Journal: Neural Regeneration Research

Article Title: Microglia overexpressing brain-derived neurotrophic factor promote vascular repair and functional recovery in mice after spinal cord injury

doi: 10.4103/NRR.NRR-D-24-00381

Figure Lengend Snippet: Microglia are essential for recovery from spinal cord injury, and their removal is detrimental to recovery from spinal cord injury. (A) Spinal cord crush injury at the T10 segment. (B) Experimental timeline of sustained microglia clearance and spinal cord injury. (C) Immunofluorescence demonstrated the efficient depletion of microglia (red) in wild-type mice. Scale bar: 500 μm. Nuclei: blue, microglia: red. (D) Microglia depletion is not conducive to recovery from spinal cord injury, based on BMS scores (*** P < 0.001, sham group vs . SCI group; ## P < 0.01, SCI group vs . SCI + PLX5622 group). (E–G) A CSF1R inhibitor reduces the number of microglia (Iba1 + cells) and CD68-positive cells after spinal cord injury on day 14. Scale bar: 1000 μm. Nuclei: blue, fibronectin: green, Iba1: red, CD68: magenta. (H–J) Microglia depletion results in a larger lesion at day 14 after spinal cord injury and increases infiltration of F4/80-positive peripheral macrophages. Scale bar: 1000 μm. Nuclei: blue, glial fibrillary acidic protein (GFAP, marker of astrocyte): green, F4/80: red, Iba1: magenta. (K) Microglia depletion reduces BDNF production on day 3 after spinal cord injury. Two-way repeated-measures analysis of variance with Bonferroni post hoc test (D); one-way analysis of variance with Bonferroni post hoc test (F, G, I, J, K). ** P < 0.01, *** P < 0.001. All mice were C57BL/6J (wild-type) mice, and there were six mice per group. BDNF: Brain-derived neurotrophic factor; BMS: basso mouse scale; D1: day 1; Iba1: ionized calcium binding adaptor molecule 1; PLX5622: colony stimulating factor 1 receptor (CSF1R) inhibitor; SCI: spinal cord injury.

Article Snippet: Adult C57 BL/6J (wild-type [WT]) mice, male and female, weighing 20–28 g and aged 8–10 weeks, were obtained from Charles River (Zhejiang, China, license No. SCXK (Zhe) 2019-0001).

Techniques: Immunofluorescence, Marker, Derivative Assay, Binding Assay

Journal: Neural Regeneration Research

Article Title: Microglia overexpressing brain-derived neurotrophic factor promote vascular repair and functional recovery in mice after spinal cord injury

doi: 10.4103/NRR.NRR-D-24-00381

Figure Lengend Snippet: Cre-dependent manipulation of BDNF expression by microglia. (A, B) Construction of transgenic mice. (C, D) Assessment of induction efficiency in the CX3:BDNF mice. Scale bar: 400 μm. Scale bar in enlarged figures: 100 μm. Nuclei: blue, P2RY12: green, mCherry (mCherry is used to label tdTomato proteins, a red fluorescent protein. mCherry labelled cells here are mainly CX3CR1 positive cells): red, Iba1: magenta. (E–I) Evaluation of BDNF expression in microglia in transgenic mice before and after spinal cord injury. Scale bar: 50 μm. Nuclei: blue, BDNF: green, Iba1: red. Unpaired t -test (D, H, I); One-way analysis of variance with Bonferroni post hoc test (F, G). *** P < 0.001. The mice used in Figure C and D were CX3:BDNF, and there were six mice in each group. In E–I, C57BL/6J (wild-type) mice were used in the sham, SCI, and WT groups, and CX3:BDNF mice were used in the SCI-CX3:BDNF group, and there were six mice in each group. BDNF: Brain-derived neurotrophic factor; Iba1: ionized calcium binding adaptor molecule 1; WT: wild-type.

Article Snippet: Adult C57 BL/6J (wild-type [WT]) mice, male and female, weighing 20–28 g and aged 8–10 weeks, were obtained from Charles River (Zhejiang, China, license No. SCXK (Zhe) 2019-0001).

