Oligodendroglia and axis cylinders in rabbits before during and after myelination.код для вставкиСкачать
OLIGODENDROGLIA AND AXIS CYLINDERS I N RABBITS BEFORE, DURING, AND AFTER MYELINATION ANATOLE DEKABAN Shaughnessy Hospital and the Department o f Medicine, Ti. B . C . Medical School, Vancouver, B. C. T W O FIGURES Oligodendrogliacytes are the most numerous cells in the central nervous system of adult mammals. Information regarding their role, however, is still scanty and based more on assumption than on objective findings. The progress in this field has been hampered, among other things, by the fact that oligodendroglia undergo rapid autolysis in a variety of pathological conditions and even during agonal state and also, because we are still lacking an appropriate staining method which would show all components of these cells consistently. Cajal ( ’13), and Hortega (’21 and ’as), were the first to describe the morphology of oligodendroglia.2 They called those cells which were accumulated in the form of more o r less regular rows along the nerve fibers “interfascicular glia, 7 ’ whereas those in the immediate vicinity of neurons they named “perineuronal satellites.” Hortega also remarked on the possible parallelism of function between oligodendrogliacytes and cells of Schwann in peripheral nerves. H e From Shaughnessy Hospital and the Department of Medicine, U. B. C., Medical School, Vancouver, B. C. This investigation was aided i n p ar t by the Vancouver Chapter of The Multiple Sclerosis Socirty of Canada and by the Federal Health Grants of Canada. I n this paper the term “oligodendroglia” drnotes oligodendroblasts and young and adult oligodendrocytes. 111 112 ANATOLE DEKABAN thought that both these elements were likely to take part in the process of myelination. The opinions of subsequent investigators regarding the role of oligodendroglia were divided. Schaffer ( '28), Meduna ( '27), Jakob ( '27), Ferraro and Davidoff ('28), Cramer and Alpers ('32), and others believed that these cells can perform active phagocytosis in clearing products of degeneration, whereas Creutzeldt and Metz ( 'as), Penfield ( 'as), Hortega ( 'ZS), Globus ( '28), and others maintained that oligodendrocytes cannot assume the role of phagocytes. More recently, Greenfield ( '33),Brain and Greenfield ( '50), and Lumsden ( '51), suggested again possible direct relationship between the function of oligodendrogliacytes and the normal state of myelin. Of great, though still theoretical interest, are the findings of Canti, Bland and Russell ('35), and Lumsden and Pomerat ( '51), that oligodendrogliacytes studied in the tissue culture by means of time lapse cinematography showed rhythmic pulsatile activity. The significance of this phenomenon, however, remains obscure. The present study is concerned with the state of the oligodendroglia and axis cylinders before, during, and after the process of myelination. Quantitative and qualitative methods were used with a hope of gaining some objective data pertaining to their possible interrelationship. MATERIALS AND METHODS Forty-six brains and spinal cords of fetal, new born, young and adult rabbits were utilized. The animals were sacrificed by an overdose of ether anaesthesia and portions of the CNS were immediately fixed in 10% formalin, formalin ammonium bromide, cobalt-calcium formalin and chloral hydrate-dichromate formalin. The sections were cut 4 and 8r.r in thickness and stained by the following methods: Nissl, Masson trichrome, Heidenhain, Pal-Kulschitzky, Bodian, Hortega 's silver carbonate, Sudan IV, Sudan black B, periodic acid, Golgi, Cajal gold chloride and silver carbonate for oligodendroglia. AXONS, OLIGODENDEOGLIA AND MYELINATION 113 A segment of the cervical spinal cord 5 mm below the obex was cut transversely in sections 4 1.1 in thickness and used for quantitative study. All oligodendroglia cells were counted in one posterior funiculus, the section being stained by Masson trichrome technique. This staining was selected for counting purposes because it outlines all glial cells present in the section, whereas metallic methods which showed more cellular details were not sufficiently reliable for quantitative study. In Masson stain it was relatively easy to distinguish microglial cells, vascular epithelium and connective tissue elements, whereas certain difficulties were encountered in differentiating