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Observations on the effects of glycerol on the cold storage of the canine liver
The Journal'of ...,. 5. . :
SURGICAL, RESEARCH VoL, V l, No, 4
April ) 966
Obsm,,ations",' on t h e Effects of Glycerol o n t h e Cold Storaze of the Canine Liver G E R A L D S. MOSS, M.D,/ PETER .C. R E E D , . M ; B , ; C h a B , , a n d . A , G, , RI DDEI,L, M,S., F,R,C,S., University qf MancJ~esler
One of the many problems0f organ ramsplantation is slorage of the organ prior {o its implantation in the hosL Th~s appliesParticUlafly to those Conditions in wllich"the organ cannot bedonated fi'esh on demand b y a live individual (as can a kidney). Unpaired organs, ~ such as the liver, wilt have .-to be preserved as and.wtien available, ' until an occasion arises When the organ is needed, This paper describes our preliminary expeciences in attempting to slore canine livers by freezing; .Though glycerol is known to protect isolated cells and small pieces of tissue agains!~he effects Of freezing and thawing, the use of glycerol to protect, whole Organs, such as the liver, remains largely unexplored. a IETItOI)
:The., experiments hdve-:been performed on healthy adultmmngrel dogs of 30 to 40 pounds we!ght, Ancsthesia was ~induced w i t h N e r o bmal. Scoline was used--as :a ~relaxant and positive respiration w i t h Oxygen a n d minimal nitrous ~ oxide was "continued: 'Siric, sleriie precautions w e r e o b s e r v e d d u r l n g ) h e surgery~ Through a midline.incision the portal'vein, inferior ' y e n : . tara. and h e p a t i c a r t e r y Were isolated, iThe liver was then cooled tO:i5 °, C, fO "20° '.C~ ..bY: Sptanchnic :co01ingi: (~hd"peri~: toneum .was irrigated with iced saline Until the: liver..rehchdd the"required."tempe(atui'e.) T h e
From.{he Depar~men| of Surgery. Onivce.dty of Manchester, 'Englarid. Presen¢ address of. D~'. Moss: Massachusetts' General (Hospital, : Bostbn~ Massa.~. chusetts~: P~seht address of I)J' Riddelh Deparlmenb iof Surgery,:Universi~y'0fBristol, Engldnd.
gallbladder contents ~were e.×pressed and.the common b i l e rdUCt was - ligated~ ,hnd" divided,: "The portal Vein was:cannulated find divided a n d the suprarenal ~.inferior.i v e n a c m / a Wag ligated and divided. Through ~a median Ster: n o t o m y t h e thoracic inferior V e n a c a v a Was cannulated and divided and this.cannula:, vas Subsequently used. as ~he. outflow, t~'-act, The: hepatic, artery was mobilized . with?ia Cuff:of aorta t o facilitate later transplantation; Th:e" remaining altachments 0f... the: liver : Wei'e divided and the organ.removed. T h e liver w a s next., t~nsferred to ~:.an :iCed saline bath and flushed via thepoft~al, vein 'arid hepatic : artery cannMas-; With.10~,. Rh60;. macrodex in normal°saline unlil: the effitient ,'an clear ,Of blood. Then iheTdrgan Wag:infiaSed with increasing concentrations ,0f: glycei~0l; at 5 ° c . This was achieved in: thi'ee: stages tising 500. Cc,. Of 10% glycerol;:.:500 re, 0f::20%. glycerol, and finhlly 33%-.glycerol: gOlntioni:,,: The solvent for the glycerol weaS :10%)::Rhe~i omacrodex in normal .saline and~h&pH/6f:th:e: solu tion : w a s a N : sted ~:to : 7,14::;iT h i s :.flush]ng procedure :took 15 to 20minutes; ' -.-:The liver :was ~then sealed -in containing 33% glycer0t .and ~SI0~ freeze a , ~20~. C...:and;. in :::som, seqOently?in :a. dry!iee gmd.iacetot ~.607C: ,This latter temiaera[i~rew; because . rewarmiogfpro,;ed;i..:e:xire!~e!)..~::;i!6~;. storag e peri0ds;varied-.i fr0m :-~z4,?,houi~s:~,f 6 2 weeks:-:..Thawing .was~ initiall ~,, piading ihe. WhOie _0rgan~ih :-.: ~te:' 40°. C : ~: H0@ever~:/:, this :~:pr ie:: *l,e:, approximalelY~1000 mi|li6smg]eslliler) 2000 milti0sm61es;qi(er;",and 3500: i~i liosmof6~lli~er~~res~e,e:2 tivety~
147
148
MOSS
ET AL,
JSR .- Vol. Vl, No. 4 -
April 1966
