Changes in rat uterus caused by freezing and thawing in the presence of glycerol

Vol. 4, No. ,3; I'.~q"

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l r a r r a n l :' ::s(,d the isometric ..ontraclfle respons~e {ff ,zuinea pig u t e r t ~ s to h i s t n m i n e to assess :Ira effectiveness of his t?reservation t e c h n i q n e s in p r e v e n : i n g d a m a g e from freezin~ a n d ll:,twing. In ihe w o r k r e p o r t e d here, we h,t"v.(., comp:tred. . . f r o z e n - t ! m w e d h o r n s .and control horns of rat, uteri b y m e a n s of the iso:onie dose-response e u r v e s to dru,*:~,..., nnd ,,~ :'ll](:t tissue ,ll..o l)v ll)~..~:.tl].S of weight eh.allKes ,.

". ,~ .~ ~.~ '~( rain ~o. f ) k .' ", T h e u l e r i were t.t,.tn,.l/c::et to ,_,'13"eerol-I >~, 20.-,. w , v , a t and incubated [or 45 rain ':t :hat, : e m p e r a t u r e . T h e t,emt?era, ,~:{: was lowered to --S C,. n~M the u t e r i were ihen t r a n s f e r r e d to glyeer01-PSS, :30% w / v , o

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A f t e r 45 rain of i n c u b a t i o n a t - - S ° C , ~he uteri were, frozen b y being p l u n g e d into ti(l~fid n i t r o g e n . R c m o P a l of qlttcerol w i t h osmotic sh.ock. T h e frozen horns were, t h a w e d by being droPlJed into .PSS at. o, C. •Fhc.' were trnnsferrcd to ftl

t ~ ~' several thnes d u r i n g the next 20 fresh .'>~ m i ~ to ~ensm'e r e m o v a l of g l y c e r o l and then

were ,fllowed 1..5 h r s to r e c o v e r with occasional repl.teement of P S S .

Removal o.f glycerol with protection, against osmotic shoct~. Frozen horns w e r e d r o p p e d into -.,"

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100

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o0% ~lveerol-I 5,.'5£ a t 16°C for 2 or o r a i n , just

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or

,.,~ of .:ltbt ..... rol s.utwutanc, ou,dv for ea.,h of (~ d a y s 'tnd killed on the. 7th d a y b y a blow c~:~ Ibe h,,ad. U~eri were r a p i d l y r e m o v e d a n d freed from a d h e r i n g fa~, and lhe horns were sep:~rat(',l, cut opm~ lengthwise, "rod t h e n • '', In ..,, l){cl .fl-......... w/v; and ineutmte,.t :tT 0, C for 45 rain. T h e t e m p e r a t u r e was ~hen ~r:~(hmlh' lowered d u r i n g 10 to 15

'

~cr is .... 1;-~6"('. which leml)era.ture is not a t P.timtidc ou :he. Parlmn dioxide freezin~ mi{:ro. ' " " , " ...... lt~m,, w~, ~./s~',:I ,,_lxc{.l...-I "~< ih{: c.rvoproleclive ., :~ent. ']'he e:tle('tic tC.'.tt~!)e.rattlre of ~lye.erol.~d,':t ! (H" i.'4." - ~ .~q,3.,'...5o r-~ k.,.

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suf[ieient time Io thaw them. Th%, were then :ransrerred {o. 20% gl.x, eerol-P~S at: 16°C for 10 rain, then to 10% glyeerol-PSS at:- 16°C for 10 rain, and finally into PSSI The temperature z during 1 : b e n e x t 10. •rain, was raised to o"~'° nnd ineu!}ation a.t...this t e m p e r a t u r e continued for a n addit;ional i h r " 2 0 min. During:.:fllis J:b~ Was changed every 10.or. 15 time, • l h e -w-,,-, '

~,-,

T h e physiologie, al salt. solut.ion (P~.,.>.) contained NaC1 (118 m s 0 , . I ( C I (4.8 m a 0 , CaCL. (1.4 :na~:}, M~(2'I: (1.1'3 ]n~.[), NaI-I.~PO, (1.16 , " - e..d ,..., <,, t-o-, ~' 1.967. I~{,,{.{.~-~ ,.cmbe~ • 8, • Part., of this work w a s s u p p o r t e d by Grant. G67-24 fl'om tim Life Insur'mce Medical Ilesearch Fund.

rain.

