Overexpress miR-132 in the Brain Parenchyma by a Non-invasive Way Improves Tissue Repairment and Releases Memory Impairment After Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) is a serious public health problem worldwide, which could lead to an extremely high percentage of mortality and disability. Current treatment strategies mainly concentrate on neuronal protection and reconstruction, among them, exogenous neural stem cell (NSC) transplantation has long been regarded as the most effective curative treatment. However, due to secondary trauma, transplant rejection, and increased incidence of brain malignant tumor, a non-invasive therapy that enhanced endogenous neurogenesis was more suitable for TBI treatment. Our previous work has shown that miR-132 overexpression could improve neuronal differentiation of NSCs in vitro and in vivo. So, we engineered a new kind of AAV vector named AAV-PHP.eB which can transfect brain parenchyma through intravenous injection to overexpress miR-132 in brain after TBI. We found that miR-132 overexpression could reduce impact volume, promote neurogenesis in the dentate gyrus (DG), accelerate neuroblast migrating into the impact cortex, ameliorate microglia-mediated inflammatory reaction, and ultimately restore learning memory function. Our results revealed that AAV-PHP.eB-based miR-132 overexpression could improve endogenous tissue repairment and release clinical symptoms after traumatic brain injury. This work would provide a new therapeutic strategy for TBI treatment and other neurological disorders characterized by markable neuronal loss and memory impairment. miR-132 overexpression accelerates endogenous neurogenesis and releases TBI-induced tissue repairment and memory impairment. Controlled cortical impact onto the cortex would induce serious cortical injury and microglia accumulation in both cortex and hippocampus. Moreover, endogenous neuroblast could migrate around the injury core. miR-132 overexpression could accelerate neuroblast migration toward the injury core and decreased microglia accumulation in the ipsilateral cortex and hippocampus. miR-132 could be a suitable target on neuroprotective therapy after TBI.

Keywords: Neural stem cells; Neurogenesis; Traumatic brain injury; miR-132; microRNA-based therapeutic.

Fig. 1

Controlled cortical impact induced cortical damage and memory impairment. A Diagrammatic view of the impacted location. B TBI-induced neuronal damage was districted in the cortex without influencing the hippocampus, revealing a moderate TBI lesion, Scale bar = 800 μm. C TBI caused observed tissue defect, n = 6, ^**p < 0.01, Mann–Whitney U test. D Novel object recognition test process. E TBI mice suffered from memory impairment, sham (n = 6) vs TBI (n = 7), ^*p < 0.05, un-paired Student's t test. Data are shown as mean ± SD

Fig. 2

TBI accelerated the hippocampal neurogenesis and migration of neuroblast toward the damaged area. A Representative photographs of Dapi (blue) and BrdU (red), Scale bar = 100 µm. B TBI increased number of BrdU⁺ cells in the ipsilateral DG, ^***p < 0.001, ^****p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. C Representative photographs of Dapi (blue) and DCX (red), Scale bar = 100 µm. D TBI increased the number of DCX⁺ cells in the ipsilateral DG, ^*p < 0.05, two-way ANOVA with Tukey’s multiple comparisons test. E Representative photographs of Dapi (blue) and DCX (green), Scale bar = 100 µm. F TBI increased the number of DCX⁺ cells nearby the impact core, ^***p < 0.001, Scheirer–Ray–Hare test with Wilcoxon rank sum test. Data are shown as mean ± SD

Fig. 3

TBI-induced microglia activation in the cortex and hippocampus. A, D Representative photographs of Dapi (blue) and Iba1 (red), Scale bar = 100 µm. B Mean fluorescence intensity was increased in the ipsilateral cortex after TBI, ^***p < 0.001, Scheirer–Ray–Hare test with Wilcoxon rank sum test. C Number of IBA1⁺ cells were increased in the ipsilateral cortex after TBI, ^****p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. E Mean fluorescence intensity was increased in the ipsilateral hippocampus after TBI, ^**p < 0.01, ^****p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. F Number of IBA1⁺ cells were increased in the ipsilateral DG after TBI, ^**p < 0.01, ^****p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. Data are shown as mean ± SD

Fig. 4

miR-132 overexpression vector was specifically expressed in neuronal cells but not glia cells. A Timeline of virus transfection experiment. B The structure of miR-132 overexpression plasmid. C Representative photographs of the whole brain, Scale bar = 2 mm. D, E Representative photographs of Dapi (blue), mNeonGreen(green) and GFAP/Iba1 (red), Scale bar = 100 µm

Fig. 5

miR-132 overexpression decreased tissue damage and restored memory function after TBI. A Representative photographs of nissl stain, Scale bar = 2 mm. B miR-132 overexpression reduced TBI-induced tissue damage (n = 5), ^*p < 0.05, Scheirer–Ray–Hare test with Wilcoxon rank sum test. C miR-132 overexpression increased recognition index after TBI (n = 8), ^*p < 0.05, ^**p < 0.01, two-way ANOVA with Tukey’s multiple comparisons test. Data are shown as mean ± SD

Fig. 6

miR-132 overexpression further promoted TBI-induced neurogenesis and neuroblast migration. A, C Representative photographs of Dapi (blue) and DCX (green), Scale bar = 100 µm. B miR-132 overexpression further increased the number of DCX⁺ cells in the ipsilateral DG, ^*p < 0.05, ^**p < 0.01, Scheirer–Ray–Hare test with Wilcoxon rank sum test. D miR-132 overexpression further increased the number of DCX⁺ cells nearby the impact core, ^*p < 0.05, ^***p < 0.001, Scheirer–Ray–Hare test with Wilcoxon rank sum test. Data are shown as mean ± SD

Fig. 7

miR-132 overexpression inhibited TBI-induced microglia activation. A Representative photographs of Dapi (blue) and Iba1 (red) of the whole brain, Scale bar = 2 mm. B, E Representative photographs of Dapi (blue) and Iba1 (red), Scale bar = 100 µm. (C, D) miR-132 overexpression reduced TBI-induced microglia activation in the cortex, ^*p < 0.05, ^***p < 0.001, Scheirer–Ray–Hare test with Wilcoxon rank sum test. F miR-132 overexpression reduced TBI-induced microglia activation in the DG, ^***p < 0.001, Scheirer–Ray–Hare test with Wilcoxon rank sum test. G miR-132 overexpression decreased TBI-induced microglia infiltration in the hippocampus, ^****p < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. Data are shown as mean ± SD