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thawingonlY while the central tissue remained solid .and, the Organ woutd:not infuse s~tisfaclgrity, Subsequehtly {he liver was allowed tO~.thaw., in ..a domestic ~.refrigerator tYom the de, Per temperature.to -13 ° C,; a t : which temPerature. . infusion ..was .. possible, ~ After thawing;-:the,tiver ~was. placed:in an iced saline bath.;'atl;O~:and the.:gIycer01 removed by.infusion~i~!Wiih~,:::ddci:easing' . concentrations. :...of gl~,)cer~lll:in: three, stages (20%...gtycdrol;: 1o% glye4m[ and ifinaliy .normal ,saline). Again 4he flUsliir{g S01dti0ns. were adjusted to.,.pH 7.47 Toienabie: the effects Of.glycerol p e r s e t o b e eluCidat~ct: a: Control series. WaS:"perf0rmed in
Whi~c~/
i :~One'eooted*~ liver, was frozen afte~ being. fieparinized~ " 2~;::;~0/neiii!;cooled*.?: liver,..:was: frozen.~, after infU~i0fi :.£VithKrebs!:: solution • 3~ on6~.i--cooled~.ii.:livet was~g!vcerolized a n d ddgi}c:eroJized Withdut freezing, 4~: Oge; :co6ted, ;:/liver ;..w a s " removed .. a n d
perfused !mmediatety on t;',e pump-oxygenator circuit ,'~s a normal conlrol, ] h e ~thawed, ':deglyceroIized" livers, other than the control ~erles, were then divided imo two :groups. One group w a s placed in an isolated pump-oxygenator circuit and perfused~ the~other group was transplanted into the. right parac01ic g u t t e r 0 f healthy, recipient dogs: T h e transplantation. technique was a modification..of:, the~ method"described by. Goodrich (1956) (Figure t): Multiple biopsies wer e, performed a{ Critical :stages during the isolation arid.slorage. RLSULI S From previous W o r k i n this department we havedocumented evidence ~of the. behavior o f isolated ", perfused • cariine livers on ,a: pumpOxyge~atioff:i~circuit, ~md i ihis ~ behavior!wasl Used as a~Yardstick:againsl which to cbmpare the present, series; COntrol ,series
*RCd~cedto 15? to 20~ by splanehnicc o n i n g .
The:nonglycerolized frozen: livers NO. t~an d-
JSR -
V o l , VI.. No, 4 -- A P r i l 1 9 6 6
No. 2 thawed 0nly after a prol0!~ged'i~enod
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and would • not infuse satisfactorily.. Nor. would
they support.. perfusion :on the:isolated pump-. oxygenator The g!~ce,'olized-"nonfrozen liver •. No.- 3 .perFused %Yell and:iPerformed Satisfa'C:iori!y on tile isotated perfusion circt, it. The addition a n d r e m o v a l of glycero!::::did. not appear to h a v e . a n y significant residual•; effec! on ttie l i v e r . T h e behavi0r-of ihis.liver: iff .lhe isolated perfusion. apparatus w a s identi7 cal. With that....of the .Untreated liver"No: 4. Livers frozen u n d e r " glYcerol. • prQtection lhawed easily and supported neffusion. •
,
Theparameters u s e d aS .criteria for Viability. w e r e oxygen up!ake, B.S.P. clearance and histological appearances. T h e s e p a r a n : a e t e r s were m e a s u r e d . on the isolal.ed, pump-oxygenator circuit at 37.5 ° C. and pH. 7.4 Usi,ng fi'esh, h eParinized , do~10f whole blood as the •perfusate.. The ••oxygen i'iptake w a s csliniaied .by measuring the.., oxygen, terision"(using :a Clarke: electrode)• ofl. t h e blood::_entering and leaving t h e isolated liVer..The difference of these.:::readings was multiplied b~! the fl0w r~lei.. O x y g e n tJptake . ( F i g u r e 2 ) . • A l t h o u g h u p take nev.er equaled control, levels, there was significant uptake i n m 0 s t cases: . .... ... B.S.P. C l e a r a n c e . (Figure 3). • The..plasrna disappearance r a t e . o f B.S.P. in •this series of" •livers indicated marked impairn!ent of ftmcti0yL. tlaough in each some B.S.P. was•removed. :
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TIME .(mi.n) Fig.~re..4. Flow rafe~: after, freeze storage," C; Normal control. C ; Cooled • f+20m.C ). .glYCe:~0.ized..and: d.eglycel .~ olized. 3; 4, 8. and. 10. After 24 hours a t , - 6 0 ? Ci..l 2i...After 7 •days at - 2 0 ° C:.: 6, C.ooi.ed (+,0.. C.)~ithoui.gl3~ce~i~i.
l!'.lowrates. (Figfire ;...4);' !":Threel."l ve"J:s:.::f,-ite~l to mainiain .sati s fac t0iT.flowrates.lfor fh6%. tha'n one hou,;2.: one"li.vei-wasabte to.: aci:iievei:c:onti-o} s"tanda rd.ifl0w:; .~and ..t W01...tl:io tigti~:.ih iti~ll!¢:i.,s.atisL' •.factory, shtJwed.:a tende6cy::..midecline:i;: ........... ... His t010g{e,d :.Appe;inince~:.::.; Th6:".d~scUS,sibn Of. hiSt01ogicat: a:p ~earance ::.will'::be":..:in"ClUde'd:: ii~ •the secder~. Of .u.,"ansplhnt ed org~ins.
