The effects of aging on axon regeneration currently remain unclear. In addition, the up-regulated expression of neurotrophic factors that occurs within one week of peripheral nerve injury has been shown to play an important role in the axon regeneration. To investigate the effects of aging on axon regeneration, the expression of nerve-specific proteins immediately after peripheral nerve injury were compared between young and aged mice. A mouse peripheral nerve injury model was prepared using the sciatic nerve compression method. In each group, Luxol fast blue staining and immunofluorescence staining were performed to assess the degree of Wallerian degeneration in the sciatic nerve, and to evaluate the expression of repressor element 1-silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), nerve growth factor (NGF), and semaphorin 3A (Sema3A) in the dorsal root ganglion, respectively. Wallerian degeneration was observed in both young and aged mice after peripheral nerve injury. Significant increases were observed in the expression of REST/NRSF (P<0.0001), NT3 (P=0.0279), and Sema3A (P=0.0175) following peripheral nerve injury in young mice, while that of BDNF (P=0.5583) and NGF (P=0.9769) remained unchanged. On the other hand, no significant differences were noted in the expression of these nerve-specific proteins in aged mice. Based on the results of the present study, compensatory changes induced by peripheral nerve injury were initiated by the up-regulated expression of REST/NRSF in young mice, but not in aged mice.
In the central nervous system, repressor element-1 silencing transcription/neuron-restrictive silencer factor (REST/NRSF) maintains homeostasis by suppressing the apoptosis of neurons (
The ability to regenerate axons damaged by peripheral nerve injury decreases with age (
The up-regulated expression of neurotrophic factors that occurs within one week of peripheral nerve injury has been shown to play an important role in Schwann cell migration and myelination during axon regeneration (
A peripheral nerve injury model in young and aged mice was used to investigate the effects of aging on axon regeneration in the present study. The degree of Wallerian degeneration and the expression of nerve-specific proteins immediately after peripheral nerve injury were compared between these groups. Furthermore, the effects of REST/NRSF on axon regeneration, which currently remain unclear, were discussed based on the expression of nerve-specific proteins.
The present study was approved by the Animal Care Committee of Juntendo University, Tokyo, Japan (registration no. 1555; approval no. 2021312).
Forty male C57BL/6 mice (Young group: 10-week-old mice, n=20; Aged group: 70-week-old mice, n=20) purchased from Japan SLC, Inc. (Shizuoka, Japan) were used. Mice were housed at 5 animals/cage in a sterile environment controlled at a temperature of 22±2˚C, humidity of 40-60%, and 12-h light and dark cycle, and were given water that was CRF-1 gamma-ray irradiated at 15 kGy (Oriental Yeast Co., Ltd.)
The Young group (n=20) and Aged group (n=20) were divided into Control and Crush groups to create four groups (A: Young control (n=10), B: Young crush (n=10), C: Aged control (n=10), and D: Aged crush (n=10)). Chronic constriction injury (CCI) is a partial nerve injury that is mostly used in rodents and is performed using a hemostatic forceps (
Under general anesthesia with isoflurane inhalation anesthetic solution (4% isoflurane for induction and 2% for maintenance) (
Luxol fast blue (LFB) staining was performed to assess the degree of Wallerian degeneration after peripheral nerve injury (
Tissue sections of the sciatic nerve in the longitudinal axis were divided into three areas (the crushed site, proximal to the crushed site, and distal to the crushed site) in the Crush groups. Similarly, tissue sections of the sciatic nerve were divided into three areas in the Control group without peripheral nerve injury. The area distal to the crushed site was used in the present study because this was the site at which Wallerian degeneration occurred. Using a light microscope (Carl Zeiss, KS400), the percentage of the area stained by LFB to the total nerve fiber area was calculated in both groups (
Immunofluorescence staining was performed to assess the expression of nerve-specific proteins after peripheral nerve injury, as described previous by Goto
In the photon counting mode, fluorescence intensity was measured at 20 randomly selected sites from the perikaryon in a region of interest (ROI) set in a fluorescence-emitting area, and mean fluorescence intensity was calculated. Fluorescence intensity measured using each antibody was compared between the four groups.
Data are presented as the mean ± standard deviation (SD) and were analyzed for significant differences using a two-way ANOVA with age and nerve injury set as two independent variables (Prism 7; GraphPad Software). After the two-way ANOVA, Turkey's multiple comparisons test was used as a post hoc test. Differences were considered to be significant at P<0.05.
In the sciatic nerve, the percentage of the area of the myelin sheath to the total nerve fiber area has been used to assess Wallerian degeneration in peripheral nerve injury (
The expression of nerve-specific proteins in DRG was quantified by immunofluorescence staining. The fluorescence intensity of REST/NRSF was significantly higher in the Aged control group (147.8±15.8) than in the Young control group (110.2±9.5) (P<0.0001) (
In the present study, we investigated whether the degree of Wallerian degeneration after peripheral nerve injury was affected by aging using a mouse peripheral nerve injury model. In peripheral nerves, the number and density of axons decrease with age (
Peripheral nerve injury always induces inflammation. It is a complex series of molecular and cellular events through the recruitment of circulating proteins and leukocytes to the injury site within hours to days after peripheral nerve injury (
The expression of neurotrophic factors has been shown to play an important role in axon regeneration after peripheral nerve injury (
There are several limitations in this study. First, in creating the CCI model for peripheral nerve injury, the compression method with a hemostatic forceps was used, so the degree of nerve injury may be different with each mice. It has been reported that the expression of neurotrophic factor varies depending on the degree of nerve injury (Neurapraxia, Axonotmesis, Neurotmesis) (
Based on the results of present study, while compensatory changes for peripheral nerve injury were initiated by the upregulation of the REST/NRSF, followed by Schwann cell migration in the Young group, these compensatory changes did not occur in the Aged group. The regulation of REST/NRSF expression appears to be essential for axon regeneration when peripheral nerves are exposed to stress. In peripheral nerves, aging-associated functional and electrophysiological disorders have been reported in clinical study (
In conclusion, the present results suggested that Wallerian degeneration occurred after peripheral nerve injury in the Young and Aged groups. On the other hand, compensatory changes for peripheral nerve injury and Schwann cell migration were initiated in the Young group, but not in the Aged group.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
HO mainly wrote the manuscript and acquired, analyzed and interpretated the data. KN wrote the manuscript and made substantial contributions to conception and design of the study, and interpretation of data. SN and KS contributed to acquisition, analysis and interpretation of data. SK, TS KG, AK, NN and YS contributed to acquisition of data. SK and TS confirm the authenticity of all the raw data. IN made substantial contributions to conception and design. MI contributed to the analysis and interpretation of data. All authors read and approved the final manuscript.
