Nerve growth factor improves functional recovery by inhibiting endoplasmic reticulum stress-induced neuronal apoptosis in rats with spinal cord injury

Abstract

Background: Endoplasmic reticulum (ER) stress-induced apoptosis plays a major role in various diseases, including spinal cord injury (SCI). Nerve growth factor (NGF) show neuroprotective effect and improve the recovery of SCI, but the relations of ER stress-induced apoptosis and the NGF therapeutic effect in SCI still unclear.

Methods: Young adult female Sprague-Dawley rats's vertebral column was exposed and a laminectomy was done at T9 vertebrae and moderate contusion injuries were performed using a vascular clip. NGF stock solution was diluted with 0.9% NaCl and administered intravenously at a dose of 20 μg/kg/day after SCI and then once per day until they were executed. Subsequently, the rats were executed at 1d, 3 d, 7d and 14d. The locomotor activities of SCI model rats were tested by the 21-point Basso-Beattie-Bresnahan (BBB) locomotion scale, inclined plane test and footprint analysis. In addition, Western blot analysis was performed to identify the expression of ER-stress related proteins including CHOP, GRP78 and caspase-12 both in vivo and in vitro. The level of cell apoptosis was determined by TUNEL in vivo and Flow cytometry in vitro. Relative downstream signals Akt/GSK-3β and ERK1/2were also analyzed with or without inhibitors in vitro.

Results: Our results demonstrated that ER stress-induced apoptosis was involved in the injury of SCI model rats. NGF administration improved the motor function recovery and increased the neurons survival in the spinal cord lesions of the model rats. NGF decreases neuron apoptosis which measured by TUNEL and inhibits the activation of caspase-3 cascade. The ER stress-induced apoptosis response proteins CHOP, GRP78 and caspase-12 are inhibited by NGF treatment. Meanwhile, NGF administration also increased expression of growth-associated protein 43 (GAP43). The administration of NGF activated downstream signals Akt/GSK-3β and ERK1/2 in ER stress cell model in vitro.

Conclusion: The neuroprotective role of NGF in the recovery of SCI is related to the inhibition of ER stress-induced cell death via the activation of downstream signals, also suggested a new trend of NGF translational drug development in the central neural system injuries which involved in the regulation of chronic ER stress.

Figure 1

Assessment of SCI model rats. A. HE staining results of SCI rat at 1, 3, 7 and 14 d after contusion. B. The BBB scores of SCI model rat at 1, 3, 7 and 14 d after contusion. The score of the sham group was 21 points (meaning normal locomotion). ** P < 0.01 versus the sham group. Data are the mean values ± SEM, n = 6. C. TUNEL apoptosis assay of model rat spinal cord lesion. Immunofluorescence results of the TUNEL assay. Bright green dots were deemed positive apoptosis cell. Motor function was evaluated by Nissl staining. Big, blue dots were deemed as motor neuron. Magnification was 20 ×. D. The analysis of apoptosis cell 1, 3, 7 and 14 d after spinal cord injury lesions. The percentage of apoptosis was counted from 3 random 1 × 1 mm² areas. ** P < 0.01 versus the sham group. Data are the mean values ± SEM, n = 6.

Figure 2

ER stress-induced apoptosis was involved in the early stage of SCI. A. Immunohistochemistry for GRP78, CHOP and caspase-12 in sham, 1, 3, 7 and 14 d after spinal cord injury lesions groups. B. Analysis of the positive cells of the immunohistochemistry results. * represents P < 0.05 versus the sham group, ** P < 0.01 versus the sham group, ^# P < 0.05 versus the 1d group, and ^$ P < 0.05 versus the 3 d group, & P < 0.05 versus the 7 d group. Data are the mean values ± SEM, n = 6.

Figure 3

BBB scores and inclined plane test of different concentration of NGF treated model rats. A. BBB scores and B. inclined plane test after spinal cord injury. *P < 0.05 versus sham group, **P < 0.01 versus sham group.

Figure 4

NGF improves the locomotor activity of SCI rats. A. The BBB scores and B. incline plane test of sham, SCI group and SCI rat treated with NGF group. * P < 0.05 versus the SCI group, and ** P < 0.01 versus the SCI group, n = 6. C. Representative footprints obtained from each group at 14 days after SCI show that NGF treated rats display fairly consistent weight-support plantar stepping and very little toe dragging. In contrast, vehicle control rats show consistent dorsal stepping and extensive toe dragging.

