From the ( ; q to- a n d Hi\tochemical l.ahoratorg, Department o f Anatomq. ITiiiversitj of Copenhagen (Prof . Haraltl Okkels) and the Neurop~thologica l Idahorator>. I<ommmuneh~is~) i ta le t , Copenhagen (‘Torhen Fog, hI.1). ant1 Svcntl Petri , h1.D.).

Inwstigation of tissucs for thrce hydrolytic enzymes : alkaline and x i d i)ho\l)hatasc, antl non-specific estcrasc, is now widely used in his t oc h c in is t r y . How cvc r , t hc g re a t in a j or i t y of c w z ym c stud ics on th c central ncrvous system (CNS) have hecn pcrforincd on aninial tissues i cf. review by Colmanf ( 1961 ) 1 , and only a fcw on inan. The distribution of thc thrce namcd cmzynics in thc adult ccrebcllar cortex has been investigated 1)y ,Schi/‘f(.r e t aZ. ( 1962) , that of the acid phosphatascs by I f ’o f f r , t nl. ( 1943) and . l o s e p h y i 194!1), and thc similarity of oecurrcnce o f this cnzyinc and lipopignient has hccn demonstrated by Stecn Olsen & Petri (1963) . On a n autopsy seric3s Fricde (1962) illustrated the d i s t r ih t ion of I)€“-diaphorasc in thc human hrain, including the ccrcbcl I uin.

I<nzyrnc histochcinical studies on foctuscs arc of interest in clucidat- ing thc hiochcmistry of thc processes of diffcrcntiation as well :IS the pathogcncsis of congt,nital disvascs and thr mcdianisrn o f thc effect of teratogcmic factors. ‘Thcrc has hccm an abundance of enzyme studies on the morphogcncsis of the CNS in various animals, mainly the rodcnts i int. al. Friedc 1957). Little w o r k has hccn done on human foetuses. From 1950 1057 :in Italian group, Rossi ef al. ( 1 9 5 7 ) , studied 19 foetuses for alkaline* and acid phosphatascs antl non-spccific cstcrascs, h i t they rtportcd no cytological details antl very little on the CNS. McKng et nl. ( 1955) h a w publishcd “histochcmical horizons” of a few, very early fovtal stagcs studictl in rtspcct to the nainctl enzymcs and 5-nuclco- titlaw. No continuous morphogcnctic cnzyine studies arc on record.

I t ha4 previously heen claimtd that autopsy material is applicahlc for thc investigation of hydrolytic cnzyitics. Giissnrr ( 1958) used the azo dye tcchniquc on human autopsy matcrial, but did not include the CNS. A review on other studies of this kind was givcn hy T.nzc~rns r.t al. ( 1962) who dcscrihcd enzymatic changcxs in thc. rabbit cc~rcbellum

Aided hy grant5 from ‘The Association for the Aid of the Crippled Children. New York. antl from the Danish Science Foundation.

124

trcatctl “ a s human autopsy matcrial might h”. Four hours after death the rabbits \vcrc’ placed in a refrigerator. Twenty hours later tissues wcrc r cmovd and invcstigated for acid phosphatascs, adenosine tri- phosph:it:tscb, and tlchydrogcnascs. The cxperimcntal conditions are not tlircctly coinl)arahlc with the usual conditions bctwccn death and autop- s y . The authors found marked cytochcrnical changes in the Purkinje cell\. Thus, the quality of cnzynic studies on autopsy material is in tlou h t .

Prosr.n t In ups t iga t ions.

’I‘hc ohjcbct of thc present study \vas tno to ld :

( 1 ) t o investigate the optimum conditions for histochemical detcr- minations of alkaline and acid phosphatascs as well as non-specific cstcraw in human tissuc-including autopsy material--by a tech- nique as simple and rcproduciblc as possible, and

( 2 j to apply such a mcthod to the study of changes in the cnzyrnatic pattern during the dcvcloprnent of thc human ccrehellum.

K r 1 j : T h e cxperimcntal stutlics w‘crc performcd on foetal material stained for the threc cnzymc’s. Interest was ccntred on thc following f:ictors :

( a ) Comlxirison hetween thc rcsults when using thc Gomori and azo (lye techniqucs rcslwctivrly for demonstrating phosphatases i n frcsh and autolyscd tissue.