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DOG •. F i g t u r e . 2. : O x y g e n , u p t a k e after.: f-eeze.isi0rag e C , . N 0 r m a l ¢0nlrol. C., Liver.cooled t o :+20 ~' C:, gtycer~fize.cl nnd •deg!ycei.'plizet],, 9, ~ f t e r . : l 4 . d a y s aii-:60 ~ :C. i7~ind. 12,L".Afler. 7 d a y s .,5t."L20 ° C,i...All.0thers,:.)~fter. 24 hours a t - 2 0 ~. ( 7 , , . . . . . . . . . .
Following thawing.and.:removal..oC: glycdriSl~. nine li V~rs:..:were."t ianspfahted..iin={6:: t h:e.::,pet~i s::?of. hea it hYl. i:e 61pientS.il.tind eF: ,stricf:: S~erii e~.!p~:e~ai:i;~ tions:and iJnderianiibiotic C0 ver!..:.Tlie::..:fiiLSiithr~"£ i~,ere .:;•an•as tom0Se'd: ::~(F igui:e i.i.::!I:.I~A):L:.Ib~..!(..p~iasi 'i~! Ca:nntitae, :.:the:.:rema ining.: .six ..?by.lefid :i:~::to~ii:~::.~ idei:: hepat ic.; .["ai-tery~! nfrarenal ::: • ri:d
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f|~E (rain) Figtire. 8 . : Cross, Circtilation series B.S,P. uptake Two: experiments ShOW Po0( ~bin significant uptake. On~ does not :show any uptake,
inffarenat,i,v.c, anastomoses (Figure I,B),. Ir~ ti-ie:first but. not the:second.gr0upl portal veir to::ilP0rtali::~,ein anastomoses.were performed. ! ri..:the :.first: group,. therefore]/iigati6n of ttle .~epatia!pediCle.0f ttie reciPienl animal allowed .[solati0n :.of-:;"the.:"trar/splan t ..:liver" for : stody, :BJS:P: uptakeS. Were-pefform:ed after One hour :di'i!icm-s~sl-.circulation:•.Tw0 of t he livers showed ;gli~'nlificght~.!-BiS;P,;:Uptakes; though very much .redUced;::and ;0ng livei~.showed;n0,uptake at all :(Fig/i're:!!5)~',:i:n: ithe, seC0nd., anastomoses :groi~p "&r.!!i;:activi{idS~:,~verei-:c6ncerned :::with.:.survival fi:ild(:0"6.B~S!P! i(stiadies' were :iperformeci,~:as this i;ivSula:: have: emaiied;.the.:intercu Pti0 n: of,both ihe:;(hejsaii~:;m~er:y.~n cl.Portal ~veidsdn, tbe~ hosi. a'nimal{ ?1[i;:.was~-:felt ' tha~t th)e-hos't.a~nimalmight :V;/ell::li6t?f0te:rat6;ttiis added .insult:None:0f the ~imals, ilin:fadt2::sUi.vived24.hOurs, ge~~ei'a}).ipfi~/homena-wei'e' observed :mer the tcaifiSpihnl/::; had))been :-"pd~rforn~dd.::::The .]iVei" t ~ e d : : from pale: pink.to(deep: red :~nda . sl0w glli:e0hsian{ O6ze began from: the.lig,er..Surface:
Aprll I966
The suture lines, which" were hitherto watertight~:.;began [0 ::leak :~'and'free' blodd in • the abdomen. failed to ~ Cl0t: Hematological assay StuiJ~es (Figure 6):show gross hemolysis and fe6ucti0n 0f,.fibr{nogenqevels/ In addition.a prof0und metab01iC. acidosis .foiI0wed. the. completion of the anas!omos~g;;Despite the correctiOn of the acidosis, the adn)inistration of .fresh blood, vitamitl K and .calcium ~to try to :atteStthe bleeding, with steroid and ~Lev0Phed to"c0mbat shock, theanimals failed t0 resp0nd and died. within Six hours, Histological Appeai:'anee, S I N U S Q I I ) S , The m0Sl Striking histological feature produced by the processes of glycer0tization, freezing and .thawing was widening of the sinusoids (Figure 7). This occurred after giycerotization of tim cooled (t 5" C,) liver but was much more mai-ked:after freezing,~ On subsequent thawing and degtycerotization this widening-was observed laNely tO resolve except in those cases in which, the architecture o f the liver was irrevocably disturbed, SectionS o f t h i s latter g r o u p stained to show reticulum indicated disruption of the. SUpporting scaffold. .Measurements of.cell diameters were performed during the Various stages of. these experiments. No statistically significant alteration in the size of the cells was discernible and so the wideningof the sinusoids was real and not apparent, .CYTOPLASM, Vacuolization..of the. cytoptasm. (present in sections of :normothermic .livers) .tended . to. increase" on cooling ..and 80 60 40 20
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Figiire 6.' CR~tdng {'actors. a, Partial thrombop|astin time: b,"Plasma c[o~ting time~, d, Prothrombid ~time,~ d, ~ Fibffnogen 'e~ Plasma hem~Oglobim
JSR -- VoL VI+aNo; 4 - - A p r i l 1966
C O L D S T O R A G E OF LIVER
151
CONCLUSIONS
F i g m e 7. Hislologica| appearance of glycerolized liver at - 2 0 ° C showing widening of the slnusoids.