The present study was approved by the Animal Care Committee of Juntendo University (Tokyo, Japan; registration no. 1555; approval no. 2021312).
Not applicable.
The authors declare that they have no competing interests.
Surgical procedure to create the peripheral nerve injury model. (A) Mouse right sciatic nerve was dissected from the surrounding tissue. (B) The sciatic nerve was crushed for 30 sec using a hemostatic forceps. (C) The chronic constriction injury was made by a hemostatic forceps.
Histochemical assessment of the degree of Wallerian degeneration in the peripheral nerve by LFB staining. LFB assesses the degree of degeneration of the myelin sheath. The longitudinal axis of the sciatic nerves in the Young group (10 weeks) and Aged group (70 weeks) were examined. (A) Young control group; (B) Young crush group; (C) Aged control group; (D) Aged crush group; (E) Comparison of the degree of Wallerian degeneration after peripheral nerve injury in the Young and Aged groups. Scale bar, 200 µm. LFB, Luxol fast blue.
Histochemical assessment of the expression of REST/NRSF in the DRG by immunofluorescence staining. The expression of nerve-specific proteins was quantified by immunofluorescence staining using a REST/NRSF antibody. DRG in the Young group (10 weeks old) and Aged group (70 weeks old) were used and fluorescence intensity was compared. REST/NRSF stained green; DAPI stained blue; scale bar, 20.0 µm. (A) Young control group. (B) Young crush group. (C) Aged control group. (D) Aged crush group. (E) The fluorescence intensity of REST/NRSF. DRG, dorsal root ganglion; REST, repressor element 1-silencing transcription factor; NRSF, neuron-restrictive silencer factor.
Histochemical assessment of the expression of NT3 in DRG by immunofluorescence staining. The expression of nerve-specific proteins was quantified by immunofluorescence staining using a NT3 antibody. In the present study, DRG in the Young group (10 weeks old) and Aged group (70 weeks old) were used and fluorescence intensity was compared (NT3 stained green; DAPI stained blue; scale bar, 20.0 µm). (A) Young control group. (B) Young crush group. (C) Aged control group. (D) Aged crush group. (E) The fluorescence intensity of NT3. NT3, neurotrophin 3; DRG, dorsal root ganglion.
Histochemical assessment of BDNF expression in DRG by immunofluorescence staining. The expression of nerve-specific proteins was quantified by immunofluorescence staining using a BDNF antibody. In the present study, DRG in the Young group (10 weeks) and Aged group (70 weeks) were used and fluorescence intensity was compared (BDNF stained green; DAPI stained blue; scale bar, 20.0 µm). (A) Young control group. (B) Young crush group. (C) Aged control group. (D) Aged crush group. (E) The fluorescence intensity of BDNF. BDNF, brain-derived neurotrophic factor; DRG, dorsal root ganglion.
Histochemical assessment of the expression of NGF in DRG by immunofluorescence staining. The expression of nerve-specific proteins was quantified by immunofluorescence staining using a NGF antibody. In the present study, DRG in the Young group (10 weeks) and Aged group (70 weeks) were used and fluorescence intensity was compared (NGF stained green; DAPI stained blue; scale bar, 20.0 µm. (A) Young control group. (B) Young crush group. (C) Aged control group. (D) Aged crush group. (E) The fluorescence intensity of NGF. NGF, nerve growth factor; DRG, dorsal root ganglion.
Histochemical assessment of the expression of Sema3A in DRG by immunofluorescence staining. The expression of nerve-specific proteins was quantified by immunofluorescence staining using a Sema3A antibody. In the present study, DRG in the Young group (10 weeks) and Aged group (70 weeks) were used and fluorescence intensity was compared (Sema3A stained green; DAPI stained blue; scale bar, 20.0 µm. (A) Young control group. (B) Young crush group. (C) Aged control group. (D) Aged crush group. (E) The fluorescence intensity of Sema3A. Sema3A, semaphorin 3A; DRG, dorsal root ganglion.
Comparison of the expression of the nerve-specific proteins between Young and Aged group.
Nerve-specific proteins | Young | Aged | P-value |
---|---|---|---|
REST/NRSF (gray values) | 110.2±9.5 | 147.8±15.8 | P<0.0001 |
NT3 (gray values) | 116.3±24.8 | 91.7±30.9 | P=0.2618 |
BDNF (gray values) | 123.3±39.6 | 116.4±38.5 | P=0.9836 |
NGF (gray values) | 135.1±17.2 | 68.1±13.7 | P<0.0001 |
Sema3A (gray values) | 147.0±12.0 | 126.5±16.1 | P=0.0266 |
REST/NRSF, repressor element 1-silencing transcription/neuron-restrictive silencer factor; NT3, neurotrophin-3; BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor; Sema3A, semaphorin 3A.