( b ) Influencc of autolysis upon the cnzyinc activity, an attempt hcing niatlc to reprotlucc thc conditions during the period clapsing from dcath to autopsy as accurately as possible.

( c ) T h c decrease in cnzynic activity in fixcd, unstained tissue storccT in it refrigerator.

K c ‘2 j : The changcs in thc cnzynic pattern i n the coursc of morpho- gcnc3sis arc’ illustrated by a description of a few typical stages, while the dynamic findings arc summcd up in a small numhcr of items. Correla- tion of tho cnzynie findings with other cytochcrnical findings will lie puhlishctl in a subscqucnt paper.

M a I fir ia I .

The material consists exclusively of human tissue, partly from foetuses and partly from infants who died during the neonatal period. Hy the time elapsing from the cessation o f the heart beat until t h e removal of the specimens, the material may he divided into 3 grcinps:

1 ) 1:resh material from 21 foetuses having the followirig crown-ramp lengths ( c r’). measured in mm prior to fixation: 16, 26.5, 39.5. 42. 54.5, 62. 64, 65, 72, 86.5,

91. 111.5, 118.5, 119, 128. 129 ( twins) . 145, 151, 155, 186. 230. In mens t rua l age the material represents t he period 7 to 2 4 weeks. The mat 11 was obtained f rom foe- tuses removed from t h e uterine cavity hy Caesarean section in the course of sterili- zation ~)rocedures, ant l the specimens were removed and fixed f rom 5 to 30 minutes later.

2 ) .Autolysrd mater ia l f rom 9 infants, born prematurely or a t term, who died in the neonatal period. Thc tissue was removed 12-24 hours after death. From a1)out 6 hours after death the bodies were kept refrigerated. On gross and microscopic exa- mination the CNS showed no pathological changes. By weight, the infan ts were di- strihutcd as follows: 1150 g, 1450 g. 1750 g. 2050 g, 2350 g, 26508 g, 3050 g, 3200' g. 3550 g, anti thus represent the 6th to 10th months of gestation.

3) Artificially autolysed mater ia l produced by let t ing foetal tissue from various stages s tand unfixed at room temperature u p to 6 hours after cessation of t he heart heat; thereupon half of t he tisyue was stored in a refrigerator at 0-4" C, while the other half was stored at room temperature. On both series enzyme studies were per- formed immediately and a t t he entl of 3, 6, 12, 24, antl 48 hours after death. A to ta l of 8 cerehella were examined.

Fixed fresh and autolytie tissue was stored in a refrigerator, partly in gum sucrose and partly sectioned and mounted on slides. At intervals of some weeks up to 6 months after t he removal of t hc specimens, sections were studied to assess the en- zymat ic act ivity.

Fix11 t io n . The standard method u a s fixation immecliatclq- a f tc r removal of approx. 2 m m

thick tissue hlices in ice-cold, 10 per cent neutral buffered formalin as advocated by Lil l ie (1954) . A t the entl of 24 hours the sections were transferred direct t o gum sucrose (0.88 m l mol sucrose containing 1 per cent gum arahic), changed several times, as the first change of gum sucrosc had a strong smell of formalin. The sec- tions were stored all t he t ime at 0-4" C except for those which were used in the experiments including long-term storage a t room temperature.

l'rc,pcrrot ion.

The sections were cut o n a n ordinary freezing microtome (Jung, Leitz), w i th pro- nounced cooling of t he knife, in a thickness of about 1 2 P and were mounted directly on slides. In the case of small foetuses serial sections w e r e made.

Mf,f hods .

1 ) The calcium-cobalt method for alkaline phosphatasc (Gomnri 1952) : Sections were incuhated for 1 to 1 ?4 hours a t 37" C. Sofistrate 8-glycc.rophosphate. pH about 9.4.

2 ) The modified coupling azo dye method for alkaline phosphatasr (Penrsc, 1961) : Incubation at room temperature and pH 9.2 wi th a-naphthyl phosphate as subs t ra te a n d Fast Itetl TH as tliazotate.

3 ) The lead nitrate method for acid phosphatase (Gomorz' 1956) : Using P-glycero- phosphate at p H 5.0 as substrate. sections were incuhated f o r %-1 hour a t 37" C.