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148

I'. ;~I. (?AI.~R()I,L A:N1) J. H. H N

D r u g respo,~se o/ mu,.clc a f t e r freczii..7 a~(l tl~awinc/ wit.D osmotic ,shoch. .:\l.lC ' " , ' 1 rell~ovat o f t h e u t e r u s f r o m the. rat., t h e ]lorns w e r e

cut, open lengthwise, one horn (the ccntro~) w , s s~ore(] at 4~'(7, in . "I ~' " ' o v e r n i g h l , :.m(l the other hc)rn was. proteet.ed with .=13.-~_.Iol,~ -,,oas above and stored in liquid nitrogen .ovei'night. The frozi::n horn wns thawed rapidly and the glycerol was removed with o~motic shock. Bo~.t~ the eolliro] ,Ildq ' the t:rc..e~.ed~ ' , ' " "" horns were ~hen s u s p e n d e d in t;issue h a t h s (50 ml) in •~:,~e ,,-,, C and allowed to re.,..~., ::it. o, c.,.over ~heir spontaneous r h y t h m i c a l contractions. Isotonic contractions were recorded with liehtly loaded levers on smoked drums. The l.i.'aSllO ]),'.TIliS W{?l'(y e()oh.,d I-/tII0OHS

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s:ponse of the museles to dn_~gs was tested. ll"eigDt a n d elect ' ' r o l .., b l e cDange,s. Fr(.'sh uterine horns were lolottecl and wei-~tied rapidly. The (:xperirrmnta] horrls were tin)leered with glycerol as above and st' ort.d,- in liquid l]il;ro~ell,. . . . T. h e y were thawed by one of the two me.t.hods, ,.rids- ihe Mveerol was removed. After a ~ce,.)~e~3. " , , c ' ~ ' ' period . r. .~ the horns of /.oh)..-., were blotIed, weighed rapidly, dried in a v a c u u m desiccator at, room tempe, rature, and analyzed. T h e control horns ~er(.--,~ incubated" at. o~°C"" for 0.75 hr without glyeerol,.eooled to 0 °, and i n c u b a t e d for 1 h r 40 rain. I f the controls were for tissues subject to osmotic shOck on removin~ ,,-)v,,e,-~,~ then t.he controls were w a r m e d to 37°C alld incut:)ated for 1.5 hrs. I f the e0ntrols were for tissues .receiving osmotic protection then the controls were w a r m e d for the a p p r o p r i a t e lengths o f time at the ,u)Pr < c.)p~""~ate, t c",m pera tu res. E l e c t r o l y t e a n a l y a e s . Analyses of so(tium, p o t a ~ i u m , calcium, a n d magnesiurn were m a d e with a U n i e a m s P g 0 0 flame s p e e t r o p h o t o m e t e r or "m E E L flame photometer on tissues which had. been dried in a v a c u u m desiccator a t room u..

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t e m p m ' a t u r e a n d t h e n digested in concentrated H N O . . Chloride was extracted from d r i e d material witt~ 0.1 :x HNO~ containing 10% acetic acid and then estiina.ted using a. B u c c h l e r - C o t lo~.e C h l o n d o m e t e r a u t o m a t m t~trator. •. T

,

t

Egect ofglycerol ra the absence Of freezing. O n e : h o r n : o f each pair w a s incubated ill !0% . . . . . . . " " o,. ' ~ 7 ° C ; Cooled to.0~C: glvcerol-PSS for 0. 7 . ~,~ hr.a~ and: incubated -in 20% glycerol for 0.75 hr and then m - o 0 7 o ~,lxcerol .for 0.75..hr, all..at 0~C. '

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Glycerol was removed l-,v one of lh,,, lwo methods des(.q'ibed. The o i h e r horn of each pe~ir .~erved as eont, rol and was (reared as (tes('ribed fc)r tho controls in the seetio1~ on wei~eht and eleel r(:,lyte chgtnm?s. ]{.E S Ut,TS

,The eotl~r,H. . . uterine horn a,~M ~hf~ ~,y'ccr<,i-~' tm)tort(,:I frozen horn whi(,t~ ",va.~.. ',hawed wi!h osmotic ~lu.ek both showed sl, rona spoll[.:!ll(' Ills rhytlm;i(,al cc',ntr::~cti(u~s a.t o, C. The subs:(:(]l.lOtll

