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Neuroscience Letters 170 (1994) 195-197
NEUROSCIENC[ lETTERS
Methylcobalamin (methyl-B12) promotes regeneration of motor nerve terminals degenerating in anterior gracile muscle of gracile axonal dystrophy (GAD) mutant mouse Kazuto Yamazaki a'c'*, Kenichiro Oda b, Chiyoko Endo b, Tateki Kikuchi c, Tsuneo Wakabayashi a ~Laboratory Animal Research Center, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki 300 -26, Japan Divisions qf ~Degenerative Disease and ~Animal Models for Human Disease, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187, Japan Received 17 January 1994; Revised version received 3 February 1994; Accepted 4 February 1994
Abstract We examined the effects of methylcobalamin (methyl-B~> mecobalamin) on degeneration of motor nerve terminals in the anterior gracile muscle of gracile axonal dystrophy (GAD) mutant mice. GAD mice received orally methyl-B~2(1 mg/kg body wt/day) from the 40th day after birth for 25 days. In the distal endplate zone of the muscle, although most terminals were degenerated in both the untreated and methyl-B.2-treated GAD mice, sprouts were more frequently observed in the latter. In the proximal endplate zone, where few degenerated terminals were seen in both groups of the mice, the perimeter of the terminals was increased and the area of the terminals was decreased significantly in the methyl-Btz-treated GAD mice. These findings indicate that methyl-B~2promotes regeneration of degenerating nerve terminals in GAD mice.
Key words. Methylcobalamin (methyl-B~2,mecobalamin); Gracile axonal dystrophy mouse; Motor nerve terminal; Degeneration; Regeneration
The gracile axonal dystrophy (GAD) mouse is an autosomal recessive neurological mutant with the gene symbol gad [19]. The progress of clinical signs of G A D (gad/gad) mice is divided into two stages: sensory ataxia (detectable around the 30th day after birth) and motor paresis stages (detectable around the 80th day after birth) [5]. Pathological examination revealed axonal dystrophy with dying back type axonal degeneration in the gracile fascicules and the posterior spinocerebellar tract [3,6,19]. In the peripheral nervous system, we observed degeneration of Ia endings in muscle spindles [10] and motor nerve terminals [5]. The degeneration of Ia fiber endings and motor nerve terminals becomes detectable around the 20th and 60th day after birth, respectively. Methylcobalamin (methyl-B~2 , mecobalamin, c~-(5,6dimethylbenzimidazolyl)-Co-methylcobamide) has been * Corresponding author. Address: Laboratory Animal Research Center, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3, Tokodai, Tsukuba, Ibaraki 300-26, Japan. Fax: (81) (298) 47-5960. 0304-3940194/$7.00 Š 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(94)00116-R
used for the treatment of disturbances in the peripheral nervous system [4,15]. It protects nerve fibers of the sural nerve against degeneration in rats intoxicated by acrylamide [11]. In the present study, we investigated the effects of methyl-B~2 on degeneration of motor nerve terminals in the anterior gracile muscle of G A D mice. Twenty-four male GAD mice and 18 phenotypically normal male littermates (gad/+ or +/+) from a congenic C57BL16-gad strain were used. The congenic strain, originally introduced from the Laboratory of Animal Genetics, Faculty of Agriculture, Nagoya University, Japan, was bred under specific pathogen-free conditions at Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan. The mice were provided with a commercial diet (CE-2; Nihon Clea, Tokyo, Japan) and water ad libitum under controlled temperature, humidity and lighting conditions (22 + 2°C, 55 + 5% and a 12-h lightdark cycle with lights on at 07:00). G A D and normal mice were distinguished at the 30-40th day after birth: the GAD mice, when hung by the tail, stretch their hind limbs upward spasmodically
196
K
Yamazaki el .L / A,euroscience Lcfter.~ 170 (19~4 ) 195 197
Fig. I. Motor nerve terminals from a control normal mouse (a), a GAD mouse treated with saline (b) and a G A D mouse treated with methyt-Bj~ (c). ZIO-silver stain. Scale bar = 20/,tin.