4 ) The naphthol-.AS-HI phosi,hate method fo r acid phosphatase (Llnrsfonr 1958) : Incubaticin at p H 5.4 for % -1 ?h hours at 37" C. Correction of t he pH was often necessary. Am sa l t s : Bed Violet LH and Fas t lied 3 GJ,.

5) The naphthal-AS-acetate method for estcrase (Gomori 1952). A m salt : Past

126

Red RC. Incuhation a t room temperature for ?LL to 1 hour at pH 6.8. The naphthol AS-acetate was dissolved i n acetone, making a stable stock solution.

Control section were incubated after addition o f fluoride or wi thout substrate. In t h e azo dye method- -which were the s tandard mcthods- -the sections on the

slides were placed, a f te r a i r drying, horizontally on two spatulas in a shallow tray, a n d the mixture of snhs t ra te and azo Yalt was dropped on to them direct from a funnel furnished wi th a filter. A l l sections were counter-stained wi th hlager's haemalum except fo r those which were stainctl b y thc Gomori technique and those wh i rh were intended for photography. None of t he methods required laboratory equipment other t h a n a freezing microtome, analytic halance, and incubator.

A s a routine, one section of each specimen was stained wi th PAS for dcmonstra- t ion of glycogen. I n th i s respect coating wi th celloidin proved of importance to the accurate intraccllular localization. In addition, one section o f each specimen was s ta ined wi th Einarson's palloc~anin-chrom.7lum for assessing the nuclcic arid ~ 0 1 1 -

tent in the rcacting cells.

KESI'I,TS

Cornpar ison of Gomori and Azo Dyc i V l i ~ l l i o d s on F m s h Fixcd 1i.ssiici:

I r i a pi ) ru i sing :in cnz y 111 c his t oc h em i c a1 mc t ho d , t h c eva 1 u at io t 1 shou Id not he basctl mcrcly ulmn the clearness of thc histological picture, but also upon thc total impression of the following factors: (1) Intensity of rcaction, ( 2 ) localization of reaction product, and ( 3 ) size and distribution of granules.

'I'hc intensity of the Goiiiori reaction varies inorc than tha t with the :izo dye technique. With the latter there is, for instnncc, maximum or sub-rnaxiinum coloration a t thc sites of acid 1)hosphat:iscs in all niaturc Purkinjc. cells, while in corrcsponding sections the Gomori method shows a 1)ronounced variation in the stainability of the same cells.

In localization too thcrc are diffcrenccs bctw ecn the Gomori and thc am dye mcthods, especially in staining for acid phosphatascs. T h c main diffcrcriccs arc staining o f nuclei anti certain proccsscs by the Gomori method nntl staining o f neuropil and whitc matter by the azo tlyc rncthotls. The differmcc.s arc presuinably tluc in part to a variation in thc scnsitivity o f thc methods nntl in par t to a dificrcnt diffusion tcn- dency of thc final reaction product: Nuclear staining by the Goniori nicxthod is obscrvctl only in cases of over-incubation nnd is considcred an artcfact. Thc processes, cspccially the tangential fibres of the haskct cclls, sonic.tiincs stain clcctivcly in thc vicinity of a Purkinjc cell which shoivh rnnximal coloration. In the niolccular layer too the Gomori mcthotl often shows inore riiarked staining of fibres. Staining o f pro- ccsscs is capricious, howe\.cr, and somewhat rcrninisccnt of the results of silver staining methods which arc also based on precipitation. Staining of ncuropil and-in cascs of over-iiicuhation- of white matter, also in the non-niyclinatcd state, iiianifcsts itself as diffuse tinging in

1 2 i

t h e azo dye Incthotls. Serial scctioris o f foetuscs staintd for cnzynics show a distinct reaction in peripheral nerves i n contrast to the cntircly negative inescnchymc---also a t a normal incubation period. This might indicate tha t the rcaction of axons is a real one arid that the ncurofibrils or the surrounding cytoplasm contain acid phosphatascs.

With thc azo dye methods thc granules arc more delicate than with thc Gomori ttchniquc, but thcrc is a clcar variation in thcir sizc. Thcy arc 1argc.r in Golgi cclls and in basket cclls than in thc Purkinje cells. The dclicatc granules givc a more physiological picture than the frc- qucntly coarsc Gomori prccil)itations, although this docs not justify any statcincnt rcgarding the corrc.ctncss o f thc picturc.