Techniques: Expressing, Transgenic Assay, Derivative Assay, Binding Assay

Journal: Neural Regeneration Research

Article Title: Microglia overexpressing brain-derived neurotrophic factor promote vascular repair and functional recovery in mice after spinal cord injury

doi: 10.4103/NRR.NRR-D-24-00381

Figure Lengend Snippet: BDNF overexpression improves motor function. (A) Experimental timeline of spinal cord injury in CX3:BDNF mice. (B–E) Footprint analysis on day 28 post-spinal cord injury (SL, step length; SW, step width). (F, G) Microglial BDNF overexpression promotes motor function recovery after spinal cord injury, based on BMS scores (*** P < 0.001, sham group vs . SCI group; ## P < 0.01, SCI group vs. SCI-CX3:BDNF group). (H–J) Rotarod analysis on day 28 post-spinal cord injury. One-way analysis of variance with Bonferroni post hoc test (C, D, E, I, J). Two-way repeated-measures analysis of variance with Bonferroni post hoc test (G). ** P < 0.01, *** P < 0.001. In B–J, C57BL/6J mice were used in the Sham group and SCI group, and CX3:BDNF mice were used in SCI-CX3:BDNF group, and there were six mice in each group. BDNF: Brain-derived neurotrophic factor; BMS: Basso mouse scale; D1: day 1; IF: immunofluorescence; r/min: revolutions per minute; SCI: spinal cord injury.

Article Snippet: Adult C57 BL/6J (wild-type [WT]) mice, male and female, weighing 20–28 g and aged 8–10 weeks, were obtained from Charles River (Zhejiang, China, license No. SCXK (Zhe) 2019-0001).

Techniques: Over Expression, Derivative Assay, Immunofluorescence

Journal: Neural Regeneration Research

Article Title: Microglia overexpressing brain-derived neurotrophic factor promote vascular repair and functional recovery in mice after spinal cord injury

doi: 10.4103/NRR.NRR-D-24-00381

Figure Lengend Snippet: BDNF overexpression reduces neuronal apoptosis and axonal necrosis. (A) Immunofluorescence was employed to detect 5-HT-positive axons on day 28 post-spinal cord injury. Scale bar: 500 μm. 5-HT: white. (B–H) Microglial BDNF overexpression inhibits neuronal apoptosis following spinal cord injury. In B, scale bar: 50 μm, nuclei: blue, TUNEL: green. In F, scale bar: 100 μm, nuclei: blue, NeuN: red. One-way analysis of variance with Bonferroni post hoc test (C–E, G, H). Error bars in all figures represent SD. *** P < 0.001. In A–H, C57BL/6J mice were used in the sham and SCI groups, and CX3:BDNF mice were used in the SCI-CX3:BDNF group, and there were six mice in each group. BDNF: Brain-derived neurotrophic factor; DAPI: 4′,6-diamidino-2-phenylindole; 5-HT: 5-hydroxytryptamine; NeuN: neuronal nuclei; SCI: spinal cord injury; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: Adult C57 BL/6J (wild-type [WT]) mice, male and female, weighing 20–28 g and aged 8–10 weeks, were obtained from Charles River (Zhejiang, China, license No. SCXK (Zhe) 2019-0001).

Techniques: Over Expression, Immunofluorescence, TUNEL Assay, Derivative Assay

Journal: Neural Regeneration Research

Article Title: Microglia overexpressing brain-derived neurotrophic factor promote vascular repair and functional recovery in mice after spinal cord injury

doi: 10.4103/NRR.NRR-D-24-00381

Figure Lengend Snippet: BDNF overexpression promotes activation of microglia to the Arg1-positive phenotype and reduces inflammation after spinal cord injury. (A–D) BDNF overexpression by microglia facilitates adoption of an anti-inflammatory type and enhances the production of anti-inflammatory factors on day 3 after spinal cord injury. Scale bar: 100 μm. Scale bar in enlarged figures: 40 μm. Nuclei: blue, Arg1: green, Iba1: red, CD68: magenta. (E–H) BDNF overexpression by microglia reduces the proportion of pro-inflammatory microglia and diminishes the production of pro-inflammatory factors on day 3 after spinal cord injury. Scale bar: 100 μm. Nuclei: blue, IL-1β: green, Iba1: red. One-way analysis of variance with Bonferroni post hoc test (B–D, F–H). ** P < 0.01, *** P < 0.001. In A–H, C57BL/6J mice were used in the Sham group and the SCI group, and CX3:BDNF mice were used in the SCI-CX3:BDNF group, and there were six mice in each group. BDNF: Brain-derived neurotrophic factor; DAPI: 4′,6-diamidino-2-phenylindole; SCI: spinal cord injury; IL-10: interleukin-10; IL-1β, interleukin-1β; ns: not significant; TGF-β1: transforming growth factor β1.