and/or clasp them tightly [10,19]. In the methyl-B~2treated mice (nine normal and 12 GAD mice), methyl-B~2 was orally given every day (1 mg/kg body wt, 0.1 ml/10 g body wt) from the 40th day after birth. Methyl-B~2 was synthesized by Eisai Co., Ltd., Tokyo, Japan. It was dissolved in nonpyrogenic saline solution (Otsuka Pharmaceuticals Co., Ltd., Osaka, Japan)just before administration. The control mice (nine normal and 12 GAD mice) received the same volume of saline (0.1 ml/10 g body wt). After 25 days, the anterior gracile muscle was removed under deep diethyl ether anesthesia and was stained with zinc iodide-osmium (ZIO)-silver [1]. The perimeter and area of the motor nerve terminals were measured by a MOP videoplan image analyser (Kontron, Germany). The muscle is divided into the proximal and distal endplate zones. In GAD mice, degeneration occurs first
m the distal endplate zone and then extends to the proximal endplate zone [5]. In the distal endplate zone, the degeneration of the terminals was prominent in both the control GAD and methyl-B~e-treated GAD mice (Table 1). >80% of the motor nerve terminals exhibited the degenerative swelling and dissolution of the axons near endplates. Degenerating terminals with sprouts were more frequently seen in the methyl-B~2-treated GAD mice than in the control GAD mice. The proximal endplate zone contained few severely degenerated terminals in both the control GAD and methyl-Bl,-treated GAD mice. However, the terminals showed a more elaborated configuration in the methyl-Bp-treated GAD mice than in the control GAD mice. The terminals in the control GAD mouse were swollen and degenerated while those in the methyl-B12-treated GAD mouse exhibited many branchings (Fig. 1). In the control GAD mouse, motor terminals in the proximal endplate zone were decreased in their perimeters (P) and increased in their areas (A) (Fig. 2, Table 2). On the other hand, the P was markedly increased in the methyl-B~z-treated GAD mouse. The P and P:A ratio were significantly increased (P < 0.01 and P < 0.05, respectively) and the A was significantly decreased (P < 0.05) in the methyl-Bl2-treated GAD mice (Table 2). Due to the complexity of the motor nerve terminals, assessment of their morphology is very difficult. Robbins and Fahim chose the P, A and P:A ratio of the motor nerve terminals as parameters to quantify degeneration and regeneration: a decrease in the P and an increase in the A occur in degeneration while an increase in the P occurs in regeneration [14]. In the present study, we used
400-'
eSeo •
300-
Normal GAD
Treatment
Saline Methyl-B~2 Saline Methyl-Br2
% terminals showing normal configuration (with sprouts)
% terminals showing degenerative changes (with sprouts)
98 97 15 22
2 3 85 78
(2) (3) (7) (10)
(0) (0) (5) (22)
o 8 &
o ira ~
200 20q
A
•
~?tlmA
O0 0000A~ 000 OCO00
Table 1 Motor nerve terminals in distal endplate zone of anterior gracile muscle in G A D mice given saline or methyl-B~2 from 40th day after birth for 25 days Phenotype
080
•
I
I
300
400
i
500 p.m
Perimeter Fig. 2. Relationship between perimeter and area of motor nerve terminals in proximal endplate zone of anterior gracile muscle in a normal mouse given saline (open circles) and G A D mice given saline (closed circles) or methyl-B~2 (closed triangles). Each mark indicates one terminal.
K. )bmacaki et aL/Neuroscience Lelters 170
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1~941 195 197
Tuble 2 Motor nerve terminals in proximal endplate zone of anterior gracile muscle in G A D mice given saline or methyI-B~ from 40th day after birth for 25 days Phenotype
Treatment
n
Terminal perimeter (P) (/am)
Terminal area (A) {,ran-')
P:A
Normal
Saline MethyI-Bp
140 140
325 _+ 35 331 _+ 39
293 + 30 3{11 + 41
1.1 +_ O. 1.2 + 0.
GAD
Saline Methyl-B~
133 133
277 + 321"* 370 + 28'
335 _+ 30 * 303 + 21 ;
I).S _+ O. 1.3 + ().
Values are expressed as m c a n + S.E.M. *P < 0.05; **P < 0.01 by Student's t test.
