Characterization of the transient middle cerebral artery occlusion model of ischemic stroke in a HuR transgenic mouse line

Abstract

This set of experiments characterizes a model of transient cerebral ischemic stroke in a transgenic (Tg) mouse line in which the glial fibrillary acidic protein (GFAP) promoter is utilized to drive expression of a human RNA-binding protein, HuR. Additionally, the effect of cerebral ischemia on the expression of endogenous Hu proteins is presented.

Fig. 2

Histology in Tg mice. (A) Neuropil rarefaction (asterisk). 1.25x; scale bar = 1 cm. (B) Red neurons (arrows) and neuropil vacuolation (asterisk). 20x; scale bar = 50 μm. (C) Hippocampal (arrows) and cortical (arrowheads) infarcts. 10x; scale bar = 500 μm. (D) High-power image of hippocampal injury. 60x; scale bar = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 3

Distribution of endogenous Hu proteins in Wt mice 24 hours after injury. (A) Hu immunoreactivity (green) with nuclear and cytoplasmic distribution (arrow) in contralateral ROIs. (B) Decrease in Hu immunoreactivity (arrow) and Map2 immunofluorescence in the infarct borderzone. (C) Lack of Map2 signal and predominantly cytoplasmic Hu immunoreactivity (arrow) in the infarct core. (D) HuR/tubulin Western blot with densitometry, HuR normalized to tubulin, n = 6, mean ± SEM, *p = 0.046 (Student’s t-test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

The effect of ischemia-reperfusion on Hu immunoreactivity in rats. Approximate locations of the images are demonstrated with asterisks in the diagrams: the stippled border represents the infarct borderzone. Top row: KSRP immunoreactivity in uninjured contralateral region (left column); periphery of injury, i.e., the region immediately adjacent to where KSRP immunoreactivity transitions to the altered pattern (middle column); and core of injury (right column). Middle row: Hu immunoreactivity in the same regions. Bottom row: merge of KSRP and Hu immunoreactivity, and Hoechst nuclear stain. In the contralateral region, there is colocalization of nuclear KSRP and Hu immunoreactivity in nuclei of most cells. Hu immunoreactivity is also cytoplasmic in some of the cells. On the ipsilateral side, at the periphery of injury, KSRP immunoreactivity attenuation appears greater than Hu immunoreactivity attenuation. In the core of injury, KSRP immunoreactivity is absent, and Hu immunoreactivity is located in the cytoplasm and processes of shrunken cells with preserved nuclei. Scale bar = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 1

Reference diagram: infarct core (blue) and borderzone (yellow) and approximate locations of ipsilateral (Ipsi) and contralateral (Contra) ROI (grey boxes), not to scale. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).