the presence of glycogen in rat liver following in vitro processing in decalcifying agents

8/2/2019 the presence of glycogen in rat liver following in vitro processing in decalcifying agents

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69 9

TH E PRESEN CE O F G LYCO G EN IN TH E RAT L IV ER FO LLOW ING I.V V ITROIRO CESS IN G IN D ECA LC IFV ING A G ENTS

J. R . TRO TTF acu lty o f D en tis try , U n iv ers ity o f M anito ba , W in n ip eg , C an ada

R eceived for p ub lica tion M ay 31 , 196 1

In earl ier attem pts to p rese rve gh ycogen im ithe m ucop erioste um o f jaw s it w as fo und th atthe m ethods used w ere inadequ ate (8 ). I f themucop eriosteum was rem ov ed from the p ala te o fa ra t, fixed in ace tic a lco ho l fo rtnah in , ands ta in ed b y- the p eriod ic- a cid S ch iff rea ctio n( PA S), larg e am ounts of g ly cog en w ere foun din the ora l e p ithelium . H ow ever , i f the jaw s d ec alcified th en th is g ly -co gen as lo st. Th ereseem ed to be three Possib ilit ies to co ns ide r.F irst , th e m e tho ds used fo r the fix atio n an dsta in ing of g ly cog en m ay b e inad equ ate , second ,if the g lycogen w as ad eqcm ate lv fixed , then theacids used fo r deca lcifica tio n m ay- be rem ovin git, o r th ird , it m ay - be nec essary fo r a m in im umam ount of g lyco gen to b e p resen t in the tis sue sbe fore it can h e dc m o tis tra ted h istoch em ica lly(1 ). The fir st o f th ese h)o ssil )ili ties has b eencomisid ered elsewhere (9). This commun i c a t i o nd eals w ith the a ction o f de calcify im ug agen ts o nglycogen in l ivers.

P rev iou s pe rtin en t pub lic atio ns in clu de th efo llow ing : S cha jow ic z and C abr in i (6 ) h aved iscus sed the e ffec t o f tu itr ic a cid , hy -droch lor icacid an d fo rm ic a cid :20 sod ium c itra teeq ual pa rts, o n th e h is tochem ical beh av io ur o fgly co gen in bo ne an d ca rtilage . Th ey poim ut ou ttha t h )erio dh ic- ac id Schiiff po sit ive su l)s tan c-tSum ud erg o li ttle m odif icat ion , exc ep t for g lyco gen ,w h ich is qu ick ly los t excep t in cartilage cclls .S ch iajow icz an d Cabrin i a lso teste d th e effect o fd iso d ium e thy len ed iam ine tetraac etate (ED TA ),at pH 7 .0 on hiisto chem ical re act io tis in bon e ,c art ilag e and kid ney . C oncern ing g ly-cog en , th eresu lts w ere th e sam e a s befo re . L oe (3 ), in h isstu dy on bone , fix ed tissu e in l0 fo rm a lin for4 8 h ours I)efO re cleca lcify in g in EDT ; itt 1) H7 .2 . .\lth ough period ic ac id S ch iiff pos itive m a-te ria l w as se em i in the cy top lasm of os teo b la sts,it is tio t t- lear w h eth er thm c au tho r con sid lers th isto b e g lycogem i or n o t.

M ETHOD s AND M ATER IAL s

Tw o exp erit iien ts w ere pe rfo rm iie(l to test tim eeffect o f dec alc ify ing so lu tio ns on the g ly co genconten t of rat liv ers (S pragu e-D aw ley stra in ).

1. A liver w a s ta kem i from a 1 m iiomm th old m im Icrat, d iv ided in to 4 p iec es and l) laced l itmm mned i-ate ly ill ac etic ac id alc oho l form iialin an df ixed fo r 2 4 hours . The pH of th e fixat ive w as4 .0 . A fter fixat ion on e p iec e of t issu e wa sproce ssed in i tiie d ia te ly as describ ed e -v ious ly (9) and sect ion s cu t at 6 Theyw ere s ta in etl b y the pe rio d ic a cid Schif fre action . O f the rem ilain ing th ree p iece s ofliver , o ne wa s p lac ed in to tr ichb orace tica cid at pH 0.6 , an o th er in fo rtn ic a cid an dsod ium citra te accord ing to the form m m laeof M o rse (5), a t. pH 2 .5 , a no the r in ED TAadjuste d w ith N aO H to pH 7.5 .

2 . A six w eek old nm a le ra te k ille d an d th eliv er ca refu lly sl iced in to p iec es ap rox i-m ate ly 3 m m th ick b efo re he itm g fix ed inac etic acid a lco ho l form ahin for 6 h ou rs, 24hours, 48 hou rs am id sev en day s.

