Toxicity and cryoprotection by dimethyl sulfoxide and by glycerol in isolated frog sciatic nerves

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Vol. 5, No t~, 1.9t59

TOXICITY

AND CRYOPROTECTION BY DBIETIIYL SULFOXIDE A N D B Y G L Y C E R O L IN I S O L A T E D F R O G SCIAIIC NERVES D B. PRIBOIg AN D,A. NA R A

Department o] BMogy, University oJ Toledo, 7"oledo, Ohio ~8606 This report is p:,rt of a long-term l)rogram for investigating the mechanism of freeze-thaw ,i:un;~ge to membr:,ne systems and the meeh.> nism of eryoproteclion from such damage. -~.ccording to lhe I-todgkm-I/uxley model for nerve conducuon, changes m the electne:~l parameters of nerve tibers reflect eh:tnges m ,structuralfunctional integnlv of tl~e :~xon m e m b r a n e s ' Therefore, ~so]aie(l nerve fibers represent ~ conven~en~ experm~enlal tool far stu(1.y~ng freezetha~ dam'~ge to :rod er3 ol)rote(:tmn of a men> l)r,~ne systt,~n \V(." chose Io .~tudy frog scm~(: n(~ves svJee linear dectrt<~l paran~eters "tre xxell e l u r : , , e t e r ~ z e d all(| ~l~(.,y are easy to manipulate. ']'h~s ~ork ~s 1)rdm~n:~r3 ta II1VCStlKaII()I'IS (if n e r x e l)r"oparalJOll ~. whero~ the response of a s~n~le :txo~ may I)e 1)hvsmlog}e'~.ll3 ~sol:tted .Bo~h dmJetl,'31 sulfoxidr, (D~ISO) '-~ and glycerol""° are know~ lr) ha,o some effect on the eleelr~c:~l par'an~et(.r- of nerve.-. Therefore, we detcrmme(l ~he effect of I)3I,'q0 or g13 eero] m aml)lnbmn l{nlger's sol~t~on on fro~ sciatic nerves ~ t h o ~ t s~l)se(l,,Cnl freezing and th'~wing. Tln.~ allox~ed us to tl~stmgmsh l)elween (l:m~age from these agents a~tl damage from the freezethaw process m the prebeuec of the-e agents. ~ [ t'rEIII.~.LS AND ~'.[ETtIODS |r ]so~aie(l frog scu t~c nerves" w~th or w~thout their permeural sheath were exposed to 5, 10, or 15% (v/v) ])5'ISO or glycerol in a.ml)M)~an ]~inger's solutmn at room temperature for 1,5 rain. In one set of experime~ls these nerves were then washed twice with Ringer's solution and then allowed to recover. In another set of experiments with d~fferent nerves exposed to D~[SO or glycerol, the nerves were frozen at --10°C in the presence of these agents, thawed, washed twice with Tlinger's solution, and allowed to recover. In both sets of experiments electrieal parameters of the nerves were measured before treatment and after 30 and 60 min of recovery

l{eceivcd December 16, 1968.

Jn tilt ]:~iliger's solution. The electrical paramelers measured were: 1) eoMuctml~ veloc,V, 2) action potelltm], ~lnd 3) absolute refractory period. Exposure of tt nerve with or without intact perJneural shettth to a, l):~rt~ctflnr concentration of ])MSO or glycerol wilh or without subsequent freezing represent,~ a particular " t r e a l m e n t " of a nerve Thins, m the first set of experiments there were () (hfferent treain~ents wah D M S O - - 3 w~ih and 3 without intact pel'ineural s h e ~ t h s ~ a n d llkewl.-,e 6 with glycerol for a total of 12 dlti'ere1~t treatments. The second set of experiments is .-m~tlar to the first except that the nerves were ..ul)seqlmntly flozen and thawed. Ill addition nerve~ w~lh or wzthout mta(,t, permeural sh~,alhs ~ere frozei~ and thawed in I~mger's solution without. D~ISO or glycerol Itence there was a total of o(~ dJtTerent tre:ltn~ents. Each type of treatment was performed on nerves from 2 frogs except for the effect of 105; gl3cerol on nerves w~th permeural sheaths, wluch was performed on 3 frogs. Thus a tolal of 53 frogs, were ~sed in these experimenls In each treatment, a control obtained from the same frog was used for eomImrison These control nerves underwent the same mechanical manipulatmns as dld the {rea ted nerves. h~strzlmc~Ttal~o~. The stmmlating ~mlt conslstod of :t Tektronix 162 wt~ve form generator in eon.iunetmn w~th two Tektromx 161 p~flse generators. The stmmlatmg unit was connected to the stimulating electrodes through a stmlulus isolator, model 112 (American Electronic Laboratories, Inc.). A standard nerve chamber was used consisting of ~ Plexiglas box w~th seven palladmm electrodes spaced 1 cm apart. The recording electrodes were connected to a low level preamphfier, Tektromx R~'I 122. This, in turn, was connected to a dual beam Tektronix 502A oscilloscope. ~l)hotographs were t:~ken of each response with a Tektronix C-12 viewing hood connected to a Polaroid Land Pack fihn camera back.

