Methylcobalamin increases neurite outgrowth and neuronal survival through methylation cycle in DRG neurons and

promotes peripheral nerve regeneration.

+1Okada, K; 1Tanaka, H; 2Temporin, K; 1Kuroda, Y; 1Okamoto, M; 1Moritomo, H; 1Murase, T; 1Yoshikawa, H
+1Department of Orthopaedics, Osaka University Graduate School of Medicine, Osaka, Japan, 2 Toyonaka City Hospital, Osaka, Japan
kokada2433@snow.ocn.ne.jp
Introduction which was not inhibited by addition of Nbtd. The apoptotic rate in
Methylcobalamin (MeCbl) is a vitamin B12 analog and is necessary for MeCbl+SAM group was decreased, which showed no significant
the maintenance of the nervous system. Although some previous studies difference from that in MeCbl group or SAM group (Fig. 2).
have referred to the effects of MeCbl on neurons, the precise mechanism The sensory function of the fourth and fifth toes in the MeCbl group
of this effect remains obscure. Here we show that MeCbl promotes improved more than that in the control group from 10 weeks post
neurite outgrowth and neuronal survival in dorsal root ganglion (DRG) operation. Motor functional recovery was better in the MeCbl group than
neurons and that these effects are mediated by the methylation cycle, a in the control group from 11 weeks post operation in the toe-spreading
metabolic pathway involving methylation reactions. We also test. The course of SFI recovery in the MeCbl group was significantly
demonstrate that MeCbl improves nerve regeneration and functional different from that in the control group at 5 weeks post operation. The
recovery in a rat sciatic nerve injury model. recovery of terminal latency was greater in the MeCbl group than in the
Methods control group (Fig. 3). The gross areas of NF-positive tissues at the 2
Cell culture mm distal and 10 mm distal sites in the MeCbl group at 12 weeks post
DRG were obtained from Wistar rats at postnatal day 9. The cells were operation were larger than those in the control group, and the gross areas
resuspended in modified Sato medium and plated on poly-L-lysine- of MBP-positive tissues at the 2 mm and 10 mm distal sites in the
coated four-well chamber slides. MeCbl group were also larger than those in the control group (Fig.4).
Neurite outgrowth assay Fig.1 MeCbl promotes neurite outgrowth through methylation cycle.
Neurons were cultured with MeCbl (10 µM; Sigma-Aldrich), S-
adenosylmethionine (SAM, 10 µM; Sigma-Aldrich), 4-nitro-2,1,3-
benzothiadiazole (Nbtd, 10 µM; Sigma-Aldrich) for 72 h and then
immunostained with anti-TuJ1 antibody. Axonal length was measured
using an image analyzer (Lumina Vision, Mitani Co.).
Neuronal survival assay
Neurons were cultured with MeCbl (10 µM), SAM (10 µΜ), Nbtd (1
µΜ) for 72 h. The neurons were visualized by staining with anti-TuJ1
antibody and apoptotic cells were visualized in the TUNEL assay. Grenn: TuJ-1. *: p < 0.05
Normal and apoptotic cells were counted in ten randomized 200× fields Fig.2 MeCbl promotes neuronal survival through methylation cycle.
using an image analyzer.
Surgical procedures
Eight-weeks-old female Wistar rats were anesthetized. The left sciatic
nerve was exposed at mid-thigh level, and cut with microscissors. The
dissection site was sutured end to end with 10-0 nylon. Continuous
administration of MeCbl (1 mg/kg/day) or PBS for 12 weeks was
performed using an osmotic minipump (Model 2ML4; Alzet), which
was placed subcutaneously in the right side of the back.
Motor and sensory functional analysis Red: TuJ-1, Green: TUNEL. *: p < 0.05
To evaluate sensory function, the toe pinch test was performed every Fig. 3 MeCbl improves the functional recovery of injured sciatic
week. To evaluate motor function, the toe spreading test was performed nerve of rats.
every week and the sciatic functional index (SFI) was evaluated every 2
weeks.
Electrophysiological analysis
Terminal latencies were obtained by stimulating the sciatic nerve at 12
weeks post operation. Terminal latency was detected and measured
using PowerLab devices and software (AD Instruments).
Histological analysis *: p < 0.05
The sciatic nerve was removed at 12 weeks post operation, sectioned Fig. 4 MeCbl increases regenerated axon and myelin of sciatic nerve.
axially at 5 µm, and mounted on a glass slide. Then the slides were
immunostained by anti- neurofilament 200 (NF) rabbit antiserum
(Sigma-Aldrich) and anti- myelin basic protein (MBP) mouse antibody
(Covance), which were measured and calculated using NIS-Elements
BR 3.00, SP3 software.
Statistics
Data are expressed as mean ± SEM. Statistical evaluation was performed
by one-way ANOVA and post hoc Student’s t-test using StatView
software.
Results
The axonal length in MeCbl was greater than that in the control group in
DRG neurons. After addition of Nbtd, an inhibitor of methionine Figures present the slices of the sciatic nerve at 10 mm distal. Red: MBP, Green: NF. *: p < 0.05
synthase in the methylation cycle, the axonal length in the MeCbl group Discussion
was decreased to the level same as that in the control group in DRG These results suggest that MeCbl increases neurite outgrowth and
neurons. SAM, the downstream metabolite of methionine in the neuronal survival through methylation cycle in DRG neurons
methylation cycle, increased the axonal length. Addition of Nbtd in the (summarized in Fig. 5). Continuous administration of high dose MeCbl
SAM group did not reduce the axonal length. In MeCbl+SAM group, increases the regenerated axon and myelin, and promotes the recovery of
axonal length was increased, which were not diminished by addition of function in a rat sciatic nerve injury model.
Nbtd. Furthermore, axonal length in MeCbl+SAM group showed no Fig. 5
significant difference from that in MeCbl group or in SAM group (Fig.
1). MeCbl decreased the apoptotic rate less than that in control group.
Addition of Nbtd raised the apoptotic rate in the MeCbl group to the
level same as that in the control group. SAM reduced the apoptotic rate,
MS: methionine synthase

Poster No. 1628 • 56th Annual Meeting of the Orthopaedic Research Society