The spec im iie tm s w ere d iv ided in to th reegro ups . T he f ir st g rou p wa s used as contro lsan d fo llow ing fixat ion w ere p roc essed asab ov e. A tio th er gro up w a.s l)lae ed in E I)TAa t 1 )H 7 .5 for 7 days . The las t g roup w asp laced in fo rm iiic a cid and soch itm m citra te ,a s in the p rev iou s ex pe rim ent , fo r 7 d ays.

c o ti tro ls, a k nown p erio d ic acid Schif f po si-t ive slide w a s alw ay s pa ssed thro ugh (lie so lu t ionsat. the sam e titne as the expe rim ental s lide s, anda slide from each experim en ta l g roup w as digestedin ch ia .stase befo re sta in ing (4 ).

G ly cog en w as es tim ated in th e sec tion s as 0,+ , + + , + + + , and + + + +. No tio te wa s mmiak eo f the den sit , distribu t ion , o r degre e o f po lariz a-t ion , as th is had b een con sid ered prev io usly (9 ).

REsULTs AND DI5CUSsmON

T he firs t req u irem ent for dcm on stra tim m gg ly co g em i in tissu es is th at i t m u st be ad led lu ate lvf ixed in the c ells . It w as found prev io usly th atfo r rou ititie roc ess itig ac ctic a cid a lcoho l fo rm ah iti


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TABLE IIPrese rva tio n o f L ive r G ly cog en a fter 7 D ay s in

D iffe ren t D eca lc ify in .g So lu tions

A ll sec tio ns show n s tained by period ic ac id S ch iff m etho d , pho to graphed a t 2 50X .FIG . 1 . C ontro l rat liv er , fixed seven day-s in ace tic a lcoho l fo rm alim i

F iG. 2 . R at l iver , fix ed six h ou rs in a cetic alc oho l form a lin , lef t s ev em i d ay -s in E I)TA so lu t ion .F to . 3 . R a t liver , fixed sev em i days in a cet ic a lco hol form alin , left. sev en c lay s in E1 .)TA solu ition .

Ftc; . 4. Ra t liv er, fixed seven days iii a cetic alc oh ol fo rm alin , left in form ic ac id an d so dium citra teseve r d irty s.

Ftu. 5 . R a t live r, f ix ed iii ac etic alc oh ol form alim i fo r tw enty --fo um r houm rs , the tu p lac ed in 5 ( trich lo r-acetic ac id fo r sevem i clay s .

FIG . 6 . A sec tion o f rat live r, fix ed in a cet ic a lco hol fo rm alin for tw en ty-four hou rs, then p la ced inE l)TA so lu tion fo r seven days. T he s ize of the sp ecim en w a.s no t co n tro lled . N otic e the p erip he rald ens ity o f g lycog en .

Fm G . 7 . A no the r i llum stration o f the sam e sec tio n as F igu re 6 , taken d eep er in the sec tion , show ingscat tere d g lycogen .

70 0 J. R . TROTT

TA BLE IP re8erva t ion o f L iver G lycogen A fter D iffere n t

T im e s o f F ixa tio n a nd M eth ods o fD eca lc i fica tio n (7 D a ys)

F ixa tion (A ceticAlcohol

Formalin)

D ecaic ify ing A gent (7 d ays)

Contro l EDTA Formic acid

6 hou rs +++ +++ 024 hours +++ ++ 048 hou rs ++++ +++ 0

7 day s ++++ ++++ 0

Solution pH Glycogen

Contro l5% Trich lorac etic A c idFo rm ic A c id & Sod ium C itra te

(Itiorse, 1945)ED TA .

0. 62. 57. 5

++++00

++ +

g av e th e m ost sa tis fac to ry resu lts in rat liv er .H ow ev er, lo nger f ix ation m ig h t b e des irab le fo rtissu es tha t a re to be (lec alc ifie d .

F rom Table I , w here the siz e of sp ecin ie n w ascon tro lled , the leng th of fixa tion d id no t app earto hav e any great bea rin g on the am ount o fgly cog em u lost du rin g the seven day s in th e EDTAso lu tio n (F igs . 1 , 2 , 3 ). It w ill he seen tha t fo rmica cid -so d ium c itra te rem ov es al l the g lycogenfrom the tissue as e ffec tive ly as a d iasta se co ntro l(F ig . 4 ). S im ilar resu lts w ere o i)ta ined from thefirst exp erim ent (Table II) , where th e size ofthe sp ecim tn w as tio t co n tro lle d . H ow ev er,there w as a ten den cy fo r the g lycogen in thesec tio n to h e un evenly d is trib u ted (F igs . 5 , 6 , 7) .Deep e r areas show less g ly -cogem i tha tu tho senear the tis sue su rface ; th e 3+ figure d oes no t

re flec t th is. T hu s it appears d es irab le no t to h av espe cim ens th icke r th an abou t 3 m m if com p le teand ad eq ua te fixa tio n of g lycog en is to o ccur .T he len g th o f fixa tio n does no t appear to m akem uch d iffe ren ce, as c an b e se en from Table I.I t is in te res tin g to o bserv e tha t even though th e1) H of the fixa tiv e is 4 .0 , g lycog en is still reta ined .