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tanmd from F. G. iloffman & Son. The frogs were kept m a. 4°C cold room and thus were m cohl torpor. The nerves were isolated at, room temperate,re and all electrical parameters were measured at this temperature range. The sciatic nerves were ~solated in the following manner. The gut was reth.,eted anteriorly to expose the nerve, insertions at, the spinal cord. From here tim commctive tlssue w'ts removed along dm entire length of the nerve to the tiblahs antieus. All branches were cut ok)so to tl~e mare trunk with the most cranial branch of the two spinal insertmn,-, being cu~ last. W~(h pra,ctme a pair of sciatm nerves could be renmved within 15 rain. A similar procedure was followed m isola,tmg nerves w~thout intact perineural shea, ths, hereJust after referred to as "desheatlmd ne~x,e~. "-" before the t~o ~Fmal') , lnsertmns were cut, the

t('mt,ial was determined i,y initmtmg z~ response whu-h apt)eared on the oseflloscolm as a bit)h:~sie wave form and then increasing the stimulus to the point just ]wfore the fl fiber responm could be observed. Absotutc refractory ported. A series of two sequenti'd stunuli ~vas applmd Io the nerve and the alnphtu(le :~nd delay betu e(m *he two stimuli were adjusted to give dual [.phasic ieq~onses of equal amplitude and :~pproximalely 7 msee apart. The delay time of one pulse genera*or then was decreased while the sensiuv~t,y of the oseflloscol~e was increased. The intervM bdt~veen the occurrence of the first response and the fi1~t point ill tinm t]mt the second nerve response did not appear was taken to be the absol~lte refraetor.~ period,

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nerve bundles and then peeled posteriorly the whole length of the nerve. :k pair of ,tesheathed nerves could be isolated nl 30 lllln. Freeze-thav' procedure. Nerve preparations were drawn lnlo dispel-able pipettes along w~th apt)roxmmtely 1 ml of It, roger's soluuon w~lh r)r withou~ prot, ect~ve agent depe~tdmg on the exper,men/. Each p~pet te was sealed with wax and the o~xst,tlhzatmn ' .... " ~a'."s' initiated by to~mhmg the sule of the pipette away from tl~e nerve wall,, a was piece of D r y lee. Once ,t~e er3,_ta[hz,ttmn ".~" " ' ' started, the Inpette w'ts rap~(lly phmgu(t into an ethanol bath gtl, - - ] 0 ° C trod kept, there for 5 mm after the lhnger's solutmn was completely frozen. The nerves were thawed by r'tpadly transferring the frozen preparations to a water ba ~}, "tt room temperature.

Mcasurcment o/ Electrical Parameters Conduction velocity. The conduction velomty was determined by recording the monophasic action potentials from two electrodes. I~mwing the sweep l,mm of the oscilloscope and the disl.ance between the two recording deetrodes, one can calculate the conductmn velocity.

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Effect o/I)M,50 on Nerves with Pcrb~cural Sheaths Comluction velocity (F~g, 1A ). The conduction velocity of control nerves dropped re 90% of the m~tial vahm after 60 mm of recovery m Ringer's solulmJ~, in nerves treated with 5, 10, or 15% D M S O the conductmn velocity dropped to 90, 8.t, and S0% of the respective iniuat wdues after 60 mh~ of recovery in Ringer's solutmn. Action potentzat (Fig. 22t). The action potential of control nerves rose to 117% of the imtiM value after 30 and 60 m m of r e c o v e w m Ringer's solution. I n nerves treated with 5% DMSO, the rise in action potentml was only d~ghtly less than that of control nerves. The action potential of nerves treated w~th 10 or 15% D M S O decreased from its initial value and thus is apprecmbly less lhml that of the control nerves. Absolute refractory period (Fig. 3A). There was very little change in the absolute refractory period of the control nerves or n e r ~ s exposed to 5, 10, o,' 15% DMSO.