decrease again on warming. T h e vacuolization was random and followed no particular pattern i n relation to the vascular radicals. T h e cytoplasm of the fi'ozen specimens was granular and denser than that of normal sections. NUCLEL Many nuclei become pyknotic after freezing though in most cases a considerable percentage ( 2 0 % ) o f histologically normal nuclei were o b s e r v e d after storage at - 2 0 + C. for 24 to 48 hours+ RETICULU~I. Most livers survived freezing and thawing without gross disturbance in the reticulum pattern. ~ w e v e r , in a few cases the scaffi31d pattern was gmss!y d i s t u r b e d : : l n these cases e x p o s u r e o f the sinusoids to arterial blood pressure resulted in further disorganization of the l i v e r architecture+ However, even w h e n the s e c t i o n consisted almost of independent cells o r cords of cells, these cells and their nuclei apffeared histolog+ Tl~e livers f:rozen " to - 6 0 ° C. suffered greater architectural d a m a g e than those fi'ozen to - . O CO . GLYCOGEN, T h e liver sections were s t a i n e d fo~ glycogen a s a crude method o f a ~ c ~ { a i n i n g : whether, m e t a b o l i s m w a s pro a c e e d i n g ' a t these l o w {emperatures, T h e r e was in all cases 'a gradual reduction in the quantity of glycogen in serial sections performed during the: period: s we took to indicate anaerobic i t y and mobilization
infused:
!. Thirty-three per cent glycerol does not appear to be toxic to hepatic cells as evidenced by the performance of the glycerolized liver No. 3 in the control series. 2. Glycerol appem+s to decrease the thawing time and facilitate rapid infusion after thawing (controls I and 2). 3. Frozen-ihawed livers pretreated with 33% glycerol and stored at - 2 0 ° C. for short periods are capable of clearing small amounts of B+S.P., supporting flow.and taking up oxygen. 4. The histological appearances are those of slow cooling, i.e.: shift of fluid extracellularly and extracellular standard microscop evidence of intracellular crystallization. The supporting reticulum of the liver stands deep freezing poorly and begins to fragment when re-exposed to arterial blood pressure. 5. Transplantation of frozen glycerolized livers results in metabolic acidosis and deterioration in the clotting mechanisms. T h e deterioration in the clotting mechanism appears to be basically a profound hemolysis. This could be due to the libet~ation of a specific hemolysin due to breakdown products a a mechanical hemolysis cau: of the normally nonwettable lining of.the liver sinusoids caused by freezing and storage procedures. T h e metabolic acidosis likewise could be due to two things, the accumulation of lactic acid and products of metabolism caused by i n a d e q u a t e cooling and anaerobic respiration, or the autolysis of liver cells. 6. Livers pretreated stored for short periods are capable of limited With further inquiry alon ticularly in the rewarming phase) and +the d i s c o v e r y of more effective a n t i f r e e z e agents, we feel lhe pl:oblems of ~ h o l e organ storage are not insurmountable. REFERENCES I. Goodrich, E. O.: Homotmnsplantation of the canine liven Surgery, 39:244. 1956. 2. Huggins. C . ~ Differential+ hepatic cooling. A , M . A i Arch. Su÷g., 74:327, 1957. 3. Jacobson, L . O.: Modifications of radiation injury in experimental animals. Am. J+ +Roentgenol.. 72:543. 1954, 4. Meryman, H. T , : Effects of freezing on liver ceils. Proc. Roy. Sue, B,, 147:4521 5. Smith, A.U.. Polge, C_ and Smiles. J . : Microscopic observations of living ceils during freezing and thawing. J, Roy. Micr. S ~ . , 71:186;. 1951:
SURGICAL, RESEARCH VoL, V l, No, 4
April ) 966