S cha jow icz & C ab rin i (7 ) sug gest tha t the reis a cap su le a rou nd th e cart ilag e ce lls w h icha llow s g lycogen to be reta ined in the cell du ringsu bsequent d ec alcifica tio n procedu res. Thisappears un like ly , because if the cap su le a llow sfree access o f fixa tiv e to the ce ll, d eca lc ify ingagen ts sho u ld a lso have access . F rom the tw oexp erim en ts th at a re repo rted , and from pre-v io us w o rk , it w ould app ea r tha t adequa tef ixa tion , fo llow ed by th e u se of ch ela tin g ag en tsfo r the deca lc ifica tion of bon e and cartilage , a reth e ne ces sary p rerequ isites if gly cog en is to bed em on stra ted a t a ll. It is dou b tfu l if L oe (3 )used the b est fix ativ e fo r the d em on stra tio n o fg lycog en , even thou gh h e used EDTA at a pHof 7. 2 f or d e ca lc if ic a ti on .

S UMMARY

It ha s been sh ow n tha t g lyco gen can be ad e-q ua tely re ta in ed in rat live rs after fixa tio n w ithacetic a lcoho l fo rm alin fo r 6 hou rs, 24 h ours ,48 hou rs o r 7 c lay s, fo llow ed by treatm en t w ithc liso cliu tn e thy henec liam in cte traa cetate ad justedto pH 7 .5 . F orm ic ac id -sod ium citra te o r 5%tr ich lorace tic ac id so lu tio ns rem ove th e g lycogenfo llow in g fix atio n .

A cknow led gm ents: This w ork w as m adepossib le by a gran t from the N ationa l R esearchC ouncil o f C a tua cla and! the au thor is g rate fu lfo r th e ir su pport. H e is a lso m uch in deb ted tothe tech n ical adv ice o f M iss M . M elv ille of theD ep artm en t o f P atho log y , F acu lty d)f Med ic i n e ,U n ive rsity of M anitob a. T he tech n ica l w ork w asdone by - M rs. D . H art, N .R .C . labo ra to ry tec hn ician , an (l ty p in g b y M iss V . Ree s .


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70 2 J. It . TROTT

1tFFhh(hNCl AI. KCGLER , J. H . AN t) 1 L K IN 5ON , W . J . (. A

re lation h)e t w ee tm to t a! g lyco gem i com item it o f oxm yocard im ttm i am md its h i isto che tm iica l d etn om m -s t rat io rt . .1 . H is to eh em . (y to ch em . 7: 398-402 ,1959.

2 . L Im ,l.mm H . I). Hmstopa t lmo log ic T echn ic a ndPrac t ica l His toehemis tr t j . B lak is ton , N ewY ork , 1 954 (P 35 .

3 . L os , H . B om m e t i ssue fo rtu ia t ion . .1 et a Odun t.Scand . 17 : 311-4 27 . 19 59 .

4 . M UM A N Is , J. F . A . AND M OW RY , H . W . S ta in ing. l le thods, Histological a nd 11 i s tochemica l .Paul B . H o eb er Im ic., N ew Yo rk , 19 60 , (p .126).

5 . M om ts A . Fo rm ic a cid-sod ium citra te (Ic -calcificat io mm a mid bu tt y l a lcoho l d eh ydra t iomm

of tee th am id h on es for sec tion ing itt l)arathin.J . D en t. Res . 24 : 1 43 -15 3 , 1 945 .

6 . S ct tA Jo wt ( Z, F. AN D CABRmNI , H . L . The e ffe ctof ac ids decalcify i tm g so lu t ion s) am id emm zy m neso tt the h isto ch em ical b ehav io um r o f i)one amidcartilage. J. H is tochem . ( y toch em . 3: 122-129,1955 .

7 . Se tts ,jow tc z , F . AND CABR IN I, R . L . C helatim m gag ents as h isto log ica l a nd his tochem icm uldeca lc i f ie rs . S ta in T echno l. 3 1: 1 29 -1 33 , 1956 .

8. TROTT , J. H . A n im ive stig ation in to th e g hycog em mconm temm t of th e g ing ivae . Dent. P ract it. 7:2 34-24 1 , 1 957 .

9 . TR 0TT , J. H . A n eva lua tion of m e tho dscom nm iuotily used fo r th e f ixa tio n am id stain im igof ghy co gen . J . H istochem . ( y to ch em . 9 :7 03 -7 10 , 1 96 1.

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the presence of glycogen in rat liver following in vitro processing in decalcifying agents