Fro. 1. The conduction velocity of frog sciatic nerves with or without permeural sheath treated with various concentrations of DMSO or glycerol. The conduction velocity was measured before treatment (initial value) and after 30 and 60 rain of recovery" in Ringer's solution. The percentage of the initial value is plotted against the time of recovery. Each treatment point is the average of determinations on two frogs; each control is the average of determinations on six frogs, two for each DMSO or glycerol treatment. Note the ,'~bsolme values of the conduction velocity before ~reatment and after 30 rain of recovery arc also shown on the graphs. A. DMSO-treated nerves with perineurM sheath. B. DMSO-treated dcsheathed nerves. C. Glycerol-treated nerves with perineural sheat, h. D. Glycerol-treated desheathed nerves.

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Effect of DMSO on Desheathed Nerves %

Condaetion velocity (Fig. 1B). The control nerves and the 5% D.MSO-treated nerves showed ,~ shght increase, and the 10 and 15% DMSOtreated nerves showed a slight decrease in conduction velomW from their respective initml values after 60 rain of recovery in Ringer's solution. Actwn potentiol (Fig. 2B). The control nerves and 5% DMSO-treated nerves showed a slight decrease in action polentml from their initial values. After 60 rain of recovery, the action potential of 10% ]D3'ISO-treated nerves was 103% of the initml value, whereas that of the 15% DMSO-tr~tted nerves w')s 82% of the initial value.

Absolute refractory period (Fig. 3B). As with nerves wilh intact, permem'al sheaths, there was very hbtle change in at,solule refractory period of control nerves and of 5, 10, or 15% DMSO-t.reated nerves,

Effect of Gtgce'rol on Ner~)es ,v~th Penncurd Sheaths Co~duction ve!ocity (F~g. IC). :Both t.he contzo[ nerves and ~he 5% glycerol-treated nerves showed a shght decrease in conduction velocity after 60 rain of recovory. The 10 and 15% glycerol-tre, tled ~:,,rves, on the o~her hand, showed a definite drop in conduct.ion veloclD~ from ~.helr mitml v~iues (~ e., about 80% of the initial value). Action potential (Fig. ?C). There was little change in action potentml of control nerves and of 5% glycerol-treated nerves after 60 rain of recovery in Ringer's solution. This is in contrast to 10% glycerol-treated nerves whose action 1)olential rose to 118% of the initial value after t30 min of recovery and 15% glycerol-treated nerves whose actmn potendat fell to 74% of the inilial value after 60 rain of recovery. Absolute re[factory period (Fig. 3C). In contrast to the control nerves for the previous experiments, ~he absolute refractory period of these controls rose to 133% of the initial va,lue.

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Tlmre was little change m the nb,~olute r~fractory period of lhe 55; glyct-rol-trealcd nerve& The absolute refractory period of 10',':-, ztyeerof treated nerves ross to !14% of the imtiat value in contrast ~o that of 15% glycerol-treated nerves, which fell to 88% of the mJtial value,

Efl. ect o/Glycerol on Desheathcd Nerves CoTtductmn veloczty (Fig. 1D), The condnclion velocity of control nerves ~howed lkt]e change, but tlmt of the 5 and I0% glyceroltreated nerves fell ¢o abom, 70% of the initial values, respectively, after 30 rain and tlte~ ro.,e to about S5% of thezc values after 60 rain of rccovory m ~,~,~t~ .~ solmion. The con&ration velomty of 15% glycerol-treated nerves dropped to and remained at 60% of the initial value. Actio'~ pote~tiol (Fig. 2D). There was Imie change m the actmn potentmt of eo.u,rol ncz-vcs. Afler 30 rain of recovery there was a considen~bly ~real, er percentage drop m aetmn potential for the 59~ glyecrol-~reated nerve~ than occurred In ~he 10% glycerol-treated nerves, but this parameter for both ~mrve preparations apl~roaehed about 90% of the initial value after G0 nun of recovery. The action potential of 15% glycerol-treated nerves, on dm other hand, fell to 33% of the initml value after 30 mm and 49% of the m~tial value after 60 mm of recovery, Absol.ute refractory period (Fig. 3D). Control nerves and 5% glycerol-treated nerves showed little change in absolute refractory period. This parameter in 10% glycerol-treated nerves dropped from 104% of the initial value after 30 rain to 89% of this value after 60 rain. C o t respondingty, this parameter in 15% glyceroltreated nerves dropped from I27% of the initial value after 30 min to 97% of this value after 60 nain.

