`JNS 4251
`Journal of the Neurological Sciei~cr,~~. I ~~ U 9y~1 ld0 I l;~
`~~ 1994 Elsevier Science B.V. All rights reserved 002'_->1(IX/44/$U7.011
`Ultra-high dose methylcobalamin promotes nerve regeneration
`in experimental acrylamide neuropathy
`Tetsuya Watanabe, Ryuji Kaji *, Nobuyuki Oka, William Bara and Jun Kimura
`Department ojNeurology, Kyoto University Hospital, Shogoin, Sakyoku, Kyoto 606-01, Japan
`(Received 11 June, 1993)
`(Revised, received 19 October, 1993)
`(Accepted 28 October, 1993)
`Key words: Methylcobalamin; Acrylamide neuropathy; Nerve regeneration; Therapy; Compound muscle action potential
`Despite intensive searches for therapeutic agents, few substances have been convincingly shown to enhance nerve regenera-
`tion in patients with peripheral neuropathies. Recent biochemical evidence suggests that an ultra-high dose of methylcobalamin
`(methyl-B12) may up-regulate gene transcription and thereby protein synthesis. We examined the effects of ultra-high dose of
`methyl-B12 on the rate of nerve regeneration in rats with acrylamide neuropathy, using the amplitudes of compound muscle
`action potentials (CMAPs) after tibial nerve stimulation as an index of the number of regenerating motor fibers. After
`into~tication with acrylamide, all the rats showed equally decreased CMAP amplitudes. The animals were then divided into 3
`groups; rats treated with ultra-high (500 µg/kg body weight, intraperitoneally) and low (50 µg/kg) doses of methyl-B12, and
`saline-treated control rats. Those treated with ultra-high dose showed significantly faster CMAP recovery than saline-treated
`control rats, whereas the low-dose group showed no difference from the control. Morphometric analysis revealed. a similar
`difference in fiber density between these groups. Ultra-high doses of methyl-B12 may be of clinical use for patients with
`peripheral neuropathies.
`In spite of the common belief that peripheral nerves
`are capable of extensive regenerative growth after in-
`jury, clinical recovery seldom is complete in patients
`with peripheral neuropathy. Various agents are known
`to enhance peripheral nerve regeneration, but not many
`have proved helpful in clinical settings (Horowitz 1989).
`Vitamin B12 has an important role in methyl trans-
`fer reactions through folate metabolism, and a defi-
`ciency of it causes hematologic and neurologic disor-
`ders such as megaloblastic anemia and peripheral neu-
`ropathy. Neurologic manifestations however are not
`always accompanied by hematologic abnormalities nor
`are they reversible by folate supplement (Shorvon 1980;
`Lindenbaum 1988). Vitamin B12 may have a specific
`action on the nervous system, in addition to its action
`on the hematopoietic system.
`Recent biochemical evidence suggests that methyl-
`cobalamin (methyl-B12) acts directly as a methyl donor
`in DNA metabolism (Pfohl-Leszkowicz 1991). More
`importantly, ultra-high concentrations (> 1 µ M or 1.34
`µg/ml of methyl-B12) up-regulate gene transcription,
`"Corresponding author. Tel.: 075-751-3770; Fax: 075-761-9780.
`which may in turn increase protein synthesis for nerve
`We have used physiological and morphological
`methods to investigate the effect of ultra-high (500
`µg/kg) versus low (50 µg/kgJ doses of methyl-B12 on
`peripheral nerve regeneration in experimental acryl-
`amide neuropathy.
`Animal treatment
`Twenty-six male Wistar rats (body weight 250-300
`g) were treated with acrylamide (25 mg/day i.p. for 5
`days a week for 4 weeks). All animals developed clini-
`cal signs of neuropathy such as hind limb weakness and
`ataxia by the end of administration (day 0). The ani-
`mals were allowed to recover for 90 days (day 0-90),
`and divided into 3 groups: 12 rats treated with uitra-
`high doses of methyl-B12 (Eisai Co. Ltd., Tokyo, Japan)
`(high-dose group; 500 µg/kg body weight/day, i.p.), 5
`treated with low doses methyl-B12 (low-dose group; 50
`µg/kg, i.p.) and 9 treated with normal. saline (control
`group). Methyl-B12 or saline was injected 5 days a
`week for 12-13 weeks after day 0.