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('}lollltc ,qt](l lO .serolollnl at 22~(] \yore "¢(:,rv sinrit.tr for bo)h .lit)r/is ... (l:i~s:.. 1 a~.,d 2). C.o~.-" ~).tmris('m of lht'. t.tc{l.l(i! sixr! t")f l}~p {?(~lt|.rgctiol.,. o f t h e ta(,,servc,,d hOHl relaiivc ~,:.~ ~]m c,,.a) • ' ,c t rc, l h o r n :~ sul/u.~aximat d,',s("s of I~1¢'s~' ' ..... d r l l u s 3~1~[ of o. 'x"x t o (",', is (zi yen in r I ' t ,,h., ~ '~.,. Simil>;r .,x,:.~ltle: were obt:iined in o i h e r ( ' x p e r } m ( ' ~ . s , ine..h_i(lin~

(,n,:,s in which t,ho t~ssues w(,rr, frozen ff)r 2t d:,.vs. _ an ~ !.h(" firs~ line o) I:fl:)le <1 tw)~M~ summ:)rizes Ta.ble 2) ..Lo~,~ ' ,' ~l~.(,.,,'" ~Ive(;ro!.. . irnpre~na.ti(),,, i'rcc,zir)~, and ih.'-~win~z with <)sm(,ti(: "P "i"-

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sllc)(~.k ~(..>/h,t.(l it/ a KI'e'IIOF tll¢'l'('t-~.~O ().f W(:q .w(:'i~|lt i h : m o(_'.(m)'rod ill i h e (:()I~irot ~isstl,:.,,. T]l(?re w a s 8Ol.~le d e c r e a s e o f d r y weight afwr

the. process, compared It) *he dry weighI of (.he con~r'ol. The .process. ...... aL.o, < res,~Ite, l i)~ an t"~lel 'eq ' e" ~; (.q" , s e of t i"s s u e sodium and a (tecr.a..t. t issu(.,, potassium. It, x~ .... m " t m a ~ e r i a l wheth¢,r .,~, t h e l ; i s s u e s were l"I' ozen ' aI ~ - 1 , 0 ( ) ' C for i d:~v er for 21 days. T a b l e ;3 s u m m a r i z e s t:h.~, resul)s of exl)erilll(?.Iltg at.teml3ting to deter~:.~ine ~l)e cause of ttie wet weimar, ; ," .~. a n d l.he (:ausc~ o f t h e ., ,IlCte,i,.(~ ionic m~ba/ance. ]xemoval of Myeero] from frozen t,~Jm, with osmotic protectmn, helped tc prevent:, the wet weigh.t inerease b u t did not.improve the electroh, te i m b a l a n c e . . 1 R e m o v a l of glycerol with osmotic shock from unfrozen tissue- w a s deleterious to tile weight b u t did not cause electrolyte imbalanee. R e m o v a l of glycerol w i t h osmotic protection TM

R~,

from unfrozen tissue helped t.o p r e v e n t the wet. weight increase and did not cause electro1) qe..... mab,tlance. " , , " " it is • seen thai; the .'[hus electrolyte in)balance r e s u l t s , f r o m t h e freezing a n d is p r o b a b l y u n r e l a t e d to the: use of glycerol, wMle the increased wet weight is due to the osmotic shock on r e m o v i n g tl!e_glycerol and is p r o b a b l y also related t o t h e freezing. I~ is p r o b a b l e t,laat g1"eater action to p~e e nvt '

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The tog dose-response curve of the conh'ol horn (®) an(! the protected frozen to ,acei;ylelioline, 1he log (to~..,response ~" . . curve of the eontrol horn (®) and the proteef.ed frozen to serotonin.

- the wet shock would further .de~rea~ea

weight.