Figure 5

Treatment with NGF improves the recovery of SCI and the survival of neurons. A. HE staining results of the sham, SCI group and SCI rat treated with NGF group. B. NeuN staining and Nissl staining results of the sham, SCI group and SCI rat treated with NGF group. C. The analysis of Nissl body after spinal cord injury lesions in different groups. ^## represents P < 0.01 versus the sham group, ** P < 0.01 versus the SCI group. n = 6. D. Analysis of the positive neurons of the NeuN staining results. ^## represents P < 0.01 versus the sham group, ** P < 0.01 versus the SCI group. n = 6.

Figure 6

NGF administration inhibits the expressions of ER stress-induced apoptosis response proteins, GRP78, CHOP and caspase-12. A. Immunohistochemistry for GRP78, CHOP and caspase-12 in the sham, 7d and 14d after spinal cord injury lesion and NGF treatment 7 d and 14d after injury groups. B. Analysis of the positive cells and C. optical density of the immunohistochemistry results. ** P < 0.01 versus the SCI (7d) group, ^# represents P < 0.05 versus the SCI(14d) group, Data are the mean values ± SEM, n = 6. D. Protein expressions of GRP78, CHOP and caspase-12 for the sham, SCI and NGF treatment groups. GAPDH was used as the loading control and for band density normalization. E. The optical density analysis of GRP78, CHOP, and caspase-12 protein. ** P < 0.01 versus the sham group. Data are the mean values ± SEM, n = 6.

Figure 7

NGF treatment increases the level of GAP43 in spinal cord lesions. A. Immunofluorescence staining results of GAP43; the nuclear is labeled by Hoechst (blue), the neurons with obvious GAP43 signals are labeled by bright right dots, magnification was 20 ×. B. The protein expressions of GAP43 in sham, SCI rats and SCI rats treated with NGF groups. GAPDH was used as the loading control and for band density normalization. C. The optical density analysis of GAP43 protein. ** P < 0.01 versus the SCI group, and ^## represents P < 0.01 versus the sham group. Data are the mean values ± SEM, n = 6.

Figure 8

NGF decreases the level of apoptosis in spinal cord lesions. A. TUNEL apoptosis assay of model rat spinal cord lesions. Immunofluorescence result of the TUNEL assay. Bright green dots were deemed positive apoptosis cell, magnification was 20×. B. The analysis of apoptosis cell of the sham, SCI and NGF treatment groups. The percentage of apoptosis was counted from 3 random 1 × 1 mm² areas. C. Protein expressions of caspase-3 for the sham, SCI and NGF treatment groups. D. The optical density analysis of caspase-3 protein. * P < 0.05 versus the SCI group, and ^# P < 0.05 versus the sham group. ** P < 0.01 versus the SCI group, and ^## P < 0.01 versus the sham group. Data are the mean values ± SEM, n = 6.

Figure 9

PI3K/Akt/GSK-3β and ERK1/2 signals are involved in the protective effect of bFGF both in SCI rats and PC12 cells under stress. A. The protein expressions of p-Akt/Akt, p-ERK/ERK, p-GSK-3β/GSK-3β in the sham, SCI model and SCI rat treated with bFGF groups. B. The optical density analysis of p-Akt/Akt, p-ERK/ERK, p-GSK-3β/GSK-3β protein. ** P < 0.01 versus the sham group, and ^# P < 0.05 versus the SCI group. Data are the mean values ± SEM, n = 6. C. MTT results of the different concentrations of TG-treated PC12 cells. D. MTT result of NGF-treated PC12 cells induced by TG. E. FACScan result of PI/Annexin V-FITC staining for cell apoptosis analysis. F. Statistical result of apoptosis rate in PC12 cells treated with TG and NGF. ** P < 0.01 versus the control group, and ^# P < 0.05 versus the TG group. Data are the mean values ± SEM, n = 3. G. The protein expressions of CHOP, GRP78, p-Akt, p-ERK1/2, p-GSK-3β in ER stress-induced apoptosis PC12 cells treated with NGF and different inhibitors. GAPDH was used as the loading control and for band density normalization. H. The optical density analysis of CHOP, GRP78, p-Akt, p-ERK1/2 and p-GSK-3β protein. * P < 0.05 versus the control group, ^# represents P < 0.05 versus the TG group, ^$ P < 0.05 versus the TG + NGF group. Data are the mean values ± SEM, n = 6.