`~- 82500 pry'kg
`~— Control
`~- n,zso~~~~xq
`E 0
` 12
`A a
` ,o
`60 d
`30 0
`Days before antl after acrylamde mioM~calion
`Fib. I. Ci~rnparison of CMAP changes :unun~ the group>. ►3;us
`inJicatc standard dcvioUon~. ""P < ILUII I. ~"P = 11.11111
`90 d
`(MAY meus~~rernrr~~
`Physiological evaluations were made before and im-
`mediately af~cr acrylamide administration and 3 times
`during the rcc~rvery phase; during the intervals from
`~{~iy 25-3~ (indicated as "day 30"), day 55-6~ ("day
`hll") and day 235-95 ("day 90").
`Rats were anesthetized with a mixture of ketaminc
`( I1)(I mg/kg. i.pJ. xylazinc UO mg~kg, i.p.) and at-
`ropinc (U.IIS m~;/kg, i.pJ. A pair of needle electrodes
`lNihon K~~hden NE233S) was placed at the ankle with
`the cathc~dc ~~n the medial aspect. A supramaximal
`~[imulating current was applied to the tibial nerve at
`the ankle with v constant voltage stimulator (Medelec
`MS6). We recorded the compound muscle action po-
`tcntials (CMAPs) from the small hind toot muscles
`with a rcce~rding needle electrode (Nihon Kohdcn
`N~233S) un the dorsum and a reference at the second
`digit CMAP amplitudes, which reflect the number of
`inncrvate~l muscle fihcrti, were measured from the ini-
`iial negative to positive peaks. Recordings usually were
`ma~lc un ~mc side, the carne side being used through-
`uut the ~Stu~ly. The other side, however, was used
`~vhenevcr the reproduci~iility of the potential hecamc
`insufficient hecausc ~~1 local hcmatoma formation.
`Repetitive ncr~e stimulation was performed a[ 3 Hz,
`whenever po~sihle, in order to assess the neuromuscu-
`lar transmission (Kimura 199). The skin temperatur~
`:it the hind limb, which was monitored throughout the
`recording ~essiun, was kept between 30 and 32°C.
`The m~~rphological study used specimens of the tih-
`ial nerve at Ih~ midcalf taken from 3 animals on day
`(~O, each specimen being representative of a specific
`group with ('MAP amplitude values within I SD of the
`mean. The an~ilysis method used has already been
`~les~ribed elsewhere (Kaji 1989).
`Statistical ,uialysis was made with a microcomputer
`(M~icin[ush II) with StatView " software (Abacus Con-
`~cpts. Inc., Berkeley. CA, USA).
`Vilumirr BlZ}~
`Scrum an~i CSF cunccntrations of vitamin BlZ were
`measured in two animals from high-dose and low-dose
`groups during the day 90 period by competitive protein
`hiding atsa~, which was performed at SMI Bristol
`Laburatoric~. 'f~~kyu. Japan, on a commercial basi.ti.
`Nurm~al ticrum levels in the rat arc 683 ± 63 pg/~11
`(mean ± SD. n ~) (Sagaw~a ct al. 197).
`1). These: amplitudes did not differ stutistic.illy among
`the groups (P = 0.8(1 for Before; P — 0.41 t, f~n~ After;
`one-way ANOVA). In the recovery phase, the high-d~>se
`group showed a significantly higher amplitudes than
`the ether groups on day iU (l' < U.U01; one-way
`ANOVA, Fisher's PLSD), day hl) (P < Il.(lU I ), and day
`9U (P = II.OUI ). The low-close and the control gr~~ups
`did n~~t show significant dift~erence (P> fl.US). Repre-
`sentativc recordings from each group arc shown in I~ig.
`2. Repetitive nerve stimulation a~ 3 Hz produced nu
`significant decrement (< 211"4) in CMAP amplitueJes,
`suggesting that the decreased amplitudes xrc nut
`caused by impaired neuromuscular transmission but
`represent the reduced number ~~f m~~t~~r fibers inner-
`vating the muscle.