DISCUSSION When

-

uterine horns

were

protected

wl th

glycerol, frozen, and thawed with osmotic shock occurring during the glyebrol removal,. t h e subsequent doserresponse curves to a eetylelmline and serotonin appeared quite n01:mal, However, the elect.rolyte analyses o f t,he control

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Im"reased sodium eliminated from tNe tissue. "['lu:, net changes ot" sodium ani.I l}o!:~ssimn wer{, l_}gdti/wed l)v t i m i I.~el'{::is(, o f lissrl,: eidoride.. {)11( * I1111~51 t it('r(;fot'o C O t l ( q t . l d o

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Fresh Wet Weight

Control ................ Frozen 1 day. ......

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1:;Ice i r o b 1,:s :

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K

Na

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:~,)!,) 2~,)S ....

7{)I 1073

323 251

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Cont;r{}t . . . . . . . . . . . . . . Frozen 1 day. ............ C o n t r o l ................. Frozen 1 dnv. ...........

Control ................... F r o z e n 13 d ' t y s . . . . . . . . . . . .

v(;rv

al"l{3r fF,'0ZiBff

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1 day ...........

Con trol ............... ]5'rozell •" o {hLv8 . . . . . . . . .

tS{i 186

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Control .................. F r o z e n 21 d ' a y s . . . . . . . . . .

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{"O!llrat:tioIt

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219 219

10.9 20.5

and protected frozen,thawed horns (Table 2) Showed >that some potassiun~ was 10st from the tissue as a r e s u l t of t h e freezing and tim.wing, a n d . t h a t i~.was not regained after 1.5 hrs of incuba, tion at..'31 C, nor x;a.., the

41,0 37. :3

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•

702

wet weight,,_ wlfieh. 4.v,as significant. When R " glycerol A u d r e y Smith u,_ea to protect t,he uterus, she 0 b r a i n e d b e t t e r recovery of uterine c o n t r a c t i l i t y by omitting calcium from the ,.olutmm-, q:~oth f o r t.i,.st~*t.le,frozeri to --79°C nild

FREEZING AND T H A W I N G OF UTERUS

151

TA I$.1.,I.1 :.I (.:~MI'AItIS~IN

()I" (711ANGIqF, ( J . \ U S I - ; D

1135

"['}m t n i l , i b e r ,,t" eXl},~rimeitt.s w l , i e h w e r e [ ) e r f o r m e d av,,r:lR,:~ x.'nltles

]?H.Ia;I,.;ZIN(; ANI} ]IY O S M O T I ( ;

is ~ i v e t l in p a . r e n t h e s e s .

~.I.I(.)(;K

]tesult.s are expressed

as

:.k N , t C . r~f l.]'lO lt'lO:lII. I :.}we~,f T,"mp~'~alure

.Me,hod'of (;lycerol ~ Rernnval

I n c r e a s e of Wt't W e i g h t

l,oss of I)ry ! 't,Vei~ht

.~ I lai*wi!

I i

E l e c t r e l y tes ".................................. ~........

--

-.(.

,.~

,,;~,

K

~

i

!

C1

i 598

=t=. 2 3

Na

mmoies.ik¢,

d r y wl

GIG

22

i

~ l<.\l.~orime,~l:tl

(i .... 196

(),,,:~r,,l

9.-1 ~-. t . 1 . ~ 2 t . : ¢ .~..- l.-t

()slm~t.i('

~ 32.1

S.1

-_k=. ~i

: 179 ~

16

±

91i2 ...-q= .:t2 I 778 ::l= 2 0

!

(",u,~r'~t ,1l'],,l,,rinm~iI:~l

~i-

:

()siil,~l.ic pi'(~-

---l!fti

(.t '

{1.7

'.1.7 ± 1 . 2 19.-1 :.*=. 2 . 7

--3..i

-t|.l

~:

21

1{13 .-.-j:: 1-I

5 0 . ¢) =t= 2 2

981

i 558

::t= 4-t ; 8 0 2

=t::

~

12 20

I0CI ion

( : , , : , ~ r , d '7~ l..]Xl!crlrllolii:t.l ..72

is {i

• ' t'~xl),"iintoni:~l

t~

()stn~tie sh~wk

()si)l()ti,:

,-I

t'~,r

9.2 .+.. 1 . 7 1-t. 1 -A:: 1 . 5

i

357 ~: !)/5 ~ 3 2 8 ± 2i

528 ± 2S 659 =k.. :!6

501 =k: 15 583 _-t=. 27

.578 651

5 3 4 :t:: 2 5 .5(hi :4- 2 0

i

t,)'()-

. . =i= 2.1.).