Aulolytic CIianges.

Sincc, the cnzymc rncthods arc* not quantitative, an asscssincnt can comprise only the coarscr diffcrcnccs in intensity and localization. (Table 1 ) .

T A H L E 1 .4rrtolyfir Chnrii/c,s: dr i t l t'hosphutcrsrs S t n i n i . t l b y t h v d z o I)yr l o c h n f q r ~ o .

~~

Immediately after removal of specimen ...........................

3 hours a t 20" ........................ 6 hours i l l 'LO" ........................

12 hours at 20" ........................ 24 h i ~ u r s a t 20" ........................ 48 hiiurs at 20" ........................

6 hours a t 20' + 6 hours at 1" 6 hours at 20" + 18 hours at 4" 6 hours :it 20" + 12 hours at 4"

+++ + -t +t

+--+ + -t +

++ f t +

- (+I (+ ) ++ ++ ++S (f) ++ ++

During thc first 6 hours post rnortem thcrc are, ticspite storage at room tctnpcraturc, only niodcratc, changes in the enzyme pattern. Hcrc- after, i f thc spcciincns are still stored a t ahout 20" C , thcrc will be a pronounccd diffusion and decrease in enzymatic activity, and 2-2 hours post rnortcrn the tissuc is greatly changed, thcrc hcing only a faint rcaction in a few cells. Storage in thc cold extcnsivcly reduces the dccoriil)osition, spccimcns placcd in thc refrigerator aftcr 6 hours a t 20" C showing only slight changcs aftcr 18 hours' refrigeration.

Di1fcrcncc.s Iwtwccn f rcsh tissuc and tissuc stored for 6 hours a t rooni tcrn1)crature and 18 hours in a rcfrigerator:

( 1 ) In thc lattcr the ticndritcs do not stain s o far pcriphcrallg, and ( 2 ) the granulcs clump.

128

( 3 ) A number of the cells which showctl merely sparse reaction a t the sites of the enzymes in frcsh tissue are now completely ncgativc. 1 his applies particularly to certain cell groups, e.g. small ner\ e cclls. In other words, ihcrc is a fairly niarltctl difference in thc sensitivity of the various types of cells to autolytic action.

r 7

A s rcgards tissue storcd for 23 hours or longer a t room tcrnpcrwturc :

i 1 ) The changcs arc similar to those in spccimens placcd in the rcfri- gcrator a t the end of 6 hours and kept thcrc for 42 hours, ( 6 + 1'1 aftcr death) but niore pronounccd.

( 2 ) There arc typical autolytic nuclear changes, i.e. pyknosis a11d karyorrhexis.

( 3 ) Only the largest, arid in fresh tissue most positively reacting cells still show soinc~ tracc of reaction.

( 1) 'There :ire marltcd artcfacts due to sprcatling, ill-defined limits, and cx t re In el y di f f u sc s ta i 11 i n g of th c t is s u c.

Thcrc is a difference in the scnsitiaity of the various enzymes to autolytic action : Alkalinc and acid phosphatases appear to be more resistant than AS-cstcrase whose activity has distinctly decreased a t thc cntl of 12 hours. Acid phosphatases in autolytic tissue apparcntly givc more diffusion than other enzymes.

On coiiiparison of the rcsults of thc azo dyc techniquc, with thosc of the Gornori technique aftcr autolysis the latter appcarcd to give morc distinct pictures, but with inorc capricious distribution o f the reaction products. The clearness is probably duc to thc failure to stain a number of lyo-enzymes whose activity has dccr cascd to such an extent that thcy arc unablr to s tar t a precipitation. This may also bc the explanation of the somewhat capricious result of thc reaction. After autolysis, the> valuc of azo dyc nicthods is pcrceptibly reduced by a tcndcncy to diffusion wh ic h affords indistinct 1, ic t u rcs . 'I'h e i ncu h a t ion ti m c must not 1) e too long.

1nfiucnc.e of Storrige Period on Enzymatic Activity. (Table 1 1 ) . Refrigeration of frcsh fixcd tissuc, partly spccimcns in gum sucrose

and partly scctions mountcti on slidcs, did not perceptibly rcduce the cmzyrnatic activity until 2 or 3 months aftcr the remoaal of the speciintns. Thc scctions 1)rcscrvctl thcir activity longer, up to 6 months aftcr thc rc- moval. Scctions in a c l o s ~ d containcr werc preserved 1)ctter than un- covered scctions, possibly hccausc thc latter undcrwent drying.-Auto- lysctl matcrial was more rapidly rcndcrcd inapplicable, so that it had to be scctioncd soon aftcr the rcmoval. In the forin of svctions it kcpt longcr, but not for longer than 4 wecks.

129

Immetliat el) + I t After 1 m o n t h t- + After 3 months + After 6 months ( + I

+++ ++ ++ +

ItEStJLTS O F MOI~PllO(;EKETIC STITUY

'I'he tlctuiletl description o f the tlcwlopnient of the ccrelwllurn 1)uh- lishcltl hy Jnkob in 1928 will form thc basis of the following description o f the changcs in the cnzymc pattcrn in thc various pcriods. (l 'ahlc 111).

Epentlq-ma1 cell

Migrat ing neuroblast

Serve cc~l ls of central nuclei

l'urkinjc cell4

(;olgi cells

Ba\kct cells

(;lia cellz o f white matter

Hergmann glia

Perivascnlar cells

Seuropil o f molecular layer

\\'bite mat te r

+++ ++ + (+)

+ ++ + + - - - I + +I+

+ ++ (+) ++

(+)

? +

+ ++ +++ ( + I

?

(+)

+ -1 c + +++ +++ -1

++ ( + ? I +++

( + I ?

( I ) In the 16 mm embryo (about 37 days) a frontal section revealed a small cerebellar anlagc. Likc the entire neural tube it was charac- terized hy activity in the matrix with numerous mitoscs in the c~pcndyinal layer. Between the newly formed cells the epentiymal cc~lls stretched out their processcs peripherally. In the ependymal cclls there was cspccially in their luminal part-a strong reaction to staining for alkaline and acid phosphatascs as well a s non- slwcific estcrascs. (Fig. 1 ) . Whilc alkaline phosphatases were found only in a zone towards the cpcndymal surface (Fig. 2 ) in a site

130

I.’i<g. I . Kpendyma. 16 m m cmhrytr: Acid phosphatases in t h e eliericlynial cel ls and thcii-

proccssea. Ko staining of the ne\vly formed neurohlasts i n 1wtwec.n.

PI</. 2. Epc.nd> rnal l ining of 4th ventricle: Alltalinc. phosphatase5 in the luminal part of the

cj)endFmal eells. Counter-.;taining of nuclei wi th haematoxylin. Foetus o f a c-r of 62 mm.

131

\\ l iorc~ PAS staining rewaled a pronounced accumulation of glyco- gvn -the other two c’nzymc\ were also visible in 1)roccsscs hctnccw the non-reacting ncurohlasts.

( 2 ) In thc proliferative phaw from the end of the 2nd month to the riiicltllc~ of thc 5th month ( c r from 26.5 lo 145 iiim, a total of 16 hrains) the following findings wcrc noted: ( a ) A market1 increase in the volume of the cerchcllar anlagc with thickening of the mantle 1ayc.r. ( 1 ) ) Dccrcasctl activity of the matrix with a parallel rcduction in the content of :ill thrcc enzymes in the ependymal cclls. IVhilc the reaction to alkalinc 1)hosphatasc staining in the luitiinal par t of the epcnt1yni:il cclls mas almost completely lost towards the cnd of the

Fiy . 3 . Diffcrentintion of nciirol)lasi\: Acid phosphatasc\ in bipolar neurohlasts arid cpen-

dymal cells. Foetus of a c-r of 128 mm.

F i y . 4 . Accumulation of neurohla5ts superficially in the maiitle layer: Gomori method for

acid phosphatases. Foetus of a c-r of 145 mm.

pcriotl there persisted, in all stages, a pronounced reaction in the vascular endothcliurn in all parts of the CNS and in the meninges. ( c ) Occurrence of bipolar ncuroblasts throughout the mantle layer, in groups as \ w l l as solitary. In addition to their shape, these cells are charactcrizc~l by their cytoplasmic hasophilia and their content of acid phosphatascs which appear first in the form of delicate granules in a zone surrounding the nucleus in order gradually to f i l l thc entire cytoplasm. (Fig. 3 ) . ( ( 1 ) Some of these ncuroblasts accumulate in a superficial layer which is sc,parutcd from the external granular layer hy a rclativcly accllular zone, thc molccular layer. (Fig. 4 ) . In the layer of ncuro- hlasts --tho latcr Purkinje laycr-thcrc is, in addition to thc cells

1 3 3

Fig. 5 . Matura t ion of the neurohlasts dur ing the prenatal period: Acid phosphatases in the Purkinje cells, vessel wall, perivascular cells, some Golgi cells, a few hasket cells.

and astrocytes (bottom left-hand corner).

containing :icitl phosphatases, a more diffuse reaction of neuropil to staining for estcrascs and acid phosphatascs.

( 3 ) After the iiiidtllc of the 5th month (c-r cxceeding 150 inrii, 4 brains stiidicd) there is a maturation of the neurohlasts, a t the site of the central cerebcllar nuclei and-somewhat later (c-r 230 inin-in thc superficial neuroblastic layer: The cells assume a iiiore tri- angular shapc, their basophilia is increased, the nuclei hecornc larger and more vcsicular. At the same tirnc, there is a marked increase in the content of acid phosphatases which now fill the cntire cytoplasm with dclicatc, densely arranged granules visible also in a few places in the proximal par t of the dcndritcs. Staining

134

F i g . 6 . P a t t e r n of acid phosphatases in a full-term in fan t : Pu rk in j e cells, hasket cells, Golgi

cells. Occasional s ta in ing i n the Ucrgmann layer.

for c \ tc raw\ gavc- -apart f rom the staining of the cytopla\ni of l ) r imit i \c n c n c cells- also diffuse staining in a rim in thc dccp part of thr molccular layer. Furthermore, a few pcrix ascular cell% take a d c c s p stain.

( 4 ) During the prcnatnl pclriod (wcight cxcwding 2000 g, 5 brains \ tu tl i c (1 ) th c rv :ire ( a ) maturat ion of Ihc nerve cells, the superficial ones gradually acquiring the shape charactcristic of the Purk in je ccll, a n d ( 1)) :ippc:irancc of gratiulcs upon staining for acid phosphatas,cs :ilw in thc srnallcr ner\.c cells: thc Golgi cclls and 1)aslict cclls. Morcwvcr, thcrc a rc a fcw granules in the glia cclls of thc white 1nattc.r. (Fig. 5 ) .

( 5) I n full-tcrm infants ( 4 brains c;tudicd) who havc diccl during thc n (10 n a t a 1 1 )c> r io tl t hc f o 11 owing d i s t ri h u t ion of acid 1) hos pha t a s c s i s w c n with the am tlyc technique: I n a few arachnoid cclls and lwri- v:i\cular cclls in the. nicningcs :I few coarse granules. No reaction

135

F i g . 7. Pattern o f esterases in a full-term infan t : Staining on15 ( i f Purkinje cells and their

dendrites. Staining of nucleolus.

in thc t.xtcrn:tl granular layer. In the molecular layer a few granules in cclls cvenly distributed throughout thc layer-possihly stcllatc cclls. In the tlccIwr part of thc molecular layer, a row of cells just pc r i 1, h c r :i I to t h e Pu r kin j c c el 1 s---- 1) resu m ah I>- h a s ke t cc 1 1 s- ~ -s h ow B numhcr of coarse gr:inules scattc.rcd in the cytoplas~n. ‘rhc Pur- kinjc cells cxhibit a pronounced cytoplasmic reaction cxtcnding into thc tlcndritcs. Rerginann’s glia cells show occasional granules in a vcry few placcs. In the internal granular layer the Golgi cclls give a fairly strong positive reaction, showing coarsc granules. (Fig. 6 ) . These granules arc-likr those of the\ basket cells-dwrlcer than the. granules of the Purkinjc cells. In thc granulc cclls thew is no reaction apar t from a few, scattcrcd granules to which we tlarc not attach any importance. 111 thc white inattcr there a r c distinct granuies in the glia cclls extending sonic way into the procvsscss. A slight, diffusc staining of thc ncuropil is not con- sidcrcd to hc a definite proof of true positivity. In cntlothelial cclls thcrc \vas in a few places slight granulation. A fc\v perivuscular cc*lls were highly positive. Thc c.stc)rascs were’ found in the sanie pcri\:nscular cclls and in Purkinjc cells. (Fig. 7 ) . Slight, diffuse staining of thc dccpcst parts o f thc niolccular I:iyer possibly represents diffusion artefact.

136

Al kaline phosphatascs 1vcr-c obsc~rvcd in almost all cndothclial cc.lls, also in thc rnrmingcs.

If “tlynainic principles” arc to he deduced from thc stages dcscrihrd above, a n ini~)rcssion may hc gained by regarding thc distribution of acid 1)hosphatases : The ncurohlasts arising froin the gcrin cells arc cn- tircly ncgativc., whilc the surrounding proccsses of cpcndymal cclls havc a high contcbnt of the enzyme. Gradually, the, rieurohlastic cytoplasm shows increasing amounts of. acid phosphatascs, first in thc largc ncrvc cclls and iinmcdi:rtcly beforc birth also in thc sinallcr ones. Thc glin cclls are latc in acquiring a contcnt of acid phosphatases.

DIS(:L~sSION

Since thc function of the enzyincs is still insufficiently known arid the cnzymc spccificity o f the methods far from clucidatcd, thc thcorcti- cnl rcflcctions based on thcse rcsults can only be hypothctical :

Alkalinc 1)hosphatascs play a rolc in the transport through nitin- hrancs of c.g. glucosc which is partly cinploycd as a source of cnergy and 1)artly dcpositcd as glycogen. The latter was dcrnonstratetl in sitcs with a high activity of alkaline phosphatase, i.e. ependyina and vessel w.nl l . The contcnt of phosphatascs places thc cndotheliuin among thc inor(’ active tissucs of thc (INS-- contrary to what inight 1)c dcducctl froin its povcrty of structure in the light inicroscope.

;-\citl 1)hosphatascs arc conccrned with thc maturation of neuroblasts, ant1 their occurrencc is parallel to cytoplasmic hasophilia, indicating their role in RNA synthcsis. Thc highest content of acid phosphatascs in newborn infants \vas found in those ncrvc cclls which must be prc- surned to possess the inaxirnurn protein synthesis : The Purkinjc cells :ind thc largc ncrvc cc,lls in the dentate nucleus.

There w ~ r c distinct differcnccs in the staining and distribution of granulcs after staining for acid 1)hosphatascs: T h e Purltinje cclls con- tained tlclicatc, bright granules lying throughout the cytoplasm, hut particularly dense around the nucleus. The granules of thc basket and Golgi cclls Lvcrc coarsc, tlarkcr, and more scattcrcd, accidcntally, show- in g an ac c u 111 u 1 at ion pe r i 1) h era 1 I y in the c y top1 as in. Scvc r al cs 1) 1 an at ions of this differcncc may be imagined:

( 1 ) A diffcrcncc in the intracellular localization of the enzyincs, e.g. bound to lysosoincs in one group of cclls, free in the cytoplasm in another.

( 2 ) A tliffcrcmcc in thc scnsitivity of thc cclls to autolysis and thereby in diffusion tcntlency. Purkinjc cclls arc known to be particularly vulncrablc to various noxae.

1 3 7

( 3 ) *4 diffcrcwce in the suhstancc dc.nionstratcd: Although the cells in both groups, in the investigation of unstained preparations, have no staining of their own, they may contain sinall quantities of lipo- fuscin in Lvhich the azo dycls may bc dissolvcd. However, nothing is known ahout the lipofuscin content of thesc cells in the foetus. 'I'hcsc problems are heing pursued at present.

Whether the demonstration of non-specific esterascs in the riiolcvxlar layer during thc prenatal period represents a sign of incipient synapsc formation cannot be decided without specific staining for cholin- estcrasc.

S I'M MARY

[ 1 ) In a material of human foetuses as well as infants who had died during the neonatal period, with an even age distribution, thc author studied allialinc and acid phosphatascs as well as non- specific cstcrascs in the cerchellum.

[ 2 ) Comparison between fresh material and autopsy material rcvcalctl that the latter is applicable. I t seeins to be important that thc bodies he refrigcrated as soon as possible.

( 3 ) Comparison of the Gornori and azo dye methods showed th. 'i t -~

especially in autopsy material-these two methods give slightly different results.

( 4 ) Alkaline phosphatascs arc demonstrated in endothelium and during the first 5 months in the luminal part of the cpcndymal cells where glycogen is also present.

(5) In the early foetal stages the acid phosphatases are localized to the ependymal cells and their processes, while later they appear in the nerve cells simultaneously with cytoplasmic basophilia. The tliffer- onces in the clistrihution of acid phosphatascs in Purkinjc, basket, and Golgi cells are discussed. This enzyme may be observed also in glia cells and in perivascular cells.

( 6 ) Esterases were found in approxiniatcly the sanie locations as the acid phosphatases, but more ample in the perivascular cells, less marked in thc small nerve cells and not present in glia.

REFERENC.ES

Burstortc:, $I. S. (1958) : Histochemical demonstration of acid phosphatasc with Naph-

Colmant , lf.-,I. (1961) : Ergehnissc der Enzymhistochemie a m zentralcn und periferen

Friedr , H. (1957) : Die histochemischc Iieifung cles Kleinhirnes der Ratte dargestellt

thol AS-phosphates. J. Sa t . Cancer Inst., 21 .523 .

Ncrvensgstem. Fortschr. Neur. Psych., 29, 61.

dni-ch das Vcrhalten der Succinodehydrase. Arch. Psychiatr., 196,196.

b’rir,t/ca, K . , B L. ,If. F/oniin(/ (1962) : A m a p p i n g o f oxitlati\re enzymes i n t h e h u m a n t)rein. .J. Scurochem., :J. 179.

G om u r i, (;. ( 1 9.52 ) : JI i cro scop i c His t och e ni i s t ry. Chicago 1 n iver s i t y 1’1.c 5 s . Coniori , C. (19.56) : I-liztwhemical mcthot ls for acid phosphatase. J. Histrichem. (:).to-

chem., %, 425.

Azi~f: ir l~stof fmcth~i~Ic . H is tochemie, I , 48. (;ijssii(,r, 1.2’. (1958) : Histcichcmischer Nach hydro ly t i scher I<nzytnc rnit Hilfe der

d / i cob , .4. (1928) : Das Klcinhirn. Handl iuch hlikr. Anat . .Rlenschen, G , 674. ,los(,ph;q, Zl. (1!)49) : Acid phosphatase i n t h e seni le hrain. Arch. Neur., 6 1 , 164.

Wnllncc,, G. W. Etlgctr, & H. W. I.o/k (1962) : E n z y m e local isat ion )ellurn a n d effect of post m o r t e m autolysis . .1. Neur(ichcm.. 9. 227.

I , i / / i r , H . I). (1954) : His t~ ipa th t i log ic ‘I’echnique and Pract ical Histcichemistry. Hlack-

McK(ry, I ) . G. , E . (:. .4drrins, .4. ?’. H(, r t i y ti S. I)trrizi<gor (1955) : Histochemical horizons

O l s r , n , S., Ly: (:. I’rtri (1963j : Histochemical 1,ocalisation of Acid Phosphatase i n t h c

I’otrrse, .i. (;. B. (1961) : Hist( ichemistry. Churchi l l Lttl., London. l iossi , F., (;. I’c,scr,fto, & E . KrcrIr, (1957) : E n z y m a t i c act ivi t ies in h u m a n ontogenesis.

. I . hiz t~ ichcm. cytiichem., 5 , 221. .Schiffc,r, I ) . , (J. ~ ‘ P S C O , 6 I,. I’irrzzrt (1962) : ( :ontr ibut ion t o t h e Histochemical Demon-

s t ra t ion o f I’hosphatases ant1 Non-specific Hsterases i n H u m a n Nervous Tissue. I’sychiatr. Neurol., Hasel, 144, 34.

Wolf‘, d . , F... .4. Ktrhnt, c t W . phatascs w i t h special reference t o t h e ncrvous system. Am. .J. Pa th . , 19, 423.

s tone (h. Inc., Philadelphia .

i n h u m a n embryos. Anat. Nee., 122, 125.

H u m a n (:erel)ellar Cortex. Acta Nerrr. Scantl., t h i s issue.

w n i t t r i (1943): 4 s t u d y of t h e d is t r ihu t ion of acid phos-

I~ece ivcd January 30, 1963. Pctvr Mryer , Ar.D., Depar tment of Anatomy, Universi ty of Copenhagen, 0.