Article Snippet: Adult C57 BL/6J (wild-type [WT]) mice, male and female, weighing 20–28 g and aged 8–10 weeks, were obtained from Charles River (Zhejiang, China, license No. SCXK (Zhe) 2019-0001).

Techniques: Over Expression, Activation Assay, Derivative Assay

Journal: Neural Regeneration Research

Article Title: Microglia overexpressing brain-derived neurotrophic factor promote vascular repair and functional recovery in mice after spinal cord injury

doi: 10.4103/NRR.NRR-D-24-00381

Figure Lengend Snippet: BDNF overexpression by microglia promotes vascular regeneration. (A, B) Blood vessel density on day 28 after spinal cord injury. Scale bar: 1000 μm. CD31: magenta. (C–F) Changes in blood flow before and after spinal cord injury. Scale bar: 0.2 cm. Low blood flow: blue, high blood flow: red. (G) VEGFA mRNA expression was detected on the third day after spinal cord injury. One-way analysis of variance with Bonferroni post hoc test (B, D–G). * P < 0.05, ** P < 0.01, *** P < 0.001. In A–G, C57BL/6J mice were used in the Sham and SCI groups, and CX3:BDNF mice were used in the SCI-CX3:BDNF group, and there were six mice in each group. BDNF: Brain-derived neurotrophic factor; ns: not significant; SCI: spinal cord injury; VEGF: vascular endothelial growth factor.

Article Snippet: Adult C57 BL/6J (wild-type [WT]) mice, male and female, weighing 20–28 g and aged 8–10 weeks, were obtained from Charles River (Zhejiang, China, license No. SCXK (Zhe) 2019-0001).

Techniques: Over Expression, Expressing, Derivative Assay

Journal: Neural Regeneration Research

Article Title: Microglia overexpressing brain-derived neurotrophic factor promote vascular repair and functional recovery in mice after spinal cord injury

doi: 10.4103/NRR.NRR-D-24-00381

Figure Lengend Snippet: BDNF overexpression primarily by microglia but not by macrophages promotes functional recovery after spinal cord injury. (A, B) Experimental timeline of spinal cord injury in TMEM:BDNF mice and CX3:BDNF + PLX73086 mice. (C, F) Detection of induction efficiency in TMEM:BDNF mice. Scale bar: 200 μm. Nuclei: blue, P2RY12: green, mCherry (mCherry is used to label tdTomato proteins, a red fluorescent protein. mCherry labelled cells here are mainly TMEM119 positive cells): red, Iba1: magenta. (D, G) Unlike PLX5622, PLX73086 did not affect the number of microglia in the spinal cord. Scale bar: 200 μm. DAPI: Blue, Iba1: Red. (E, H) PLX73086 exhibits effective scavenging of peripheral macrophages. Scale bar: 500 μm. Nuclei: blue, F4/80: red. (I, J) BMS scores after spinal cord injury in the TMEM:BDNF and CX3:BDNF + PLX73086 groups (I, *** P < 0.001, sham group vs. SCI group. ## P < 0.01, SCI group vs. SCI-TMEM:BDNF group; J, *** P < 0.001, sham group vs . SCI group. ### P < 0.001, SCI group vs. SCI-CX3:BDNF + PLX73086 group). Mean ± SD (F). Unpaired t -test (G, H). Two-way repeated-measures analysis of variance with Bonferroni post hoc test (I, J). *** P < 0.001 in G. The mice used in C and F were TMEM:BDNF, and there were six mice in each group. The mice used in D, E, G, H, and F were C57BL/6J mice, and there were six mice in each group. In Figure I, C57BL/6J mice were used in the Sham group, SCI group, and WT group, and CX3:BDNF mice were used in the SCI-TMEM:BDNF group, and there were six mice in each group. In Figure J, C57BL/6J mice were used in the Sham, SCI, and WT groups, and CX3:BDNF mice were used in the SCI-CX3:BDNF + PLX73086 group, and there were six mice in each group. BDNF: Brain-derived neurotrophic factor; DAPI: 4’,6-diamidino-2-phenylindole; D1: day 1; SCI: spinal cord injury.

Article Snippet: Adult C57 BL/6J (wild-type [WT]) mice, male and female, weighing 20–28 g and aged 8–10 weeks, were obtained from Charles River (Zhejiang, China, license No. SCXK (Zhe) 2019-0001).

Techniques: Over Expression, Functional Assay, Derivative Assay