression may be upregulated. Serum superoxide dismutase (SOD) activity decreased and serum malondialdehyde (MDA) levels increased with increasing reperfusion time, indicating that the redox potential increased following MCAO. Serum SOD and MDA measurements may, therefore, be useful as biomarkers for the early detection of ischemic injury in a clinical setting.

Figure 1

Necrotic zone assay of rat brain tissue sections stained with TTC. (A) Brain sections stained with TTC from the sham, 0 h reperfusion and 32 h reperfusion groups. (B) Histogram for comparison of the necrotic zones in different brain sections, indicating that necrotic zone areas increased with increasing reperfusion time and different brain slices exhibited different necrotic zone (P<0.01). TTC, Triphenyltetrazolium chloride.

Figure 2

Morphological structure assay of the hippocampus using hematoxylin and eosin staining. (A)–(G) Morphological structure changes in the hippocampus in the sham group and following different reperfusion time periods (0, 4, 8, 16, 32 and 64 h; magnification, x200).

Figure 3

Morphological structure assay of the cortex using hematoxylin and eosin staining. (A)–(G) Morphological structure changes in the cortex in the sham group and following different reperfusion time periods (0, 4, 8, 16, 32 and 64 h; magnification, x200).

Figure 4

Cortical neuroglobin expression in ischemic and non-ischemic regions following induced focal cerebral ischemia, as determined by immunohistochemistry. (P<0.05 and P<0.01, compared with sham control).

Figure 5

HIF-1α and NGB mRNA expression levels in ischemic and non-ischemic regions, as determined by reverse transcription-quantitative polymerase chain reaction. (A) NGB mRNA level in the treatment and sham groups. (B) HIF-1α mRNA level in the treatment and sham groups. (P<0.05 and P<0.01, compared with sham control). HIF, hypoxia inducible factor; NGB, neuroglobin.

Figure 6

HIF-1α and NGB protein expression levels in ischemic and non-ischemic regions, as determined by western blot analysis. (A) NGB protein expression level in the treatment and sham groups. (B) HIF-1α protein expression level in the treatment and sham groups. HIF, hypoxia inducible factor; NGB, neuroglobin.