Obsm,,ations",' on t h e Effects of Glycerol o n t h e Cold Storaze of the Canine Liver G E R A L D S. MOSS, M.D,/ PETER .C. R E E D , . M ; B , ; C h a B , , a n d . A , G, , RI DDEI,L, M,S., F,R,C,S., University qf MancJ~esler
One of the many problems0f organ ramsplantation is slorage of the organ prior {o its implantation in the hosL Th~s appliesParticUlafly to those Conditions in wllich"the organ cannot bedonated fi'esh on demand b y a live individual (as can a kidney). Unpaired organs, ~ such as the liver, wilt have .-to be preserved as and.wtien available, ' until an occasion arises When the organ is needed, This paper describes our preliminary expeciences in attempting to slore canine livers by freezing; .Though glycerol is known to protect isolated cells and small pieces of tissue agains!~he effects Of freezing and thawing, the use of glycerol to protect, whole Organs, such as the liver, remains largely unexplored. a IETItOI)
:The., experiments hdve-:been performed on healthy adultmmngrel dogs of 30 to 40 pounds we!ght, Ancsthesia was ~induced w i t h N e r o bmal. Scoline was used--as :a ~relaxant and positive respiration w i t h Oxygen a n d minimal nitrous ~ oxide was "continued: 'Siric, sleriie precautions w e r e o b s e r v e d d u r l n g ) h e surgery~ Through a midline.incision the portal'vein, inferior ' y e n : . tara. and h e p a t i c a r t e r y Were isolated, iThe liver was then cooled tO:i5 °, C, fO "20° '.C~ ..bY: Sptanchnic :co01ingi: (~hd"peri~: toneum .was irrigated with iced saline Until the: liver..rehchdd the"required."tempe(atui'e.) T h e
From.{he Depar~men| of Surgery. Onivce.dty of Manchester, 'Englarid. Presen¢ address of. D~'. Moss: Massachusetts' General (Hospital, : Bostbn~ Massa.~. chusetts~: P~seht address of I)J' Riddelh Deparlmenb iof Surgery,:Universi~y'0fBristol, Engldnd.
gallbladder contents ~were e.×pressed and.the common b i l e rdUCt was - ligated~ ,hnd" divided,: "The portal Vein was:cannulated find divided a n d the suprarenal ~.inferior.i v e n a c m / a Wag ligated and divided. Through ~a median Ster: n o t o m y t h e thoracic inferior V e n a c a v a Was cannulated and divided and this.cannula:, vas Subsequently used. as ~he. outflow, t~'-act, The: hepatic, artery was mobilized . with?ia Cuff:of aorta t o facilitate later transplantation; Th:e" remaining altachments 0f... the: liver : Wei'e divided and the organ.removed. T h e liver w a s next., t~nsferred to ~:.an :iCed saline bath and flushed via thepoft~al, vein 'arid hepatic : artery cannMas-; With.10~,. Rh60;. macrodex in normal°saline unlil: the effitient ,'an clear ,Of blood. Then iheTdrgan Wag:infiaSed with increasing concentrations ,0f: glycei~0l; at 5 ° c . This was achieved in: thi'ee: stages tising 500. Cc,. Of 10% glycerol;:.:500 re, 0f::20%. glycerol, and finhlly 33%-.glycerol: gOlntioni:,,: The solvent for the glycerol weaS :10%)::Rhe~i omacrodex in normal .saline and~h&pH/6f:th:e: solu tion : w a s a N : sted ~:to : 7,14::;iT h i s :.flush]ng procedure :took 15 to 20minutes; ' -.-:The liver :was ~then sealed -in containing 33% glycer0t .and ~SI0~ freeze a , ~20~. C...:and;. in :::som, seqOently?in :a. dry!iee gmd.iacetot ~.607C: ,This latter temiaera[i~rew; because . rewarmiogfpro,;ed;i..:e:xire!~e!)..~::;i!6~;. storag e peri0ds;varied-.i fr0m :-~z4,?,houi~s:~,f 6 2 weeks:-:..Thawing .was~ initiall ~,, piading ihe. WhOie _0rgan~ih :-.: ~te:' 40°. C : ~: H0@ever~:/:, this :~:pr ie:: *l,e:, approximalelY~1000 mi|li6smg]eslliler) 2000 milti0sm61es;qi(er;",and 3500: i~i liosmof6~lli~er~~res~e,e:2 tivety~
147
148
MOSS
ET AL,
JSR .- Vol. Vl, No. 4 -
April 1966
\
/
\
.
F
(
\
~epatit I A~rta al
zt.itizc afleqt
~epalie p.e~lid
I.V,C.aria
~ z
/
f
\,
,
~\ k
A
B Figure t, " A, Cross circt~lalion series. B, Transplantation series.
