Serum response factor promotes axon regeneration following spinal cord transection injury

Abstract

Studies have shown that serum response factor is beneficial for axonal regeneration of peripheral nerves. However, its role after central nervous system injury remains unclear. In this study, we established a rat model of T9-T10 spinal cord transection injury. We found that the expression of serum response factor in injured spinal cord gray matter neurons gradually increased with time, reached its peak on the 7^th day, and then gradually decreased. To investigate the role of serum response factor, we used lentivirus vectors to overexpress and silence serum response factor in spinal cord tissue. We found that overexpression of serum response factor promoted motor function recovery in rats with spinal cord injury. Qualitative observation of biotinylated dextran amine anterograde tracing showed that overexpression of serum response factor increased nerve fibers in the injured spinal cord. Additionally, transmission electron microscopy showed that axon and myelin sheath morphology was restored. Silencing serum response factor had the opposite effects of overexpression. These findings suggest that serum response factor plays a role in the recovery of motor function after spinal cord injury. The underlying mechanism may be related to the regulation of axonal regeneration.

Keywords: axon; growth associated protein 43; motor function; myelin sheath; neuron; regeneration; serum response factor; spinal cord; spinal cord transection.

Figure 1

SRF expression in spinal cord tissue in normal and SCT rats. (A) SRF in the spinal cord in normal (N) and SCI rats at different time points (1, 3, 7, 14, 28 days) after SCT by immunohistochemistry. SRF (black arrows) was expressed in gray matter neuron-like cells, showing brownish-yellow staining. Scale bar: 100 μm. (B) The number of SRF-positive cells in the spinal cord gray matter 5 cm distal from the injured section, which increased gradually after SCI and reached a peak on the 7^th day. (C) qPCR confirmed the mRNA expression of SRF. The data were normalized to GAPDH. (D, E) Western blotting of SRF protein expression. Data were normalized to GAPDH and represented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, vs. normal group (one-way analysis of variance followed by Tukey’s post hoc test). qPCR: Quantitative polymerase chain reaction; SCI: spinal cord injury; SCT: spinal cord transction; SRF: serum response factor.

Figure 2

Identification of lentivirus transfected into PC12 cells and spinal cord tissue. PC12 cells were used to screen the optimal titer of the lentivirus. (A) PC12 cells shown by optical microscope. (B) High expression of green fluorescence protein (GFP) was detected in PC12 cells transfected with lentivirus under a fluorescence microscope. (C) Morphology of longitudinal section of spinal cord tissue 5 cm from the cross section, 1 week after the rats received SCI and injection of lentivirus expressing GFP, observed by ordinary light microscope. (D) GFP was observed under a fluorescence microscope in the same spinal cord tissue. Scale bars: 500 μm in A and B, and 100 μm in C and D.

Figure 3

SRF expression after lentivirus injection into spinal cord tissue. SRF expression was determined after lentivirus injection. (A) qPCR results showed there was no significant difference between the negative control (NC) group and normal (N) group. SRF was upregulated and downregulated in the overexpressed (LV-srf) and silenced (srf-RNAi) groups, respectively. (B) Expression of SRF protein shown by western blotting. Data normalized by GAPDH are represented as mean ± SD (n = 3). **P < 0.01, vs. N group (one-way analysis of variance followed by Tukey’s post hoc test). SRF: Serum response factor; qPCR: quantitative polymerase chain reaction.

Figure 4

SRF improves motor function in rats with SCI (A) Skin was incised in the middle back and the spinal cord was exposed. (B) Illustration showing the spinal cord transection site and virus injection site. (C) BBB scores were used to evaluate motor function recovery. Data are represented as mean ± SD (n = 10). **P < 0.01, vs. NC group (one-way analysis of variance followed by Tukey’s post hoc test). BBB: Basso, Beattie, and Brenham; NC: negative control; SRF: Serum response factor.

Figure 5

SRF regulates axonal regeneration after SCI. Biotinylated dextran amine (BDA) injection was used to anterogradely track the growth of the corticospinal tract nerve fibers in the distal end of spinal cord cross sections. In the NC group, very few short and loose nerve fibers were observed 28 days after SCI. More nerve fibers were observed in the LV-srf group. No visible brown-yellow staining was observed in the srf-RNAi group. Images in the bottom row are enlargements of boxes in the images above. Black arrows indicate descending tracts. Scale bars: 500 μm (upper) and 50 μm (lower). NC: Negative control; SCI: spinal cord injury; SRF: serum response factor.

Figure 6

SRF promotes axon regeneration after SCI, shown by electron microscope. Loose and disordered myelin sheaths surrounding nerve axons were observed in the NC group, which appeared even worse in the srf-RNAi group. However, a higher density of myelin sheaths around axons were observed in the LV-srf group. Scale bars: 2 μm (upper) and 1 μm (lower). NC: Negative control; SCI: spinal cord injury; SRF: serum response factor.

Figure 7

Overexpressed SRF accompanied by upregulated GAP43 after SCI. (A) Representative photomicrographs of spinal cord labeled by GAP43 (green, Alexa Fluor 488) and Nogo A (red, Alexa Fluor 594) in the caudal end of the injured spinal cord tissue. Newborn axons identified by GAP43 were increased in the LV-srf group compared with the findings in the NC group. Nogo A was displayed by red fluorescence. Scale bars: 200 μm. (B) Western blot of GAP43 protein expression. (C) Quatification of GAP43 protein expression. Data are represented as mean ± SD (n = 3). **P < 0.01, vs. NC group (independent samples t-test). GAP43: Growth associated protein 43; NC: negative control; SCI: spinal cord injury; SRF: serum response factor