Effect of Freeze-Thaw Process on Sciatic Nerves 0nly desheathed nerves frozen in the presence of DMSO survived ~he freeze-thaw proce~. Al!

Fro. 2. The action potential of frog sciatic nerves with or without perincural sheath treated with various coneentrat,ions of DMSO or glycerol. The action potenual was measure([ before treatment (initiM value) and after 30 and 60 rain of recovery in Ringer's solution. The percentage of the initial value is plott, ed againsi, the time of recovery. Each ?rea[racnt, point is the average of determanations on two frogs: each control is the average of dcterminations on sLx frogs, two for each DMSO or glycerol treatment. Note the absolute values of the action potentml before treatment and after 30 rain of rccovew arc also sho~,ra on the graphs. A. DMSO-treated nerves with perineural sheath. B'. DMSO4reated de.. sheathed nerves. C. Glyccrol-trezted nerves with perineural sheath. D. Glycerol-trealed desheathed nerves.

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CI~YOPROTECTION OF F R O G N E R V E S other nerve preparations wlmn frozen at --10°C did noi, regain their excitability after 30 and 60 miil of recovery hi Ringer's solution at room temperalm re.

Effect o] Freeze-Thaw Process on DMSOtreated Desheathed Nerves

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h~fl'ttence o / t h e Perineural Sheath In many articles discussing the role of the exgenml sheath surrounding sciatic nerves, this sheath is referred to as a connective tissue sheath or as t h e e p i n e u ~ m n . '~' '" In m~y ease, there is

The desheathed D M S O , treated nerves did survive the freeze-thaw process, as indicated by their recovery of excitability. However, whereas some of the electrical parameters meqsured Jndmat,d that recovery of the freeze-thawed "* ) ~ O' nerves was coml lete---e.~., the absolute refractory period and tile conduct.ran velocity for nerves frozen in ,5 or 10% DS,iSO (see Fig. 8 and Fig. 4A)--other parameters such as the action potential .... ~,,,-~,,~ ",. -~, ~.~....v ttmt~ lecox,~r3 was far from comt)lete (Fig. ,IB). Also the extent of recovery depended on the concentration of 135ISO used. The lower the D M S O concentration the greater was the recovery of the ireezc-tha.wed nerves

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considerable evidence that tiffs sheath acts as a diffusion barrier?' '' =~' ~' A recent analysis by Shantiiaveerappa indicates that this sheath is ma.de up of an epineurium composed only of a lacework of eonneetive tissue fibers and a. perineurium which is composed of an outer network of connective tissues a~(1 an inner multiple layered epithelimn. ~'~ Thus i t appears that it. is the perineuml epithelium that controls diffusion into nerves since it contains continuous sheets of squamous cells in contrast to t h e discontinuous connective tissue. While desheathed nerves do gain weighl; owing to the inflow of Ringer's solution into the nerve bundle, the eleetriesl parameters are not appreeis,bb' altered. TM In comparing the average initial value of 12 control nerves with perineural sheath to tha~ of 1.2 desheathed control nerves, we found that desheathed nerves have a slightly lower conduction velocity and action potential. There was very lit fie difference in t.he absolute refractory period. In general our results suggesg that d e s h e a t h e d nerves behave in a manner similar to nerves with intact, perineural sheath~.