these parameters in combination with the ZIO-silverstaining method to evaluate the degree of distal axonopathy in G A D mice. Subacute combined degeneration of the spinal cord is caused by vitamin B~a deficiency, known as pernicious anemia [2,12,13,16,17]. Pathological changes are detected in the posterior and lateral columns of the spinal cord. Degeneration in the peripheral nervous system was further reported in some patients and in vitamin B~:deficient monkey [2,16]. Thus, there is a possibility that G A D mice are genetically vitamin Bp-deficient and that the regeneration of motor nerve terminals observed in the present study in the methyl-B~2-treated G A D mouse may be a result of replenishment of vitamin Bi2. Vitamin Bi, concentration in the serum and cervical cord of the G A D mice, however, are not lowered as compared with that in normal littermates (unpubl. data). Methyl-B~2 plays an important role as a coenzyme in synthesizing methionine from homocysteine through transfer of the methyl group [7,8]. R N A and protein syntheses are also promoted by methyl-BL2 [9]. Therefore, methyl-B~2 appears to work on motoneurons to activate protein synthesis. In addition, it was reported that methyl-Bi2 enhances protein metabolism in Schwann cells [18]. Thus, methyl-Bl, may act on both motoneurons and Schwann cells to promote axonal regeneration. This study was supported in part by a grant from the Japan Human Science Foundation. [I] Endo, C., Oda, K. aud Shibasaki, H., hnproved visualization of motor and sensory nerve terminals by a combination of zinc iodide-osmium and silver stains, Biomed. Res., 12 (1991) 279 283. [2] Greenfield, J.G. and Carmichael, E.A., The peripheral nerves in cases of subacute combined degeneration of the cord, Brain, 58 (1935) 483-491. [3] Kikuchi. T., Mukoyama, M., Yamazaki, K. and Moriya, H., Axonal degeneration of ascending sensory neurons in gracile axonal dystrophy mutant mouse, Acta Neuropathol., 80 (1990) 145 151. [4] Kobayashi, H., Aoyagi, M., Suzuki, H., Kikuchi, A., Watanabe. J. and Koike, Y., The clinical effects of inecobalamin on the peripheral facial palsy, Otol. Fukuoka, 26 (19801 968 972. [5] Miura, H., Oda, K., Endo, C., Yamazaki, K., Shibasaki, H. and Kikuchi, T., Progressive degeneration of motor nerve terminals in
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G A D inutant mouse with hereditary sensory axonopathy, Neuropathol. Appl. Neurobiol.. 19 (19931 41 51. M u k o y a m a , M,, Yamazaki, K., Kikuchi, T. and Tomita. T., Neuropathology of gracile axonal dystrophy (GAD) mouse, Acta Neuropathoh, 79 119891 294 299. Nakazawa, T., Yoshiba, K.. Takasugi, M., Muraoka, T. and Kaziro, Y., Vitamin Bp-deficient methionine biosynthesis in brain cells, cultured in vitro, Vitamins (Japan), 41 (19701 333 337. Nakazawa, T., Yoshiba, K. and Takasugi, M., The activity of methyl-transferase induced hv ~itamin B~: in cell line (NTS) established from rat brains, Vitamins (Japan), 42 119701 193 197. Nakazawa, T.. Komiya, H. and Kanno, T., The pi'climinary repor! of R N A and D N A content> in rat spinal ncurons of vitamin Bp and folio acid deficiency, Vitamins (Japan), 42 (1970) 275 279. Oda, K.. Yamazaki, K., Miura. H.. Shibasaki, II. and Kikuchi. T., Dying back type axonal degeneration o[ scnsory ncrvc terminals in muscle spindles of tile gracile axolml dystrophy (GAD) mutant mouse, Neuropathol. Appl. Neurobiol., 18(1992) 265 281. Ohnishi, A., Kuroiwa. Y. and ()hgo, T., The ell'cot of methylcobalamin on the experimental acrylamide neuropathy m o r p h o m etric study. Jpn. J. Clin. Pllarmacol. 1-her., 11111979) 247 251. Oxnard, C.E. and Smith. W.T.. Neurological dcgeneration and reduced serum vitamin B~- Icxels m captive monkeys. Nal ure ( Loudon). 210(1966} 507 51)9. Pant, S.S..Asbury. A.K. a n d R i c h a d s o n , E . P . , T h c m y e h ) p a t h y o f pernicious anemia. A neuropathological reappraisal, Acta Neurol. Scand., 44. Suppl., 35 { 19681 3 36. Robbins, N. and Fahim. M.A.. Progression of age chauges ill n l a t u r e n l o u s e n l o t o r i l e r v e tcrl/lillals a n d its l e l a l i o n Io l o c o n l o l o r
activity, J. Neul-ocytol., 14 (1985) 1019 1030. [I 5] Waniguchi. tl., Ejiri, K. and Baba. 5:,.. hnprovement of autonomic neuropathy after mccobaJamin [re~ltlnell[ ill uremic patients on hemodialysis, Clin. Ther., 9 119871 607 614. [16] Torres, l., Slnith, W.T. and ()xnard, ('.E., Peripheral neuropathy associated with vitalnin Bl: dcficienc~ in capti,,e monkcys, ,I. Pathol., 105 (19711 125 146. [17] Victor. M. and Lear, A.A.. Subacute combined degeneralion of the spinal cord. ('urrent concepts of thc disease process. Value of serunl vitamin [i t. determinations in clariling some Of the COlllnlon clinical problems. Am. J. Mcd.. 20 (1956) 896 911. [181 Yamatsu. K., Kaneko, T., Kitahara, A. and Ohkawa, 1.. Pharmacological studies on degeneration and regener;dion of peripheral nerves {111. Effects of methylcobalamin on mitosis of Schwarm cells and incorporation of radio-active leucine into protein fraction of crushed sciatic nerxe m rats, Folia Pharmacol. Jpn., 72 (19761
259 2/,s. [19] Yamazaki, K.. Wakasugi, N.. Tomita, I-., Kikuchi, T., M u k o y a m a . M. and Ando, K., (iracilc axonal dystrophy (GAD), u nev~ neurological mutant m the mouse. Proc. Soc. Exp. Biol. Mcd., 187 119881 209 215.