`Body wei,~~h~
`The three groups showed body weight change~ which
`did not significantly differ during the experiment
`(ANOVA Y > 11.05; Fig. 3>_
`CMAP CH/~NGES (mean+SD. mV')
`Dav 311
`pay 611
`Day 90
`7.7U± I.I1
`1 I.a I ±I)J9
`High ~li~x
`I Lh7+11.19
`I'_.5 ~~ ILO};
`9.~1 ±11.9
`I I.~)~± II.-t(,
`CMA P cu>>plrlu~les
`The ('MA)' amplitudes decreased sharply after
`acrvl;imidu treatment (Before and After in Fig. 1, Table
`P-value cif thr stalis~ical significance in Ihr ~i(fcrcnrc .~mun~ 3
`groups (~mc-way ANOVA). n: numhrr ~~I' sample,. tinmc :inimuls
`were liis~ due to enetithetfc accidents during Ihr nhsei~-atiun pc-


`500 yp/Ng
`50 yp/Mq
`.c,yum~as ~ —
`~ _---
`~o.y o~
`o.y ao —'
`— ~~~-- -- •—~—
`Yti' ~
`. s ~
`~. R ~
`~: ~ ~'~
`°~ a o~ r F'
`~ ~e n u u .. m
`~1!! L~
`~. gig, °~
`~~~ ~a~~ d ~
`~D''dlF,,r' ¢ ~,a ` ,
`.~a~, ~
`_~' ~ ~► ~
`Fig. 4. Morphometric study in the groups in day 60. Representative
`micrographs used for analysis are shown on the left.
`compared to the controls and those treated with a
`low-dose of methyl-B12. Because the CMAP amplitude
`represents the number of innervated muscle fibers, its
`prompt recovery indicates accelerated reinnervation of
`muscles by aaconal regeneration in the ultra-high dose
`group. In fact, nerve specimens from representative
`animals in each group provided morphometric evi-
`dence of increased numbers of large-diameter fibers in
`that group. Although the animals lost body weight after
`acrylamide intoacication, the high dose of methyl-B12
`did not act through the improvement of the general
`nutritional conditions, because the body weight recov-
`ery curve did not differ among the three groups.
`The response of a neuron to axonal damage is best
`exemplified by wallerian degeneration.. Soon after ax-
`onotomy, the soma undergoes a process called central
`chromatolysis, which probably represents a metabolic
`shift from supporting transmitter production and
`synaptic function to production of materials needed for
`nerve regeneration (Seckel 199U). This change should
`involve a surge in transcription of an entirely different
`set of genes.
`Acrylamide neuropathy is regarded as a prototype
`of distal axonal neuropathy (Spencer and Schaumberg
`1974). It pathogenic mechanism has been intensively
`studied (Pleasure et aL 1969; Prineas 1969; Spencer
`and Schaumberg 1974; Spencer and Schaumberg 1977).
`A demise of the metabolism in the soma results in a
`length-dependent dying back of the distal axon (Prineas
`Fig. 2. Representative serial recordings of CMAPs in the same
`Results of the morphometry performed on each
`animal whose CMAP is shown in Fig. 2 are given in
`Fig. 4. Fiber densities were 2125/mmZ in the control,
`3290/mm2 in the low-dose, and 5360/mm2 in the
`high-dose groups. The histograms showed increased
`numbers of small and medium-sized myelinated fibers
`with two prominent peaks at 4 and 9µm in the
`high-dose group, as compared to the other groups. The
`m~imum fiber diameter was 15 µm in the high-dose
`group, the largest among the groups.
`Vitamin B12 lei~els
`Serum levels of vitamin B12 were 2500 µg/ml in a
`rat from the high-dose group, and 180 µg/ml in one
`from the low-dose group. CSF levels were 15.2 µg/ml
`in the former, and 1.2 µg/ml in the latter.
`We have shown here that animals treated with an
`ultra-high dose of methyl-B12 promptly recovered
`CMAP amplitude after acrylamide intoxication, as
`t 81250 µgikg
`60 d
`30 d
`Oays bebre and after acrylamide intoxicatbn
`Fig. 3. Body weight change in three groups during the experiment.