thawingonlY while the central tissue remained solid .and, the Organ woutd:not infuse s~tisfaclgrity, Subsequehtly {he liver was allowed tO~.thaw., in ..a domestic ~.refrigerator tYom the de, Per temperature.to -13 ° C,; a t : which temPerature. . infusion ..was .. possible, ~ After thawing;-:the,tiver ~was. placed:in an iced saline bath.;'atl;O~:and the.:gIycer01 removed by.infusion~i~!Wiih~,:::ddci:easing' . concentrations. :...of gl~,)cer~lll:in: three, stages (20%...gtycdrol;: 1o% glye4m[ and ifinaliy .normal ,saline). Again 4he flUsliir{g S01dti0ns. were adjusted to.,.pH 7.47 Toienabie: the effects Of.glycerol p e r s e t o b e eluCidat~ct: a: Control series. WaS:"perf0rmed in
Whi~c~/
i :~One'eooted*~ liver, was frozen afte~ being. fieparinized~ " 2~;::;~0/neiii!;cooled*.?: liver,..:was: frozen.~, after infU~i0fi :.£VithKrebs!:: solution • 3~ on6~.i--cooled~.ii.:livet was~g!vcerolized a n d ddgi}c:eroJized Withdut freezing, 4~: Oge; :co6ted, ;:/liver ;..w a s " removed .. a n d
perfused !mmediatety on t;',e pump-oxygenator circuit ,'~s a normal conlrol, ] h e ~thawed, ':deglyceroIized" livers, other than the control ~erles, were then divided imo two :groups. One group w a s placed in an isolated pump-oxygenator circuit and perfused~ the~other group was transplanted into the. right parac01ic g u t t e r 0 f healthy, recipient dogs: T h e transplantation. technique was a modification..of:, the~ method"described by. Goodrich (1956) (Figure t): Multiple biopsies wer e, performed a{ Critical :stages during the isolation arid.slorage. RLSULI S From previous W o r k i n this department we havedocumented evidence ~of the. behavior o f isolated ", perfused • cariine livers on ,a: pumpOxyge~atioff:i~circuit, ~md i ihis ~ behavior!wasl Used as a~Yardstick:againsl which to cbmpare the present, series; COntrol ,series
*RCd~cedto 15? to 20~ by splanehnicc o n i n g .
The:nonglycerolized frozen: livers NO. t~an d-
JSR -
V o l , VI.. No, 4 -- A P r i l 1 9 6 6
No. 2 thawed 0nly after a prol0!~ged'i~enod
100
and would • not infuse satisfactorily.. Nor. would
they support.. perfusion :on the:isolated pump-. oxygenator The g!~ce,'olized-"nonfrozen liver •. No.- 3 .perFused %Yell and:iPerformed Satisfa'C:iori!y on tile isotated perfusion circt, it. The addition a n d r e m o v a l of glycero!::::did. not appear to h a v e . a n y significant residual•; effec! on ttie l i v e r . T h e behavi0r-of ihis.liver: iff .lhe isolated perfusion. apparatus w a s identi7 cal. With that....of the .Untreated liver"No: 4. Livers frozen u n d e r " glYcerol. • prQtection lhawed easily and supported neffusion. •
,
Theparameters u s e d aS .criteria for Viability. w e r e oxygen up!ake, B.S.P. clearance and histological appearances. T h e s e p a r a n : a e t e r s were m e a s u r e d . on the isolal.ed, pump-oxygenator circuit at 37.5 ° C. and pH. 7.4 Usi,ng fi'esh, h eParinized , do~10f whole blood as the •perfusate.. The ••oxygen i'iptake w a s csliniaied .by measuring the.., oxygen, terision"(using :a Clarke: electrode)• ofl. t h e blood::_entering and leaving t h e isolated liVer..The difference of these.:::readings was multiplied b~! the fl0w r~lei.. O x y g e n tJptake . ( F i g u r e 2 ) . • A l t h o u g h u p take nev.er equaled control, levels, there was significant uptake i n m 0 s t cases: . .... ... B.S.P. C l e a r a n c e . (Figure 3). • The..plasrna disappearance r a t e . o f B.S.P. in •this series of" •livers indicated marked impairn!ent of ftmcti0yL. tlaough in each some B.S.P. was•removed. :
-
i.
1.
0-1
,
1
J
•
:10'
I
20"
TiHE (rain) " F i g u r e ". 3.". B, S, P. : c learance.. after > freeze ::Storage.:/, C . N 0rm;al 6~nirot. C , C 0 o i b d (4:20 ~' c )i.glydei:'6i i2ed-~ihd=i, .deglyce=:olizcd. ' A , "His,mlogic:"ally..dead :.i.livetL..? B:;::i#Meart " Cle:arance .Of four..perfu~edliver#!'afler 2 4 houi'~"':at:~20~LC,:."
40O
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8
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;: .200
3O
6O
9O
TIME .(mi.n) Fig.~re..4. Flow rafe~: after, freeze storage," C; Normal control. C ; Cooled • f+20m.C ). .glYCe:~0.ized..and: d.eglycel .~ olized. 3; 4, 8. and. 10. After 24 hours a t , - 6 0 ? Ci..l 2i...After 7 •days at - 2 0 ° C:.: 6, C.ooi.ed (+,0.. C.)~ithoui.gl3~ce~i~i.
l!'.lowrates. (Figfire ;...4);' !":Threel."l ve"J:s:.::f,-ite~l to mainiain .sati s fac t0iT.flowrates.lfor fh6%. tha'n one hou,;2.: one"li.vei-wasabte to.: aci:iievei:c:onti-o} s"tanda rd.ifl0w:; .~and ..t W01...tl:io tigti~:.ih iti~ll!¢:i.,s.atisL' •.factory, shtJwed.:a tende6cy::..midecline:i;: ........... ... His t010g{e,d :.Appe;inince~:.::.; Th6:".d~scUS,sibn Of. hiSt01ogicat: a:p ~earance ::.will'::be":..:in"ClUde'd:: ii~ •the secder~. Of .u.,"ansplhnt ed org~ins.
