Reduced infarct size in neuroglobin-null mice after experimental stroke in vivo

Abstract

Background: Neuroglobin is considered to be a novel important pharmacological target in combating stroke and neurodegenerative disorders, although the mechanism by which this protection is accomplished remains an enigma. We hypothesized that if neuroglobin is directly involved in neuroprotection, then permanent cerebral ischemia would lead to larger infarct volumes in neuroglobin-null mice than in wild-type mice.

Methods: Using neuroglobin-null mice, we estimated the infarct volume 24 hours after permanent middle cerebral artery occlusion using Cavalieri's Principle, and compared the infarct volume in neuroglobin-null and wild-type mice. Neuroglobin antibody staining was used to examine neuroglobin expression in the infarct area of wild-type mice.

Results: Infarct volumes 24 hours after permanent middle cerebral artery occlusion were significantly smaller in neuroglobin-null mice than in wild-types (p < 0.01). Neuroglobin immunostaining of the penumbra area revealed no visible up-regulation of neuroglobin protein in ischemic wild-type mice when compared to uninjured wild-type mice. In uninjured wild-type mice, neuroglobin protein was seen throughout cortical layer II and sparsely in layer V. In contrast, no neuroglobin-immunoreactive neurons were observed in the aforementioned layers of the ischemia injured cortical area, or in the surrounding penumbra of ischemic wild-type mice. This suggests no selective sparing of neuroglobin expressing neurons in ischemia.

Conclusions: Neuroglobin-deficiency resulted in reduced tissue infarction, suggesting that, at least at endogenous expression levels, neuroglobin in itself is non-protective against ischemic injury.

Figure 1

Neuroglobin (Ngb) expression and infarct distribution. Ngb expression (brown) and infarct distribution on Haematoxylin counter stained sections 24 hours after permanent middle cerebral artery occlusion (pMCAo). A. Uninjured, B. wild-type (WT) pMCAo and C. Ngb-null pMCAo. Note that there is no Ngb staining in Ngb-null mice, and that staining in cortex is very limited. The infarct is marked with red lines on the sections within red squares, which are shown in higher magnification in Figure 2.

Figure 2

Ngb expression in the ischemic penumbra. Panel A-C shows representative sections of Ngb expression (see arrows) and infarct distributions delineated by red lines in uninjured (sham), WT and Ngb-null pMCAo mice respectively. The bottom part of the panel shows high magnification images of the area in the white squares where black arrows point at Ngb expressing neurons and white at necrotic neurons. The areas above the red lines are tissue undergoing liquefactive necrosis identified by pyknosis, karyolysis, karyohexis and rapid tissue dissolution consistent with necrotic ischemic brain injury. Note how no increase in Ngb expression is seen within or adjacent to the penumbra area (Panel A-B bottom) in WT pMCAo compared to sham. Also please note how no Ngb staining is seen in the infarcted area (Panel B bottom) suggesting no selective sparing of Ngb expressing neurons. In D a western blot quantification of Ngb expression in sham (SE ± 0.15) and pMCAo WT (SE ± 0.092) mice relative to beta-actin is shown. A significant (p = 0.038) down regulation of Ngb was observed in the pMCAo group compared to the sham operated group. Lines indicate mean values. Scale bar 150 μm.

Figure 3

Infarct volumes 24 hours after permanent middle cerebral artery occlusion. Infarct volumes 24 hours after pMCAo measured with 2D nucleator and Cavalieri’s principle. Infarct volume in cortex was significantly larger in WT mice (n = 6, 7.6 mm³ SE ± 0.35 mm³) compared to matching Ngb-null (n = 9 5.7 mm³ SE ± 0.41 mm³) littermates. p < 0. 0076. Lines indicate mean values and * denote significance.

Figure 4

Immunohistochemical staining for Ngb in the brain. Immunohistochemical staining for Ngb in the brain of Ngb +/+ and Ngb -/- mice. In house made rabbit antibody 4836/5 showed specific (Ngb) staining (black arrow, signifying specific staining) in Ngb +/+ mice (A, C), which was abolished in Ngb -/- mice (B, D). Sigma rabbit Ngb antibody N7162 stained most neurons of cortex, hippocampus (white arrows, signifying unspecific staining) and neurons of the laterodorsal tegmental nucleus (LDTg) (black arrow) (E, G). Only the staining in the large LDTg neurons was abolished in Ngb -/- mice (F, H). No specific staining was observed using the Santa Cruz sc-22001 antibody (I-L). Dentate gyrus (DG). Scale bar 50 μm.