`90 d


`1~16y). Impairccl axuplasmic flow m<iy result in patho-
`lugic changes most pronounced in the distal part of the
`.ixon (Ple~istu~c et al. 19C~~)). ~Acrylamide may directly
`~lam~igc the ax~mai membrane (Spencer and Schaum-
`hcrg 1977). Whatever the mechanism, the neuron
`,h~~uld tiusta~n a metabolic shill similar to thal after
`wallcrian ~Icgcncrafi~m after acrylamidc intoxication.
`-I'hr rule u~~ ~~itamin B1? in neuronal metabolism is ~i
`mattrr <~t c~~nlr~~vcrsy. The ~iissoci~ttion of neurc~lugical
`.u~cl hemat~~lo~ic~il improvements after folate supple-
`mtnt in vit~imin t31'_ deficiency (Shorvon 1980; Linden-
`hxum l~)~~) i~ unexplained because most of the previ-
`~~u~l~~ kn~~wn metabolic pathways requiring Bl? arc
`thrciugh transme~hylalion utilizing Ietrahydrofolatc and
`S-~idenc~s~~lmethi~~nine. A recent biochemical study has
`~Icmunsu;ite~i that methyl-BIB acts directly ati a methyl
`~i~m~~r in L)NA methylation which normally down-regu-
`latcs ~cn~ rxpressii~n (Ccclar 198H; Pfc~hl-Leszkowicz
`I~)~)1). Intcrestingly, at high concentration (> ] mM or
`I.?~4 µg/mU, methyl-B I'_ may up-regulate gene tran-
`~cription p~~~~sihly by competitive inhibition of DNA
`mcthylati~m ~~~i~h .S-adcn~~sylmethic>ninc (Pfohl-Lcszk~~-
`wicz 19~)I ). f h~ CSF Icvels ~~f vitamin B12 in low and
`high ~io~~ ~ruu~s in our study fell exactly inter the
`ranges th,~t ~1uwn- or up-regulate gene expression, re-
`Although the precise biochemical mechanism ~~f ar-
`ti~~n of ultra-hikh doses of methyl-BI7. is unknown, we
`interpret the prc~~nt results as reticcting its important
`physi~~lc~gir uctiun ~~n mctab~~lism. l3~cause acrylamide
`ncurutoririh~ i~ nut likely tip be thr~~ugh the known
`m~tahulic pathways of vitamin BIZ, we speculate th~it
`the cflcct ~~I~ ultra-high dose methyl-BI? is not limited
`to acrylami~ic neuropathy but is seen in other axon<<I
`"l~hc high but not the low dose group showed ~iccel-
`rr,itccl nrrvc~ rcgen~ratiun. The biologic activity cif
`methyl-E31? miry not be limited to that of a vitamin
`which ce~rrects metabolic derangements only when there
`is a deficiency u( iL Methyl-B12 also may have a
`profound effect on metabolism when present in sufii-
`cient am~~unts. Results ~~I recent clinical trials (Okawa
`1991U that used high doses of vitamin B12 ~o treat
`sleep-wake rhythm dis~~reler have indicated i[ has bi~~-
`lugic ~ic~i~m even in the non-deficient condition.
`We conclude that ultra-high ~usc~ of methyl-B!?
`may provide a therapy that enhances peripheral nerve
`regeneration in various human ncurc~p~ithics.
`Acknowledgments This ti~ork ~:i~ ~~upp~~rlc~l h~ S~icnlifi~ R~sc;u~ch
`gr.inly (A-UId409(,. C-I)467(14i+7. X1-114-lOJO-1:1) Ir~~m the lapancs~ Min-
`istry i~f [duc:itiun. Science and CulWrc ;~nii (~r,inL-in-~Ai~l lur :~my~
`~~U~rphic luter:~l sclrmtiis ~ntei peripher+J n~un~~aih~ Innn the
`Japane>t Minititry i~Y lleallh anal H'ell~ire. ~'dc :n~r fndchlc~l lu Ur.
`Yl. K.ime~ama lur helpful ,id~'ice.
`Ce~~r. }1. 119K~) DNA methylati~m :in~l ~cnr ~iclfcitc. Cell. ~3: 3--4.