, 12.7

.

. 1.7

.~:.~ -~- lf; 2 7 S :~= 2 0

=t= 14 _~ 8

lecti,)ll

•

I

, t/s,,i(,

~,~:(m,,J . . . 1,~. .-÷...q'~(-":~

I~).t~,.I1:, ....~ 1,

ill

tz.ll] . . . . ~.

,>xper.,.))~('n~s witl~ ,,,~i,)ea ,;i~ ,l,..eri and di.me)hvI s~l[l:{)xi(t:"

])l'~)tc(:li,:,li.

t'()llll(t

(}JC'V

tmderwent

a

~,n)l~)nm~,t votl~r.'.~cu~m on th.twinff, ,vhieh could },e :'ev,,rs,.d !,v a,t~tin~ ~thvlonedi:mlineietr:l:lt't'!:II("

ttI"

~'l')tlltt

]10

J'~I'(!V('I}*(7:(t

bV

Oltlil tin~

I+v ha(fly ¢{:,m:,~ed. Th,., normal drug-induced e,>.nlr;,cTions also imldy l llat~ the membrane is in ~o~)(1 condition. The quosf,ion which now oeetlpies us is this: t.< ~tw ionic in~lmhtnee lho restflt of p e r m a n e n t ~l:una~e t,-~ ttW memhrano which is not subseq,u?lltlv repaired 1.w ihe cell: or is i,c the result of ~emt~orarv membrane deformations o0('llrrin,~ (hlrin,z tim a.tual
,:'~}~'i~n. Sinii!-~r rt,.,ull.-.-: were also O1.)ttliIlo([ wl~,,n lhe tissues w~.re n o t frozen.: Thus i.t -~:v:i> vot'v in-ol,ail~le iimi ille " .rC,I " ~(. .... of 111t7. ,i~,,,e. .. c:~h.'ium whi,::.h is l~re'mnw~l. .... to ha.re. (w(,'rr,,t in tl,o c..xnt~rm~enls of Smith and I:::,rr:~,~t. a n ~ l Whi(:Jl oee.~rred to :..1 small (.,Xl:elll; m ~mr ext).'.,rin,?nts {Ta}de '21 was d u e t o d a m (I _L. f¢ ? 1.0 l}lt'~ l H ( ? l l l b r ( l l I O oe0urrhlg as the result SUMiMARY e l ¢?Slt'tf~[l., st:,o(.'k and ttlso (II10 10 daln/t,:re o c c u r rin,, ,,,: ilm result of ",',,,,',;.',~ We have demon]Tat. uterine horns were protected with ,~ll'g.llt~(] i.h:ll i11~? incre~tsed wet. wei,..r.qit.._, of th:.o gly-eero], froze,b and ~hawed. Studies of the lissome is~ {lie. r0s,,ti of osmoiie shook when the contractions: t() drug~ and of electxolyte and ,.zlv,.x~r0] is t-~c.,,in~ removed, and ~s probably .:ll.o weight ehan~e:s shbvCe.ci that the I.mtassium_ affi~,,,ed b," f,'eezin~. We have also dt.mon~t~d.ted e o m e m of lhe tissue was a more sens}tive ,lmr the so~lium-i~otassium imbalance i.s the indie.".ttor of danmge than were: the drug-ind,J~ ~ed re<
"

]

"

"*c

~-,

152

P. M. C A R R O L L AND J. H. I,IN

]:~EFEI~ENCES 1. Bauer.• H., Coodtord, ' " i'. J,, and H" uml, " • J. Tim calcium content and %alcium uptake o f the smooth muscle of the guinea,pi'g taenia coll. J. Physiol.,176: 163:179; 1965. 2. Farrant, J. T h e calcium-dependent contraction of s m o o t h muscle after freezing in the pres-

mle~!~ of dimethyl sulpl,~xid~,.J. Physiol., 170: 33P-34 P. 196.t. 3. l:arrant, .J. Meclmnian of c(,ll damage during fr(:,(~zing mM thawing and its I)rtPct'fl/iOtl. Nature, '205: 12S4-1287, 1965. -l. Smith, AI 15. }3io!ogical effo,!ts of fre::,zing aml sup~,r(,ooling. Arnold. Lon,l~m, t961.