T,
,,co
~ i~o.:
...
I s o l a t e d P c ( f u s i o n Series •
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-
:Trat~l~lat~t. Series •
7
Ct3
4
7. 8
.
.
.
9 1210
"
DOG •. F i g t u r e . 2. : O x y g e n , u p t a k e after.: f-eeze.isi0rag e C , . N 0 r m a l ¢0nlrol. C., Liver.cooled t o :+20 ~' C:, gtycer~fize.cl nnd •deg!ycei.'plizet],, 9, ~ f t e r . : l 4 . d a y s aii-:60 ~ :C. i7~ind. 12,L".Afler. 7 d a y s .,5t."L20 ° C,i...All.0thers,:.)~fter. 24 hours a t - 2 0 ~. ( 7 , , . . . . . . . . . .
Following thawing.and.:removal..oC: glycdriSl~. nine li V~rs:..:were."t ianspfahted..iin={6:: t h:e.::,pet~i s::?of. hea it hYl. i:e 61pientS.il.tind eF: ,stricf:: S~erii e~.!p~:e~ai:i;~ tions:and iJnderianiibiotic C0 ver!..:.Tlie::..:fiiLSiithr~"£ i~,ere .:;•an•as tom0Se'd: ::~(F igui:e i.i.::!I:.I~A):L:.Ib~..!(..p~iasi 'i~! Ca:nntitae, :.:the:.:rema ining.: .six ..?by.lefid :i:~::to~ii:~::.~ idei:: hepat ic.; .["ai-tery~! nfrarenal ::: • ri:d
IS0
M O S S ; E T .AL,
JSR - V o t . Vl, N 0 . 4 . -
IO0
10
£
llti
f|~E (rain) Figtire. 8 . : Cross, Circtilation series B.S,P. uptake Two: experiments ShOW Po0( ~bin significant uptake. On~ does not :show any uptake,
inffarenat,i,v.c, anastomoses (Figure I,B),. Ir~ ti-ie:first but. not the:second.gr0upl portal veir to::ilP0rtali::~,ein anastomoses.were performed. ! ri..:the :.first: group,. therefore]/iigati6n of ttle .~epatia!pediCle.0f ttie reciPienl animal allowed .[solati0n :.of-:;"the.:"trar/splan t ..:liver" for : stody, :BJS:P: uptakeS. Were-pefform:ed after One hour :di'i!icm-s~sl-.circulation:•.Tw0 of t he livers showed ;gli~'nlificght~.!-BiS;P,;:Uptakes; though very much .redUced;::and ;0ng livei~.showed;n0,uptake at all :(Fig/i're:!!5)~',:i:n: ithe, seC0nd., anastomoses :groi~p "&r.!!i;:activi{idS~:,~verei-:c6ncerned :::with.:.survival fi:ild(:0"6.B~S!P! i(stiadies' were :iperformeci,~:as this i;ivSula:: have: emaiied;.the.:intercu Pti0 n: of,both ihe:;(hejsaii~:;m~er:y.~n cl.Portal ~veidsdn, tbe~ hosi. a'nimal{ ?1[i;:.was~-:felt ' tha~t th)e-hos't.a~nimalmight :V;/ell::li6t?f0te:rat6;ttiis added .insult:None:0f the ~imals, ilin:fadt2::sUi.vived24.hOurs, ge~~ei'a}).ipfi~/homena-wei'e' observed :mer the tcaifiSpihnl/::; had))been :-"pd~rforn~dd.::::The .]iVei" t ~ e d : : from pale: pink.to(deep: red :~nda . sl0w glli:e0hsian{ O6ze began from: the.lig,er..Surface:
Aprll I966
The suture lines, which" were hitherto watertight~:.;began [0 ::leak :~'and'free' blodd in • the abdomen. failed to ~ Cl0t: Hematological assay StuiJ~es (Figure 6):show gross hemolysis and fe6ucti0n 0f,.fibr{nogenqevels/ In addition.a prof0und metab01iC. acidosis .foiI0wed. the. completion of the anas!omos~g;;Despite the correctiOn of the acidosis, the adn)inistration of .fresh blood, vitamitl K and .calcium ~to try to :atteStthe bleeding, with steroid and ~Lev0Phed to"c0mbat shock, theanimals failed t0 resp0nd and died. within Six hours, Histological Appeai:'anee, S I N U S Q I I ) S , The m0Sl Striking histological feature produced by the processes of glycer0tization, freezing and .thawing was widening of the sinusoids (Figure 7). This occurred after giycerotization of tim cooled (t 5" C,) liver but was much more mai-ked:after freezing,~ On subsequent thawing and degtycerotization this widening-was observed laNely tO resolve except in those cases in which, the architecture o f the liver was irrevocably disturbed, SectionS o f t h i s latter g r o u p stained to show reticulum indicated disruption of the. SUpporting scaffold. .Measurements of.cell diameters were performed during the Various stages of. these experiments. No statistically significant alteration in the size of the cells was discernible and so the wideningof the sinusoids was real and not apparent, .CYTOPLASM, Vacuolization..of the. cytoptasm. (present in sections of :normothermic .livers) .tended . to. increase" on cooling ..and 80 60 40 20
400[
''
200
t
~s ~ 0
1
2
3,
HOURS
Figiire 6.' CR~tdng {'actors. a, Partial thrombop|astin time: b,"Plasma c[o~ting time~, d, Prothrombid ~time,~ d, ~ Fibffnogen 'e~ Plasma hem~Oglobim
JSR -- VoL VI+aNo; 4 - - A p r i l 1966
C O L D S T O R A G E OF LIVER
151
CONCLUSIONS
F i g m e 7. Hislologica| appearance of glycerolized liver at - 2 0 ° C showing widening of the slnusoids.