Effect o] Various C,o~Tce~ztrations o] DMSO or at~ycero~ on ;sctatzc N er~,es A 5% D M S O solution brought a b o u ~ rela' tively little change in the electrical p a r a m e t e r s of nerves with o r without perineural sheathsl A 0'7o glycerol solution also had littl,e:effect o n nerves with perineural sheaths, but it did:alter the electrical parameters of desheathed nerves, Iligher concentrations of D M S O or glycerol re, suited in changes.in t h e electrical txuameter~' • ' " ' ." of

]he,. 3. The absolute refractory period of frog sciatic nerves with or w i t h o u t perineural sheath treated witl~ various conce:ltrations of DMSO or glycerol, The absolute refractory period was measured before t,reatment (initial value) and after:30 and 60 min of recovery in. Ringer's solution. The• percentage of tile initial value i s p l o t t e d against tlie time o f re, covery, Each treatment point is: the average of determinations o n two frogs; each control is the '~verage o f determinations on six frogs, two for eacli I)MSO or glycerol treatnient. Note the absolute values of the absolute refractory period before treatment and after 30 mill: of reeovmT are also shown on the graphs. A: I)MSO-treated nerves with perineural sheatti. B, DMSO,l!reated desheathed nerves; C. Glycerol'treated nerves with perineural sheatlL D. Glycerol4reated desheathed nerves.

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FIG. 4. Tim effect of freezing at - 1 0 ° C for 5 rain on |he eondlwi, im~ velocity and ac,(,i~m potenti.d (,f DS[SO-pre(reated fr()g sci~tic nerve'.~ witlmut, perineural sheath. The concentrations of DM, O used were 5, 10, and 15~fj (v/v) i~J llilJger's so~l! i(~n. The electrical IJarame/ers were measured before treatment, (initial value) and ,ffter 30 and (i0 rain of recovery iv~ Ringer's s,dm, i(m. Th(, l)ereeniage ,)f the nnitiM v.tlue is pl~tted against the time (,f recovery. E~eh t r e a t m e n t 1)oinL is the ~vcrage of determinaimns ,;n 2 fr~)ge,; e~'h cent r()l poi~,t is the :tver:lge of (leicrmvm~tions on 12 frogs. Note. the absolute values of the eleclrical parameters before tret~tment ~nd afler 30 m ~ of ~eeovery are :d.,~,, :~hvw~: ~,n tlm g r a p h s . . l . Condu(.tmn vel(,city. B. Action potential.

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F t . 5. The eff(,ct of froezing aL - 1 0 ° C for 5 rain on the "0~solute refractmT period of DSISO-l)rettealt,d frog semtic nerves w i t h o u t permeural sheath. The eoncentr'ltions of D M 8 0 u~t'd wine 5. 10. and 15% ( v / v ) in Ilingm's solution. The absolute refractory permd ~ as mea.-ured b.efore tleatment, (miIitfl value) and after 30 and 60 rain of recovery in Iling-

or',-..~olu:;on The percentage of the iniUal value is plotted against the time of recovew. l,'m,h trt,nl xnenl, point, ~s tho qvor,tge of determma(mns on 2 frogs, each control t)omt is the :tvorag,, of d,.termlnntmns on 12 frogs. Note that the ab.-olute values of tl,e ,'efraetoD" period I~efor(. treat men t and aft(.r 30 mm of recover,," are also ~-hown on tim graphs.

both typ(.s of nerx os, and t.hesc changes were to some exl(,nt, m'eversd.)le. The ehrmges relative to the con~,rols in the eleetru.:~l par'm~eters brought on by DMSO on nerves with or without perineural sheaths were lees than the corresponding changes brought on by g15 eerol. In other wonls, Ihe effects of DMSO were more teverslble than those of glycerol. A second difference between DMSO and glycerol is that the variation of electrical parameters with concentration was more consistent with glycerol than with DMSO. Th,lt is, for glycerol-treated nerves--partmularly tI~e desheathed nerves--the higher the concentration, the greater was the change in the electrical par'm~eters.

Freezing without Protective Aget~t Nerves with or without perineural sheaths frozen at --10°C in the absence of any protective agent were apparently irreversibly damaged by the freeze-thaw process. Most of the axons of the sciatic nerve have myelin sheaths which probably have a low permeability to water as compared to a single plasma membrane. Therefore, upon freezing of the bathing Ringer's solution and/or the fluid ~4thin the nerve bundle, the individual axons would not rapidly dehy&ate as a, result of the concentrated salt solu-

lions which develop during freezing. Conscq u e n t l y , the axoplasm of these fibers probably also freezes spontaneously, anti, as with other cells, ~': tlns produces irreversible dam-~ge.