`Har~~witz. (19H91 Ther:ip~ulir ~Ir,iie~ics in ~irum~riin~ prriphcr;~l
`nerve rcgeneratii~n. Muscle Nervy. 13: ?1-1-3??.
`Kaji, R.. L.iu. Y., Ducke[L S. and Sumnrr. iAJ. (1`12;91 Slew recovery
`of cenlr,~l ~is~~ns in ❑cryl.imidc ncum~alhc. Muscle Ncrvr, I~:
`Kimura..1. 11951) Electro~i.ignusi, in Discati~:~ of Nrn~ .mJ Mu~cic.
`F.A. D:i~i,. Philadelphia. Pi\.
`Lin~enh:wm. J.. He.iltun. E.H.. S;ivuee. I).G.. I3ru,i..L('.M.. G,n~rett.
`T.J.. Puilell. E.R., Marcell, P.f~., St.ihlcr. S.P. anil ,~\Ilan. K.H.
`U`)881 Neuru~spchi~~u'ic di,~irdcr, cauvril c~~h;il:imin dclicicncy in
`the ahsencr ~~I anemi,i ~x m,icmryio.i,. Nrti~ Fn~L J. Mr~L. 31~+:
`~ ~~n- i ~3r,.
`Okaw.i. h1.. Mishima. 7.. N:mami. l.. Shimizu. S.. lijim:i. S..
`tlishikaw;~, Y. ~md Takahashi. I~. 119y11) Vitamin RI'_ ue~wnrnt
`cif sleep-wake rhythm disurder~. tileep. I ~: I~-_'?.
`Pfuhl-Leszkowicz. A., Keith. (i. antl llinc~imrr. G. (I'~911 Eflccl of
`a,halamin Jcrivatives on in ~~ftm cnz>~m~iiic DN:~ mclhyl.ui~in:
`methvlcuhnl.imin ran act as a mrth~~l ~lunur. Bi~~nc~misln. 311:
`r~i is>-xi ~; i .
`Pleasure, l)., h1fxxhler. K. ei ,~L 119h91 ~lsunal Irons~i~~rl ut ~ru~cin~
`in r~prrinirntal nruri~palhie~. Jcicnc~. Ihh: >~~}--'-'i.
`Prinra~. J. 119!,9) The pa~h~igencsi, ail ih~ing-hark piilyncur~~p~thic,.
`11. An uhrastruclural study ul rxperimrnt,il arryl;imid~ inluxica-
`~i~m in the r,il. J. Ncurupath~~l. Lzp. N~un~l.. '_2; ;~)h-f,~l.
`Sag,iw;i. N.. Nakamurti. K. el ,il. ll`)2i'1 El~fcci u( ~~itamfn ILL'
`deficient dirt on luny and hrp,ilic rhusphc~lipiil> in the r~~t trlu,.
`h1cthyl 1312. Kyoto Syntprnium. Kyow;i Kikaku. Tuky~~~. p. 71.
`Seckel. f3.R (1990) Enhtinc~men~ ul' periphrral nervy rcEcnrr;ui~m.
`Muscle Nervy. I~: ?RS-tillll.
`Shonnn. ti.D.. C't~mry. M.W.P.. ('han.u'in. I. ❑n~i Rryn~~lds. G.I1.
`U9R11)'The neuropsychi.~trV ~~f mc~aluhl,i~lic anemia. 13r. MrJ. J..
`Spencer_ P. anJ Il. Sch.iiimhrre U~17~1) iA rrvirH ~~i~ :~cryl,imi~lc
`neurote~xicity. IL Experimental .inimal ncinotusicily unJ ~~:ithu-
`luei~ mcch;inisms Can. J. Neural. Sci.. I: ICI I(i~~.
`Spencrr. P. ,inJ IL Schaumherg (14771 Ultrasirur~ural ~tudirs nl the
`Ch'ing-b.ick process. IV. Dit(erenliul ~~ulner.~hilily ~~f PNS :end
`('NS liherti in ~xperim~nt:il ccnlr:il-~~criphcr;il Jist:il ;is-
`~m~~p.~lhics..L Neu~t~path~iL F,~p. Ncurul.. ;1,: ~UII- ;_'ll.

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