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. 2014 Oct 24:4:6718.
doi: 10.1038/srep06718.

miR-21 improves the neurological outcome after traumatic brain injury in rats

Xin-Tong Ge  1 , Ping Lei  2 , Hai-Chen Wang  3 , An-Ling Zhang  4 , Zhao-Li Han  5 , Xin Chen  1 , Sheng-Hui Li  1 , Rong-Cai Jiang  1 , Chun-Sheng Kang  4 , Jian-Ning Zhang  1
Affiliations

miR-21 improves the neurological outcome after traumatic brain injury in rats

Xin-Tong Ge et al. Sci Rep. .

Abstract

The expression levels of microRNAs (miRNAs) including miR-21, have been reported to change in response to traumatic brain injury (TBI), suggesting that they may influence the pathophysiological process in brain injury. To analyze the potential effect of miR-21 on neurological function after TBI, we employed the fluid percussion injury rat model and manipulated the expression level of miR-21 in brain using intracerebroventricular infusion of miR-21 agomir or antagomir. We found that upregulation of miR-21 level in brain conferred a better neurological outcome after TBI by improving long-term neurological function, alleviating brain edema and decreasing lesion volume. To further investigate the mechanism underlying this protective effect, we evaluated the impact of miR-21 on apoptosis and angiogenesis in brain after TBI. We found that miR-21 inhibited apoptosis and promoted angiogenesis through regulating the expression of apoptosis- and angiogenesis-related molecules. In addition, the expression of PTEN, a miR-21 target gene, was inhibited and Akt signaling was activated in the procedure. Taken together, these data indicate that miR-21 could be a potential therapeutic target for interventions after TBI.

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Figures

Figure 1
Figure 1. Altered miR-21 level in the traumatic foci after TBI and intervention with miR-21 oligomers.
The temporal profile (from 0 h to 14 d post-injury) of miR-21 level (a) and microvascular endothelial cells (MVECs) – expressed miR-21 level (b) in the traumatic foci determined by qRT-PCR. The quantitative data of a and b were analyzed using the 2−ΔΔCt method, in which the miR-21 levels of the sham group (presented as the dotted line) were used as controls. The quantitative data of miR-21 immunopositive neurons (c), astrocytes (d) and microglias (e) at 3 d post-injury detected by combined miRNA in-situ hybridization and immunofluorescence staining. The data are expressed as mean ± SD. (n = 6) (*P < 0.05, **P < 0.01, ***P < 0.001 versus the injury ctl group. #P < 0.05, ##P < 0.01, ###P < 0.001 versus the sham group. Specifically, the pound signs in 1a represent the injury ctl group versus the sham group.)
Figure 2
Figure 2. Representative images of the immunostaining of in-situ miR-21 expressed by neurons, astrocytes and microglias in different areas of brain.
The combined staining of miR-21/MAP-2 (a), miR-21/GFAP (b), miR-21/Iba-1 (c) lesioned boundary (LB, scope see Figure 3e) of cerebral cortex. The combined staining of miR-21/MAP-2 (d) and miR-21/GFAP (e) in the CA1 (a subdivision of Ammon's horn). The combined staining of miR-21/GFAP (f) in the CA3 (a subdivision of Ammon's horn). The combined staining of miR-21/GFAP (g) and miR-21/Iba-1 (h) in the dentate gyrus (DG). In each set of the figures, the immunostained areas in the white box were magnified from Figure A to B and C. A group of figures (Figure C, D, E) in the same location were presented to better illustrate the effect of combined immunostaining. All figures were captured from the sham group. Scale bars: A–B, 50 μm; C–E, 20 μm. (n = 6).
Figure 3
Figure 3. The impact of regulating brain miR-21 level on the long-term neurological function, brain edema and histopathological outcomes of TBI rats.
The long-term neurological function evaluated by the mNSS test (a) (n = 10) and Morris Water Mass test (n = 6), which includes the spatial acquisition trial (b) and the probe trial (c). The quantitative data of brain water content measurement for lesioned cerebral hemisphere and cerebellum at 72 h post-injury (d) (n = 6). The H&E staining (e) and the quantitative data of lesion volume (f) at 14 d post-injury (n = 10). The data are expressed as mean ± SD (a, c, d, f) or mean ± SEM (b). Scale bars in 3e: 100 μm. (*P < 0.05, **P < 0.01 versus the injury ctl group).
Figure 4
Figure 4. The impact of miR-21 on cellular apoptosis in brain after TBI.
The immunostaining of apoptotic cells in the lesioned boundary (LB) of cerebral cortex and the dentate gyrus of ipsilateral hippocampus (DG) at 72 h post-injury (a). The quantitative data of immunostained apoptotic cells in a (b, c). The immunoblotting (d) and quantitative data (e) of apoptosis-related molecules (Bcl-2, Bax and cleaved Caspase-3) acquired from the traumatic foci at 72 h post-injury. The data are expressed as mean ± SD. Scale bars: 50 μm. (n = 6) (**P < 0.01, ***P < 0.001 versus the injury ctl group).
Figure 5
Figure 5. The impact of miR-21 on angiogenesis in brain after TBI.
The immunostaining of microvessels marked by CD31 in the lesioned boundary (LB) of cerebral cortexand the dentate gyrus of ipsilateral hippocampus (DG) at 7 d post-injury (a). The quantitative data of immunostained microvessels in a (b, c). The immunoblotting of angiogenesis-related molecules (VEGF, Ang-1 and Tie-2) acquired from the traumatic foci at 7 d post-injury (d). The immunoreactive area located on the vessels (white arrow) and the extracellular space of endothelial cells (white triangle). The quantitative data of immunostained molecules in d (e). The representative images of immunostaining of VEGF, Ang-1 and Tie-2 in the LB of the agomir group (f). The quantitative data of immunostained VEGF, Ang-1 and Tie-2 in the LB (g) and the DG (h) at 7 d post-injury. The data are expressed as mean ± SD. Scale bars: 50 μm. (n = 6) (*P < 0.05, **P < 0.01, ***P < 0.001 versus the injury ctl group).
Figure 6
Figure 6. The role of Akt signaling under miR-21 regulation in the traumatic foci after TBI.
The quantitative data of PTEN mRNA expression at 3 d and 7 d post-injury determined by qRT-PCR (a). The immunoblotting (b) and quantitative data (c, d) of PTEN and p-Akt at 3 d and 7 d post-injury. The data are expressed as mean ± SD. (n = 6) (*P < 0.05, **P < 0.01 versus the injury ctl group, #P < 0.05 represent 3 d post-injury versus 7 d post-injury).
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