Problems encountered when immunocytochemistry is used for quantitative glial cell identification in autoradiographic studies of cell proliferation in the brain of the unlesioned adult mouse.

We have used sections of adult mouse brain to determine whether antibodies specific for oligodendroglia (anti-carbonic anhydrase II, CA II; anti-galactocerebroside, GC; anti-myelin basic protein, MBP) and astroglia (anti-glial fibrillary acidic protein, GFAP; anti-S 100 protein) are suitable for quantitative studies of the proliferation and subsequent differentiation of these cells. Unlesioned adult mice received a single injection of 3H-thymidine (TdR) and were killed between 1 h and 70 days later. Quantitative evaluations of autoradiographs of 2-microns-thick serial sections stained immunocytochemically with the antibodies mentioned above or with Richardson's method for histological control led to the following conclusions. Anti-GC and anti-MBP stained only the oligodendrocytic processes and, thus, cannot be used in well-myelinated brain areas. Anti-CA II stained only a portion of the differentiated oligodendrocytes, but no proliferating cells. Anti-S 100 protein recognized all the astrocytes, but also many (interfascicular) oligodendrocytes. Anti-GFAP stained only a few astrocytes in the unlesioned mouse; all astrocytes may become GFAP-immunopositive only after wounding the brain. Thus, in contrast to in vitro studies, immunocytochemical studies with these antibodies on sections of adult animals cannot be recommended for the quantitative analysis of cell proliferation. In addition, our results show that differentiated glial cells proliferate in adult mice. Astro- and oligodendrocytes divide with the same cell cycle parameters and mode of proliferation up to about 1 month after 3H-TdR injection. In contrast to oligodendrocytes, some astrocytes might re-enter the cycle after a few weeks of quiescence.

[Changes in the neurite-glial interrelations under the influence of antiserum against galactocerebrosides (a vital tissue culture study)]. / Izmeneniia neirit-glial'nykh vzaimootnoshenii pri vozdeistvii antisyvorotki k galaktotserebrozidam (prizhiznennoe issledovanie kul'tury tkani).

Two--five-day-old culture of 10-11-day-old chick embryos has been used. Antiserum against galactocerebrosides (anti-GalC) is added to the nutrition medium before cultuvation. In the presence of anti-GalC the growth and gliocyte migration zone decreases, the area index becomes essentially small (3.5 +/- 0.7 in the control, 1.5 +/- 0.5 in the experiment). This is connected with inhibition of migration and proliferation of gliocytes during first 48 h of cultivation, while intensity of neuron regeneration remains unchanged. Alterations of the neurit-glial relations are investigated by means of the vital phase-contrast microscopy. Effect of anti-GalC to peripheral gliocytes is accompanied with a decreasing adhesive ability of their plasmolemma. This makes difficult their flattening on the neurit membrane, formation of contact membranous neurit-glial relations and formation of glial membranes. On the 3d day formation of nervous fasciculi is retarded, as well as their fusion into trunks and plexuses. On the 5th day, unlike the control, a continuous "epineural" covering of neuritic plexuses does not form. Round retractile and defective (with protrusions) forms of gliocytes predominate. These data demonstrate inhibitory effect of anti-GalC on the structural-functional maturation of the glia and on formation of neurit-glial relations in the culture of the sensitive ganglion.

The interrelations between high- and low-molecular weight forms of normal and mutant (Krabbe-disease) galactocerebrosidase.

Galactocerebrosidase (beta-d-galactosyl-N-acylsphingosine galactohydrolase; EC 3.2.1.46) activity of brain and liver preparations from normal individuals and patients with Krabbe disease (globoid-cell leukodystrophy) have been separated by gel filtration into four different molecular-weight forms. The apparent mol.wts. were 760000+/-34000 and 121000+/-10000 for the high- and low-molecular-weight forms (peaks I and IV respectively) and 499000+/-22000 (mean+/-s.d.) and 256000+/-12000 for the intermediate forms (peaks II and III respectively). On examination by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the high- and low-molecular-weight forms revealed a single protein band with a similar mobility corresponding to a mol.wt. of about 125000. Antigenic identity was demonstrated between the various molecular-weight forms of the normal and the mutant galactocerebrosidases by using antisera against either the high- or the low-molecular-weight enzymes. The high-molecular-weight form of galactocerebrosidase was found to possess higher specific activity toward natural substrates when compared with the low-molecular-weight form. It is suggested that the high-molecular-weight enzyme is the active form in vivo and an aggregation process that proceeds from a monomer (mol.wt. approx. 125000) to a dimer (mol.wt. approx. 250000) and from the dimer to either a tetramer (mol.wt. approx. 500000) or a hexamer (mol.wt. approx. 750000) takes place in normal as well as in Krabbe-disease tissues.