Freezing o/Nervcs with Perbwural Sheaths Neither DMSO nor glycerol exerted any cryoprotectn'e action on sciatic nerves with intact perineural sheaths. The, re Is considerable evidence that nerve sheaths are a permeability barher to ions ~"~' ~' "aid to glycerol." In spite of the high degree of permeabihty of many membrane systems to DMSO, ~'s k seems reasonable to assume that the nerve sheaths are impermeable t o D~I:SO as well as glycerol. If this is the case, then when these pretreated nerves are frozen in the presence of DMSO or glycerol, the individual axons do not contain any of these agents and thus freeze spontaneously. As in the case of freezing with no protective agent, the nerves are irreversibly damaged by the formation of intraccllular

ice.

Freezing o/Dcsheathed Nerves IL was somewhat surprishlg to find t h a t glycerol did not protect deshe~tthed nerves against freeze-thaw damage. However, Lovelock obtained similar results with human b!ood pre-

364

D . B . PRIBOR AND A. NAR~k

tr~'~ted with glycerol plus 3 × I0 "~ 5[ copper runs at 0°C. Such pretreated blood was severely damaged by the freeze-thaw process. The copper ions prevented glycerol from peuet.n~ting the red cells at 0°C, and, at least, for slow coohng rates, glyi:erol must penetrate ~hc cells m order to exert eryoprotection? In m~ analogous manner the myehn sheaths of the in(tiv~dual axons may be impermeable to glycerol, thus exphining tim lack oi cryoprotection by glycerol. DMSO protected desheathed nerves .'~gainst freeze-thaw damage, but the recovery of all the electrical parameters measured was not complete (see Fig. 4). Further, DMSO-~rcated nerves recover to a greater extent than DMSO-pretrcated freeze-thawed nerves. The freeze-th'lw process does bring about some irreversible damage to DMSO-pretreated nerves. This ~s partmularly evident for 15% DMSO. After 60 rain of recovery tt~c conduction velomty of ]5% DAISOtreated nerves is 100% of the mmal value, whereas tlmt of 15% Dh[SO-treated freezethawed nerves is 7659 of the m~tml v:~lue. L~kew~se the aetmn potential of ~he mrmer ~s ,~_, of the mitml value, whereas that of the la~er is 20% of the imtial value. The large mcrdasc in concentration of DMSO which occurs during freezing may itself be damaging to these nerves.

nerves, 5% glycerol gave very little cryoprotection to rat's superior gangha. The first two difference% at leasl, may be explained on the basis of the presence or absence of the myelin sheath. In ~he absence of protective agents the fibers in the rat superior ganghon may cryodehydrate and therefore super-cool r, tthez than freeze internally. This may account for some of them sllrv~ving temperatules down to --15°C ,is opposed to dm complete d.lm'lge of frog scmfie nerves at. --10°C. Also the rat's re.rye.-, probably are permeable ~o glycerol, wlmreas, because of the myehn shen~h.,., lhe frog :~xons prolmbly arc not permeable to glycerol. tIcncc, gl3eerol ~s a good proteetzxe agent in l:l~e one case but not m the ,,,fl~er. S u MM'.\ Jt Y

Isolated frog scmtic nerves with or w~thout iniact porineural she'~ths welt expo.~e,I to 5, 10, or J5% dnnethyl stufoxld,3..(])M,_.O) or ~lveelol in l{mger's solutmn for 1.5 ram. The~l elliwr tile nerves were allowed l.'~ recox ¢r ~J ~ormal ]~mger's sohl/lon, or the) were frozen 'tl --10°C m the presence or absence of ~hese l)rot('et~e agents and then ~llowed to recover m normal ]linger's solution. ]~ly this procedure th(, tox:eitv of the protective agents was (lisiJngUlS}md from freeze-thaw damage. The tolluwmg electrical Frog Sciatic Nerves vs. Mammalian Nerves t),~rameters were measured before tre:t~ tllpll I :tl]~-] after 30 and 60 rain of recovery m :t-{lT~g¢'r'-.'soluOur results dealing w~th myelm~lted nerves differ in a number of ways from Paseoe's result.s ~° tmn: 1) conduction veloelt.y, 2) action l~olentz.ll, dealing with nomnyehw~ted postganghonic fibers and 3) absolute refractory period. The results may be summarized as follows. 1~ Neither of the rat ganglion. First, we umained no survival of nerves when frozen m the absence of DMSO nor glycerol severely damaged the nerves, but the effect of each was (hfferenr m some of protective agents: ~-l~ereas he found that some the parameters measured. In general, a) the nerve preparations survived freezing at --12°C effects of DS[SO were less dramatic :rod more and one nerve preparation elicited a small action reversible than the effects of glycerol: b) except potenfia[ even after 12 hrs at --15°C. Second, for the eonduetmn velocity, the eff('ets of DMSO m our results glycerol did not protect nerves with or without perineura[ sheaths from freeze- appeared to be independent of the presence or thaw damage, whereas Pascoe found that 15% absence of the nerve sheath, whcreas the changes glycerol in Kreb's solution (v/v) gave complete wrought by glycerol were greater m desheathed protection against such injury. Third, whereas nerves. 2) All nerves frozen without protective in our results increased concentration of DMSO agents no longer responded to external stimuli. as the.cryoprotective agent brought about de- 3) Glycerol did not, protect nerves with or withcreased degree of recovery, in Pascoe's results out intact perineural sheaths from freeze-thaw increased concentration of glycerol as the cryo- damage. 4) DMSO did not protect nerves w~th protective agent brought about increased degree intact sheaths bu~ did proteet~ desheathed nerves of recovery. In fact, whereas 5% DMSO gave from freeze-thaw damage. 5) The cryoprotection almost, complete cryoprotection to frog sciatic by DMSO was not complete; i.e., freeze-thawed

