A Note on the Glial Fiber the Nature of So-Called Glial Fibers Has Been

A Note on the Glial Fiber

Hiroshi Hosokawa

Department of Anatomy, University of Tokyo Faculty of Medicine, Hongo, Tokyo

The nature of so-called glial fibers has been interpreted in two different ways. Several years after the discovery of the " Nerven- kitt " or " neuroglia " by V ir cho w (1846, '51), Clarke (1859) studied this tissue and stated that it was composed of cellular and fibrous elements which were independent of each other. D e i t e r s (1865), who teased small blocks of the brain fixed in chromic acid solution, found the dissociated " Zellaequivalente " or D e it e r s' cells, which clearly correspond to the astrocytes of today's termino- logy, and stated that these elements were furnished with long, fibrous processes extending radially from the cell body. Apparently he took the fibrous structures of the glial tissue for protoplasmic or cellular expansions. Golgi (1894), the inventor of the epoch- making silver impregnation method, thought in the same way that the glial as well as the nerve cells have long cellular processes. On the other hand, Ranvier (1883) dissociated blocks of the spinal cord in 33 per cent alchohol, stained the sediments with picrocarmin, and was led to the conclusion that the fibrous elements of the glial tissue had -essentially nothing to do with the cellular elements or the De it e r s' cells, although the former were found sometimes to penetrate through the cell body of the latter. We i g e r t (1895) studied the glial fibers with his new staining method, and insisted strongly upon their extracellular nature. Ac- cording to him, these fibers were morphologically as well as chemi- tally quite different structures from the glial cells, representing intercellular, extraplasmic fibers just like the collagenic or elastic fibers in the ordinary connective tissues. Thereafter Kölliker

1) This study was supportcd by a research grant from the Education Ministry of Japan. 2) Dedicated to Prof. Dr. T. Oga w a for his Sixtieth Birthday.

315 316 Hiroshi Hosokawa

(1896) proposed a compromise that, whereas the glial fibers were continuous with the cytoplasm of glial cells in the embryonal stages, they became gradually emancipated and remained independent from the latter. According to H e 1 d ('03, '09), glial fibers represent the differen- tiated product of the protoplasm, and they remain permanently wrapped in a thin sheath of cytoplasm continuous with the cell body of the astrocytes. H e 1 d assumed further that the astrocytic processes formed three-dimensional syncytial lattice works every- where in the central nervous system. So, he thought, the glial fibers were intraplasmic structures, not necessarily found in relation to nuclei. H e 1 d's syncytium theory was supported and extended by Bauer ('53). Two different opinions about the glial fibers, both the extracellular and the intraplasmic interpretations, have found supporters until recently among histologists and pathologists. T a f t and Ludlum ('29), Wilke ('51), Wilke and Kircher ('52) and others supported the extracellular theory, while Schmidt ('42) and Bair at i ('58) believed that the intraplasmic interpretation was the correct one. Recent advances in electron microscopy have revealed very clearly that the central nervous system is filled with protoplasmic cells and processes, leaving very narrow intercellular or extraplasmic spaces of only 100-200A in width, where no extraplasmic fibers or fibrils are shown, although fine fibrils called glia filaments are sometimes seen in the cytoplasm of fibrous astrocytes. Thus it may be said that the extracellular theory of the glial fiber has been proved to be erroneous. As stated by Palay ('58), they must be understood to be nothing but the cytoplasmic extensions of the glial cells, although it is still difficult to exclude completely the possibility of extraplasmic emancipation of glial fibers, especially in some histopathological cases (B ielschowsk y, '35). At the same time, however, it is also true that the so-called glial fibers present characteristic properties somewhat different from the ordinary cytoplasm of the astrocytes. So the question remains as to whether or not some special cytoplasmic differentia- tion is really concerned with the formation of glial fibers. If so, glial fibers are strictly speaking not the synonym of the mere fine fibrous processes of astrocytes. Wei g e r t (1895) stressed the differ- ence of glial fibers in the specific staining method introduced by A Note on the Glial Fiber 317 himself. By means of roentgenographic study, Wilke and K i r- c h e r ('52) maintained that glial fibers seemed to be identical in composition with the fibrin. The physical and chemical properties of glial fibers have been subjected to detailed investigations by Schmidt ('42) and Bairati ('58). Bairati stated that the double refracting substance found in glial fibers is apparently a kind of scleroprotein, keratin. According to him, " it appears likely that neuroglia cells, being of ectodermal origin, undergo a partial cytomorphosis as epidermal cells do."

Glial fibers in relation to the morphology of astrocytes Ordinarily the astrocytes are classified into two major types, the protoplasmic and the fibrous. The protoplasmic astrocytes are furnished with thick, branched processes which are entirely protoplasmic. The nuclei are large and somewhat oval in shape. They are abundant in the gray matter. On the other hand, the fibrous astrocytes are found mainly in the white matter. The cell body is smaller, enclosing an oval or spherical nucleus. Characteristically they are supplied with long, slender processes which do not show extensive branching. These fibrous expansions are demonstrated by silver impregnation and other methods as fine fibers or fibrils arranged in lattice works throughout the central nervous system. In addition to the typical protoplasmic and fibrous astrocytes, it is well known that there are many transitional forms between these two. As a matter of fact, it is sometimes very difficult to draw a clear line between protoplasmic and fibrous astrocytes. For the purpose of making the morphology of astrocytes more intelligible, the author examined carefully a great number of astrocytes in his preparations. The materials employed comprised the brain and spinal cord of man and various animals. The preparations were stained mainly with the Cajal's silver method for macroglia as well as Hortega's silver carbonate methods. Astrocytes of various forms were examined, sketched, and photographed (Figs. 10- 17). A series of transitional astrocytes thus appeared to " bridge " between the typical protoplasmic astrocytes on the one hand and the fibrous one on the other. Some types in the series are shown diagrammatically in Figure 1. Types P and F represent typical 318 Hiroshi Hosokawa

Fig. 1. Diagrammatic illustration of the transitional series of astrocytes, which are arranged according to the grades of fibrization. Typical protoplasmic (P) and fibrous (F) astrocytes are on the extremities, atypical protoplasmic (P', P") and fibrous ones (F", F') being situated in between. protoplasmic and fibrous astrocyte respectively. The transitional forms in between are shown as types P', P", F", and F', the number of apostrophes indicating the grades of deviation from the typical forms. When the series P-P" is examined, it will be noted that the changes include the following points : 1) The branching of processes decreases. 2) The processes become thinner and straighter. 3) The argyrophilia of the protoplasmic processes increases, and (as if a kind of condensation of the protoplasm takes place here) the granular appearance in the typical protoplasmic processes is superseded by the dense, fibrous appearance. A Note on the Glial Fiber 319

These changes progress further in the series F"-F. Here the cellular processes exhibit distinct fibrous appearance, and the protoplasmic condensation occurs even in the perinuclear cell body, the granular portion being gradually reduced. The condensation in the perikaryon takes place at first along the surface or borders of the cell body. Then it goes on apparently through the cell body (Fig. 2). In the extremely fibrous astrocytes the cytoplasm is almost lost to sight, and often the nucleus seems to be free or naked in the meshes of the glial fibers (Figs. 16, 17).

Fig. 2. An astrocyte from the white substance of the human spinal cord. Cajal's silver stain for macroglia. Drawn by camera lucida. The condensedor dark-staining protoplasm of the fibrous processescontinues and passes through the cell body. Thus it is clear that the most important change in the series of the astocytes lies in the transformation of the protoplasmic processes into fibrous ones. This phenomenon or tendency to become fibrous processes may be called fibrization. Neither the nature nor the mechanism of this transition is clear, although it is doubtless that some metabolic factors are concerned with this phenomenon in the so-called gliosis, where the fibrization is accerelated excessively. If Bair a t i's opinion is right, it is possible that a kind of keratinization or cornification plays a very important role in this process. Granules like keratohyalin in the corium were, however, 320 Hiroshi Hosokawa not encountered in the astrocytes. Is it a kind of keratinization similar to that in the nail and hair, and why does it occur more markedly in the white matter ? The author does not mean to imply that the individual astrocytes change their shape and are transformed in one or the other direction in the series mentioned. This is rather an attempt to establish a morphological standardization of astrocytes, which arose from the observation of the varieties of astrocytes in the central nervous system. It may be useful for detailed analysis of astrocytes in various parts of the brain and spinal cord, as well as for analysis of pathological changes in the astrocytes. Recently N a k a i ('62) observed in tissue culture that a protoplasmic astrocyte with broad membranous expansions changed into a stellate fibrous astrocyte. At that time, according to him, radiate folds appeared in the membrane and became thicker, and the membrane between folds was broken irregularly which changed into finer secondary processes. N a k a z a w a et al. ('62) cultivated brain tissue of the kitten at first in the roller tube, and transfered it into suspended culture. Then repeating to transfer the material into new cultures successively, they found that at the 5 th passage the protoplasmic astrocytes and oligodendrocytes began to decrease in number remarkably, and that at the 6th passage only the fibrous astrocytes remained, implying the lower rate of metabolism in the latter than that of the protoplasmic astrocytes.

Arrangement of glial fibers in relation to the nerve elements

Regional differences in the distribution of glial fibers in the central nervous system have been studied by W e i g e r t (1895), Mailer (1900), Spielmeyer ('22), Schroeder ('35), Glees ('55), et al. Apart from details of the glioarchitecture, W e i g e r t stressed the following points concerning the general topography of glial fibers : 1) Subependymal layer of the ventricles and the central canal is furnished with especially dense networks of glial fibers. 2) The surface layer of the central nervous system is also rich in glial fibers, which often form the condensed " Rindenschicht " or "superficial glial layer ". 3) Glial fibers are well developed where there once was a surface in the embryonic stage, for example, in the vicinity of the A Note on the Glial Fiber 321

Fig. 3. Glial fibers in the gray substance of the human spinal cord. Cajal's silver stain for macroglia. Drawn by camera lucida. Especially fine glial fibers are seen in the vicinity of the nerve cell, which is shown vaguely in the middle of the figure.

Fig. 4. Cross-section of white matter of ox's spinal cord. Cajal's silver stain for macroglia. Drawn by camera lucida. As—Astrocyte, Gf—Glial fibers, Nf—Nerve fibers. 322 Hiroshi Hosokawa

dorsal septum of the spinal cord. 4) Wherever there is a kind of boundary surface inside the central nervous system, glial fibers are apt to flourish. For instance, around relatively isolated nerve fiber bundles, around larger nerve cells such as cells of the anterior horn (" Neuroglia- kOrbe " or " glialbasket "), or surrounding the perivascular spaces glial networks are well developed. Employing principally Cajal's silver method for macroglia, the present author examined the distribution pattern of the glial fibers, especially in relation to the nerve elements. Materials used included the spinal cord of man and of animals such as oxen, rabbits and rats. First of all it should be emphasized that, as shown in Figures 2-4, 18, 19, glial fibers are well developed in both the gray and white matter, although it is generally believed that the former contains few fibrous astrocytes. There are, however, some differences in the arrangement of glial fibers between the gray and white matter. In the former individual fibers run in quite irregular

Fig. 5. Course and direction of glial fibers running between nerve fibers. Drawn by camera lucida. Circles represent cross-sections of nerve fibers. A Note on the Glial Fiber 323 directions and they form, all together, dense lattice works in the meshes of which nerve cells and processes are to be supported. In the white matter the glial fibers are arranged so to say somewhat more regularly. It is likely that their distribution is to some extent subject to influence by the regular arrangement of nerve fibers. They are disposed either horizontally, vertically or obliquely. It is known that the radial arrangement is the original mode of distribution of glial fibers in the embryonic spinal cord. Figure 19 shows the glial fibers in the boundary zone between the gray and white matter. One astrocyte is seen nearly on the boundary line, sending processes into both the gray and white matter. Glial fibers in the gray matter are generally somewhat thinner than those in the white matter. In order to see the detailed relationship between glial and nerve fibers in the white matter, the courses of individual glial fibers

Fig. 6. Glial fibers traversing between Fig. 7. Glial fibers surrounding single nerve fibers. Drawn by camera lucida. nerve fibers which are seen lengthwise. Drawn by camera lucida. 324 Hiroshi Hosokawa

Fig. 8. Diagrammatic Fig. 9. Supposed situation of fibrous astrocytes illustration of the rela- which are disposed in the white matter of the central tionship between glial nervous system. Nf —Nerve fibers, By—Blood vessel . (Gf) and nerve fibers (Nf).

are traced and analysed (Figs. 5-7, 20-23). It was observed that, as a rule, each glial fiber is disposed to run always in a definite direction, without change, although it takes usually a wavy course between the nerve fibers. It does not show special orientation to particular nerve fibers, for instance by running spirally around the latter. So the morphological relationship between glial and nerve fibers is, as shown diagrammatically in Figures 8 and 9, just like the relation between the passers-by on the street. There is not such one-to-one relationship as seen between the neurilemma and nerve fiber in the peripheral nerves.

Summary The so-called glial fibers, of which the nature is still not well known, are discussed in relation. to the morphology of astrocytes. The astrocytes can be arranged in a transitional series bridging the typical protoplasmic one on one side and typical fibrous one on the other (Fig. 1). Thus the form or type of each astrocyte is A Note on the Glial Fiber 325 determined by the grade of " fibrization " or " the tendency to become fibrous " of the glial processes, although it is unknown what kind of factors are concerned with this phenomenon. The histological arrangements of glial fibers especially in relation to nerve elements are also examined, and it was revealed that the former do not show particular one-to-one relationship to the latter.

References

1) B a i r a t i, A. (1958). Fibrillar structure of astrocytes. Windle's Biology of Neuroglia. p. 66-72, Charles C. T h o m a s, Springfield. 2) B a u e r, K. F. (1953). Organisation des Nervengewebes und Neurencytiumtheo- rie. Urban & Schwarzenberg, Miinchen & Berlin. 3) B i e l s c h o w s k y, M. (1935). Allgemeine Histologie und Histopathologie des Nervensystems. Bumke u. Foerster's Handbuch d. Neurol., Bd. 1, Springer, Berlin. 4) Clark e, J. L. (1859). Philosophical transactions. 1859, P. 437. (cited from Weigert, 1895). 5) D e i t e r s, 0. (1865). Untersuchungen iiber Gehirn und Ruckenmark des Men- schen und der Saugetiere. Vieweg, Braunschweig. 6) Glee s, P. (1955). Neuroglia, morphology and function. pp. 111, Blackwell, Oxford. 7) G o 1 g i, C. (1894). Untersuchungen ii.ber den feineren Bau des centralen und peripheren Nervensystems. Fischer, Jena. 8) He 1 d, H. (1903). Ueber den Bau der Neuroglia und fiber die Wand der Lymph- gefdsse in Haut und Schleimhaut. Abhandl. d. mathem.-physische Klasse d. KOnigl. Sachsisch. Gesellsch. d. Wissensch., 28 : 196-318. 9) -- (1909). Ueber die Neuroglia marginalis der menschlichen Grosshirnrinde. Monatsch. f. Psycho'. u. Neurol., 26 : Ergdnz.-Heft, 360-416. 10) Koel I ike r, A. (1896). Handbuch der Gewebelehre des Menschen. Bd. 2, Leipzig. 11) M U 11 e r, E. (1900). Studien tiber Neuroglia. Arch. f. mikr. Anat. u. Entw.- gesch., 55: 11-62. 12) N a k a i, J. (1962). Transformation and multiplication of neuroglia in tissue culture. Proceed. IV. Internat. Congress of Neuropath., 2: 241-246. 13) Nakazawa, T., J. Tominaga & K. Yamauchi (1962). Morphological concepts of astrocyte based in tissue culture. Ibid., 2 : 246-247. 14) Pala y, S. L. (1958). An electron microscopical study of neuroglia. Windle's Biology of Neuroglia, p. 24-38. 15) R a n v i e r, L. (1883). De la nevroglie. Arch. de physiol. normale et pathol., 1883. (cited from W e i g e r t, 1895) 16) Schmid t, W. J. (1942). Zur Doppelbrechung des Gliagewebes, insbesonders der Miillerschen Stiitzfasern der Netzhaut. Zool. Anz., 138: 93-96. (cited from Bairati, 1958) 17) S c h r o e d e r, A. H. (1935). Gliaarchitektonik des Zentralnervensystems. Bumke u. Foerster's Handb. d. Neurol., 1: 791-810. 18) S p i e 1 m e y e r, W. (1922). Histopathologie des Nervensystems. Springer, Berlin. 326 Hiroshi Hosokawa

19) T a f t, A. F. &- S. D e W. Ludlum (1929). On the nature of two forms of neuroglia. J. nerv. meat. Dis., 10: 360-368. 20) V i r cho w, R. (1846). Ueber das granulierte Ansehen der Wandungen der Ge- hirnventrikel. Aug.l Z. f. Psychiat., 3: 242-250. 21) (1851). Uaber Blut, Zellen, Fasern. V i r c h o w's Arch. f. path. Anat., 3: 228-248. 22) W e i g e r t, C. (1895). Beitrage zur Kenntnis der normalen menschlichen Neuro- glia. Frankfurt a. M. 23) Wilk e, G. (1951). Ueber Gliafaserbildung als intercellularer Vorgang. Deutsch. Z. f. Nervenh., 166: 447-464. 24) Wilk e, G. & H. Kircher (1952). Ueber rOntgenographische Untersuchungen zur Frage der Gliafaserbildung. Ibid., 161: 391-406.

Explanation of figures

Plate I Astrocytes arranged in the transition series from protoplasmic to fibrous type. The pictures are taken from preparations of the gray matter of human spinal cord stained with Hortega's silver carbonate method (Figs. 10-15) or Cajal's silver method for macroglia (Figs. 16-17). Protoplasmic condensation, implying the formation of glial fibers, are seen to advance gradually from the processes to the cell body.

Plate II Fig. 18. Glial fibers in the gray matter of human spinal cord. Cajal's silver stain for macroglia. Fig. 19. Glial architectonic in the boundary zone between the gray (on the left side) and white matter (on the right) of the human spinal cord. An astrocyte is seen just on the boundary line, sending expansions both into the gray and white matters. Fig. 20-23. Relationship between the glial and nerve fibers. White substance of ox's spinal cord. Cajal's silver stain for macroglia. A nerve fiber bundle deriv- ing from the dorsal root is cut lengthwise in the middle of Figures 20 and 21, and fine glial fibers are seen to surround these nerve fibers. In Figures 22 and 23, a single wavy glial fiber is shown to traverse between cross sections of nerve fibers. 327

Plate I

111 11

12 13

14 15

16,

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Plate II

•18 19

I-20 '21

22 '23

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