decrease again on warming. T h e vacuolization was random and followed no particular pattern i n relation to the vascular radicals. T h e cytoplasm of the fi'ozen specimens was granular and denser than that of normal sections. NUCLEL Many nuclei become pyknotic after freezing though in most cases a considerable percentage ( 2 0 % ) o f histologically normal nuclei were o b s e r v e d after storage at - 2 0 + C. for 24 to 48 hours+ RETICULU~I. Most livers survived freezing and thawing without gross disturbance in the reticulum pattern. ~ w e v e r , in a few cases the scaffi31d pattern was gmss!y d i s t u r b e d : : l n these cases e x p o s u r e o f the sinusoids to arterial blood pressure resulted in further disorganization of the l i v e r architecture+ However, even w h e n the s e c t i o n consisted almost of independent cells o r cords of cells, these cells and their nuclei apffeared histolog+ Tl~e livers f:rozen " to - 6 0 ° C. suffered greater architectural d a m a g e than those fi'ozen to - . O CO . GLYCOGEN, T h e liver sections were s t a i n e d fo~ glycogen a s a crude method o f a ~ c ~ { a i n i n g : whether, m e t a b o l i s m w a s pro a c e e d i n g ' a t these l o w {emperatures, T h e r e was in all cases 'a gradual reduction in the quantity of glycogen in serial sections performed during the: period: s we took to indicate anaerobic i t y and mobilization
infused:
!. Thirty-three per cent glycerol does not appear to be toxic to hepatic cells as evidenced by the performance of the glycerolized liver No. 3 in the control series. 2. Glycerol appem+s to decrease the thawing time and facilitate rapid infusion after thawing (controls I and 2). 3. Frozen-ihawed livers pretreated with 33% glycerol and stored at - 2 0 ° C. for short periods are capable of clearing small amounts of B+S.P., supporting flow.and taking up oxygen. 4. The histological appearances are those of slow cooling, i.e.: shift of fluid extracellularly and extracellular standard microscop evidence of intracellular crystallization. The supporting reticulum of the liver stands deep freezing poorly and begins to fragment when re-exposed to arterial blood pressure. 5. Transplantation of frozen glycerolized livers results in metabolic acidosis and deterioration in the clotting mechanisms. T h e deterioration in the clotting mechanism appears to be basically a profound hemolysis. This could be due to the libet~ation of a specific hemolysin due to breakdown products a a mechanical hemolysis cau: of the normally nonwettable lining of.the liver sinusoids caused by freezing and storage procedures. T h e metabolic acidosis likewise could be due to two things, the accumulation of lactic acid and products of metabolism caused by i n a d e q u a t e cooling and anaerobic respiration, or the autolysis of liver cells. 6. Livers pretreated stored for short periods are capable of limited With further inquiry alon ticularly in the rewarming phase) and +the d i s c o v e r y of more effective a n t i f r e e z e agents, we feel lhe pl:oblems of ~ h o l e organ storage are not insurmountable. REFERENCES I. Goodrich, E. O.: Homotmnsplantation of the canine liven Surgery, 39:244. 1956. 2. Huggins. C . ~ Differential+ hepatic cooling. A , M . A i Arch. Su÷g., 74:327, 1957. 3. Jacobson, L . O.: Modifications of radiation injury in experimental animals. Am. J+ +Roentgenol.. 72:543. 1954, 4. Meryman, H. T , : Effects of freezing on liver ceils. Proc. Roy. Sue, B,, 147:4521 5. Smith, A.U.. Polge, C_ and Smiles. J . : Microscopic observations of living ceils during freezing and thawing. J, Roy. Micr. S ~ . , 71:186;. 1951: