In vitro growth of gomori-positive glia

BRAIN RESEARCH

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G R O W T H OF GOMORI-POSITIVE G L I A

ZBIGNIEW SREBRO* ANDANNA MACIlqSKA Department o f Btologv, Instztute o f Bwmorphology, Medwal Academy, Krak6w (Poland)

(Accepted September 8th, 1971)

I NTRODUCTION The so-called Gomorl-positlve glxal cells are characterized by the presence of cytoplasmic granulations strongly staining with G o m o r f s chrome haematoxyhn and aldehyde fuchsln following oxidation with acid permanganate This tyl:e of cell is present m the brains of most mammals investigated as yet 1,2,5,7,9,14,15,17, and in some submammahan species s,9 The most characteristic feature of the topographic distribution o f the Gomorl-pOSltlVe gha tn s t t u is their predominantly penventrlcular locahzatlon The Gomorl-positive granulations o f the perlventrlcular gha have been shown hlstochemlcally to be unusually rich m thlol groups u,12 We have reported recently in a prehminary communication 18 that Gomorl-posmve ghal cells can be found in m vttro cultures of foetal brain tissue of the mouse In the present report we present more detailed data on the growth of the Gomorl-positlve ghal cells tn vttro The results show that their differentiation and appearance in large numbers is one of the most characteristic features of m vttro cultures of mouse foetal brain tissue. The results further show that the hlstochem~cal properties of the Gomori-positive ghal cells grown m vttro are the same as those observed m s t t u The Gomon-posltive ghal cells m w t r o display a tendency to occupy sites in the cultures analogous to the penventncular layer tn s t t u METHODS Pregnant A K R female mice on day 18 of pregnancy, counting the day of sperm in vagina as day l, or pregnant golden hamster, M e s o c r w e t u s a u r a t u s , females on day 13 of pregnancy were killed with ether, the foetuses qmckly removed from the uteri, and brain tissue fragments of the foetuses excised under a stereoscopic microscope Smallfragments ofperlventrlcular bralntlssuefromthedlencephalonorrhombencephalon, approximately 2 mm × 2 mm, were cultivated m vitro in Lelghton's tubes using a modification of the method employed by Katuza and StefanlckaWlechowa a The tissue fragments were placed m a drop of cock's plasma coagulated * Present address Department of Biology, Medical Academy, Kopermka 7, Krak6w, Poland Bram Research, 38 (1972) 27-33

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SRkBRO A N D A MACIN~,KA

by the addition of a drop of 200/0 embryonal extract, on a cover shde The cover glass with the explant was immersed in a medmm composed of Parker's fired (82",,), calf serum (10%), glucose (1%) and embryonal extract (7%) This hquld culture medmm was changed every third day durmg the first days of culture but at later stages every fourth or fifth day only The cultures were fixed for histological examination after 8-30 da2cs of cultwatton Gomorl's chrome alum-haematoxyhn-phloxlne method following fixation m Bouln's fluid was used as a standard procedure For the detection of disulphide and sulphydryl groups the performlc acld-Alclan blue (PFAAB) of Adams and Sloleer and the DDD method of Barnett and Sehgman were adopted 6 The hlstochemlcal tests were performed on both fixed and unfixed material N-ethyl malelmlde served as blocker of sulphydryl groups m the control RESULTS

Ftrst two weeks oJ culture

During the first week of culture cells m~grated out from the explant formmg a monolayer These cells were of various morphological types The most common ones were ghoblasts characterized by large size and very large nuclei These cells had clear cytoplasm forming two or more short, strong prolongations The second type, also very frequently found, were macrophages Groups of neuroblasts could occasionally be observed The neuroblasts had long thin prolongations, dark cytoplasm, and round nuclei with large nucleoh Pyknosls and signs of autolysis were frequently observed Simultaneously, the central part of the explants underwent cell necros~s, cell debris along wxth numerous macrophages being present in the necrotlzlng parts of the explants Cell debris was ingested by the macrophages which frequently contamed hpofuscln At the end of the second week m vttro the explants developed a characteristic orgamzatlon, being transformed into defimtxve cultures Thlrd to fifth week of culture

Fig 1 is a schematic drawing of a definitive culture derived from foetal brain tissue explant The general form of the culture was circular due to the presence of a round central space devoid of cellular elements Thls central cell-free part derived from the necrot~zed central part of the original explant The culture proper could be divided roughly into 3 zones zone A which was the internal part of the culture, formlng its internal wall, intermediate zone B, and external zone C Zone A was formed by several layers of narrow, elongated cells closely adhering to each other These cells were the first to show Gomon-posmve granulations which occurred on the fourteenth day of culture The number of cells containing Gomorl-posmve granulations later steaddy increased, as d~d the amount of the Gomon-posmve material present m mdwldual cells At late stages of the cultures during the fourth and fifth week the majority of cells in zone A contained Gomon-posmve granulations (Figs 2, 3a, b) Bram Research, 38 (1972) 27-33

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@0 ¢ Fig 1 Schematic drawing showing the orgamzatlon of a culture more than 14 days old The central part (left) contains few or no cellular elements A, Internal zone composed of small, elongated cells closely adherent to each other Zone A is multllayered Most cells m th~szone contain Gomon-posmve granulations (stlpphng) B, Intermediate zone formed by a monolayer of heteromorphlc cells, generally of ghoblast type Many cells contain Gomorl-posltwe granulations C, External zone composed of loosely &strlbuted large cells of ghoblast type Cells m zone C show Gomon-pomtlve granulations only occamonally

The Intermediate zone B was formed by a monolayer o f polygonal or elongated cells, mostly of ghoblast type The morphology o f cells of that zone vaned considerably Frequently cells with Gomon-pomtlve granulations were observed (Fig 3c). The external zone C was composed of a monolayer o f loosely distributed cells of the ghoblast type They were generally very large, had clear cytoplasm and a very large nucleus Occasionally, cells containing Gomorl-pomtlve granules were observed (F~g 3d) A picture mmllar to that described above was observed with hamsLer brain tissue explants The differences f r o m what was seen m the mouse cultures consisted mainly m the number of the Gomorl-pOSltlve ghal cells In hamster cultures the Gomorl-pOSltlve ghal cells were much less numerous The Gomon-posltlve granulations present m gllal cells grown m vitro were strongly positive in both the performlc acld-Alclan blue method (Figs 2, 3a, b) and m the D D D procedure, indicating a very high content of sulphydryl groups Generally speaking, our method of m vttro cultwatlon of foetal brain tissue Brain Research,

38 (1972) 27-33

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Z SREBRO AND A MACINSKA

Fig 2 Zones A and B of a 28-day-old culture Performlc acld-Alclan blue without counterstmn The dark colour is due specifically to the Gomon-posltlve material Note the strong posltlvlty of the Gomorl-posmve materlal m th,s procedure and the large number of the Gomorl-positlve ghal cells m z o n e A × 500

is very suitable for growing the Gomon-posltwe ghal cells which in these conditions appear in very large numbers, the hlstochemlcal properties of these cells being the same as observed m that type of cell m s t t u DISCUSSION

Three points emerging from the present results should be particularly stressed (1) The appearance of the Gomorl-posmve ghal cells m m v i t r o cultures of mouse and hamster foetal brain tissue is a constant characteristic The Gomon-posmve ghal cells were observed in each culture which had been grown m v ~ t r o for more than 14 days, and at later stages they outnumbered other cell types (2) The regional distribution of the Gomorl-posmve ghal cells m the cultures was also very characteristic This cell type occupied sites analogous to the perlvenBrain Research, 38 (1972) 27-33

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Fig 3 a, b, Gomon-posmve ghal cells m zone A, PFAAB without counterstaln Only the Gomonpositive material ~s vlsuahzed In b arrows indicate very large Gomorl-posmve granules × 1100 c, d, Gomon-posmve ghal cells from zone B and zone C, respectwely Chrome haematoxyhn-phloxm × 1100

tncular layer of normal brains Gomorl-posltlve granulations m s t t u are most frequently found in periventrlcular gila and in ependymocytes, these same granulations m vitro being most numerous m cells of the Internal zone A bordering the cell-free central part This gra&ent of density o f the Gomon-posltlve ghal cells in the cultures may be an expression of a more advanced state of &fferentmt~on in their central part or it may be the result of d~fferences m nutrient concentration However, the most probable explanation is that the Gomon-posltlve ghal ceils tend to occupy sites s~mdar to those where they occur m s t t u Bram Research,

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SREBRO AND A MACINSKA

(3) The time of appearance of the Gomorl-pOSltive granulations m ghal cells grown in i,ttro is strictly defined, being 14 days in brain tissue explants taken from 18-day-old foetuses This means that the differentiation of the Gomorl-poslttve glial cells m w t r o IS considerably faster than is observed m Vtl,O G o m o r l - p o s m v e ghal cells were not observed m mice younger than 28 days a Although the cells containing the Gomori-posltIve granulations are clearly of ghal type, a striking diversity of morphological types was observed in cells which showed this property This may mean that various cell types of glloblast o n g m can produce the Gomori-positlve granulations or that one cell type can take various morphological forms depending on the actual microenvironment of the culture The Gomorl-pOSltlVe gha is definitely not of the mlcrogha type, being negative for lyso-

somal enzymes 12,13 The results obtained in thls study confirm the carher h~stochemlcal observatlons that the ghal Gomon-positive granulations are unusually rlch in sulphydryl groups The biological role of the Gomorl-positlve gha is unknown VIgh et al i~ have assumed that the ghal Gomon-posltxve materlal has a secretory function Recently, a protective role of the Gomori-posltlve gha has been postulated I° According to the latter hypothesls the Gomorl-pOSltlVe gha form a protectlve coat around the brain ventricles trapping various local or blood-borne toxlns, particularly of hpld peroxide type Iz It has been already shown by Wislockl and Leduc I~ that silver granules accumulate in perlventrlcular ghal cells ol rats given sllver mtrate for prolonged time pcrlods The present results show that diffcrcntlatlon of Gomorl-posltive ghal cells ~s a constant and characteristic feature of mammalian foetal brain t~ssue grown m vitro Thls may be another in&catlon of an important biological role played by thls type of gha SUMMARY

Chrome alum haematoxyhn-posmve granulations regularly appear in ghal cells derived from mouse and hamster foetal brain tissue explants grown for 14 days m vitro The Gomon-posItlVe ghal cells are very numerous during the later stages of the m vitro cultures, t e , during the fourth and fifth week of cultivation The Gomorlpositive glial cells form the majority of all cell types present in the internal layer of the cultures which borders a cell-free center The granulations of the Gomorl-posttlve ghal cells grown m vitro are very rich in sulphydryl groups, thus showing the same hlstochemlcal properties as those of these same cells m sztu

REFERENCES

1

CRESWELL, G

F, REIS, D J , AND MACLEAN,P D , Aldehyde-fuchsm posmve material m the brain of squirrel monkey, Amer J A n a t , 115 (1964) 543-558 2 DmP•N, R , ENGELHARDT,F , UND SMITH-AGREDA,V, ~ber Ort und Art der Entstehung des Neurosekrets lm supraoptlco-hypophysaren System bel Hund und Katze, Anat A n z , 101 (1954) 276--286 Brain Research,

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3 KALUZA, J , AND STEFANICKA-WIECHOWA,A , The radlomlmetnc effect of actmomycm C on

tumors of ghal origin cultured m vitro, Arch Immunol Ther Exp, 18 (1970) 220-224 4 MAKSYMOWICZ, K , AND SREBRO, Z , Gomon-posiUve gha in the mouse ontogeny and topo-

graphic distribution, Foha blol (Krak6w), 20 (1972) in press 5 NODA,H , On the gomonphll findings other than the neurosccretory system m the observation of the hypothalamo-hypophyseal system, Gunma J reed Scz, 8 (1959) 223-232 6 PEARSE,A G E , ttlstochemlstry Theoretwal and.4pphed, Churchill, London, 1960, pp 806-808 7 SMITH, S W , The correspondence between hypothalamlc neurosecretory material and neurohypophyseal material m vertebrates, Amer J Anat, 89 (1951) 195-231 8 SREBRO,Z , New cytoplasmic structures m the forebram of Rana esculenta, Foha blol (Krakdw), 10 (1962) 137-141 9 SREBRO, Z , A comparative and experimental study of the Gomorl positive gha, Foha blol (Krakdw), 17 (1969) 177-192 10 SREBRO, Z , The epcndyma, the cysteine-nch complex-containing penventncular gila, and the subformcal organ m normal and X-irradiated rats and mice, Foha blol (Krakdw), 18 (1970) 32%334 11 SREBRO, Z , X-ray induced increase m amount of cysteine-rlch periventncular ghal cells m the rat, Experwntta (Basel), 27 (1971) 945-947 12 SREBRO, Z , AND CICHOCKI, T , A system of per~ventncular gha m brain characterized by large, peroxlsome-hke cell organelles, .4eta hlstochem (Jena), 41 (1971) 108-114 13 SREBRO, Z , AND MACIglSKA,A , The growth of mouse foetal brain tissue m vitro wRh particular reference to 'Gomon-posmve' ghal cells, Foha blol (Krakdw), 19 (1971) 409-413 14 SREBRO, Z , AND ~LEBODZII~SKI,A , Penventrlcular Gomori-posmve gha in the hypothalamus of the rabbit, Foha blol (Krakow), 14 (1966) 391-395 15 VIGH, B , AROS, B , KORITS~NSZKY, S, WENGER, T , AND TEICHMANN,I , Ependymosecretlon (ependymal neurosecreUon) V The correlation between ghal cells containing Gomon positive substance and ependymosecretlon m different vertebrates, .4eta Blol ,4cad Scl hung, 14 (1963) 131-143 16 WISLOCKI, G B , AND LEDUC, E H , Vital staining of the hematoencephalic barner by silver mtrate and trypan blue, and cytological comparisons of the neurohypophysls, pineal body, area postrema, mtercolumnar tubercle and supraoptlc crest, J comp Neurol, 96 (1952) 371-414 17 WISLOCKI, G B , AND LEDUC, E H , The cytology of the subcommissural organ, Reissncr's fiber, per~ventncular ghal cells and posterior colhcular recess of the rat's brain J comp Neurol, 101 (1954) 283-309

Brain Research, 38 (1972) 27-33