CRYOPROTECTION nerves did not recover to the s~qme extent as nerves only exposed to DMSO. I?,EFERENCES 1. Asahina, E. Freezing proce~ and injury in isolated animal cells. Fed. Proc., Z~(suppl.): S-183-187, 1965. 2. Asahina, E., and Emura. 5'i. Types of cell freezing and the post-flmwmg survival of mature'titan ascites sarcoma cells. Cryobiology, 9 : 256-262, 1966. 3. Caldwe!l, P. C. Factors governing movement and distribution of inorganm ~ons m nerve anti muscle. Pl,ysiol. Rev., .~8: 1--6~t, 1968. 4. ];eng. T. P., and Lm, "Y. M. The connect;ire tissue sh~-ath of the nerve as effective diffusion bmrier. J. Cell. Comp. Physiol.. 8.~: 1-16, 1949. 5. -Franz. T. J., and "Van J3nlggen, J. T. A po~lble meeh'mlsm of action of I ) M S O . A n n . N . Y . Aead Sel, 1 M " 302-309, 1967. 6. Freeman. A. R.. et al. Osmotically determined ehalactermtlcs of the cell membrane of sqmd and lol,sl er giant axons. ,l. Gem Physiol., 80: 423-]45. 1966 7. Hodgkm, A. L.. and fhlxley, A. F. A quantitatrio descnpl~on of membrane current and us al~I,l:cation ~, to conduetmn and excitation in nerve. J. PiJvsml., 117 : o )0-044, 1952.

OF 1,'ROG N E R V E S

,3t;,5

8. Jacobs, S. W.. Bisclwll. 5I.. and ile, sch,:r. R. J. Dilnetllyl sulfoxide: effects on the permeability of biologic membram:s Curr. Ther. Res., 6: 193-198, 1964. 9. Lovelock, J. E. The mechanism of ~he protective action of glycerol against haemolysis by freezing and thawing. Bmchim. Blophys. Acta, 11: 28-36, 1953. 10. Paseoe, J. E. The ~.urvival of the rat~ superior cm~,ieal ganglion afler cooling" to --76~C. Proc. Roy. Sot.. Scr. B., 1~7 : 510-519. 1957. 11. Sams. M. W. The effect of dimethyl stflfoxidc oa neive conductmn. Ann. N. Y. Acad. Sci., l ~1: 242-247, 1957. 12. Shanes, A. M Effects of sheath removal on bullfrog nerve. J. Cell. Conlp. Phy,-.'iol., 4J: 305-311, 1953. 13. Shancs, A. M. Sodia~m exchange through the epineurium of the bullfrog s,.iaUc. J. Cell. Coral,. Physiol., 43: 99-105, 1954. 14. Slmnes, A. M., and J3erman. hi. D. Penetration of the intact frog nerve trunk t)y potassluln, sodium chloIMe and sucrose. J. Cell. Con:tp. Physml., .~1 : 419-149. 1953 15. Shanthavec'ral~pa. T. l(.. and ]3ourne-. G. l-i. Pe~meural eplthehum: a new conc'ep~ of its role m the integrity of the peripheral nervous system. Science, !.5.1,: 1464-1467, 1966.