between oligodendroglia and astroglia. Therefore, these last elements were counted together with the aid of Whipple’s micromatic disc. The adjacent sections were stained with Cajal gold chloride and with Hortega’s silver carbonate method for astrocytes, these cells in one posterior funiculus were counted and their number subtracted from the combined oligodendroglia and astroglia counts. The degree of myelination in the posterior funiculus of the respective adjacent sections from the same blocks of cervical spinal cord was estimated by means of transmitted light absorption, using Photovolt readings under precisely the same conditions. The values of light absorption by sections stained f o r myelin were expressed in percentages of relative absorption units. With the aid of an ocular filar micrometer and using sections stained by Bodian’s method, the average diameter of medium sized axis cylinders in the posterior funiculus was estimated in different stages of development. All values of counts and measurements stated in the description of the results are averages calculated from at least 6 estimations. Since no corrections for the shrinkage of neural elements during fixation and dehydration were made, the linear and square measurements have only relative significance, comparable to each other. Qualitative changes occurring in the oligodendroglia before, during, and after myelination, were studied by means of the variety of stains employed. 114 ANATOLE DEKABAN RESULTS I n a fetal rabbit of 25 days’ gestation age, there was no evidence of myelination in the posterior funiculus. I n the fetus of 29 days’ gestation age the myelin in this location was just beginning to show in the form of widely scattered, dark bluish, tiny streaks as seen in the myelin stain. I n the full term new born rabbit (31 days’ gestation age) the myelination was slightly more advanced though still in the initial stage. As outlined before, the counting of oligodendroglia and of astroglia cells together in sections cut 4 p in thickness and stained with Masson trichrome method in the whole series of maturing rabbits was performed. I t was found that in fetuses and very young animals the cellular elements in the spinal cord were very densely distributed. With the growth of the animal and increase in thickness of its spinal cord, the density of those elements decreased. This fact can be best appreciated by the inspection of figure 1A and B, which shows the relationship of the number of oligodendroglia per t m m 2 in the posterior funiculus to the total transverse surface of the spinal cord in the series of rabbits studied. However, by counting all oligodendroglia cells in a transverse section of one posterior funiculus it was possible to estimate their true numbers before, during, and after myelination. In the fetal rabbit of 25 days’ gestation age there was no evidence of myelination in the posterior funiculus and the axis cylinders were very small in diameter. At this stage the distribution of cellular elements in this location was only moderately dense as compared with that of a new born animal. Analysis of these elements by examination of sections stained with Masson, Cajal gold chloride, silver carbonate, and Golgi methods showed that the predominant cell type in the posterior funiculi of this fetus was spongioblast, which is a precursor for both oligodendroglia and astroglia. The presence of mitotic figures in spongioblasts indicated active proliferation. Although no accurate counts of different types of cells in this fetus were possible, the approximate ratios of AXONS, OLIGODENDROGLIA A N D M Y E L I N A T I O N 115 spongioblasts to oligodendrogliablasts to oligodendrogliacytes to astrocytic series were as follows : 2.5 : 1.1:0.6 :0.1. The total cell count (spongioblasts, oligodendroglia and astroglia) in one posterior funiculus was 84. I n the fetal rabbit of 29 days' gestation age, when the myelin was just beginning to make its appearance, the average diameter of medium sized axis cylinders approximated 0.6 1-1. B TI. sc CT. or. s?. C O R D IN ao.nn. 4000- I A a 800 boo0 - ?.a00 - 2000 - 1500 I000 - 800 210. A - Shows the rate of increase in size of transverse sections of the spinal cord in square millimeters during the maturation of rabbits. B - Shows that the density of oligodendroglia cells per 2 mm2 of the posterior eolunin is progressively decreasing during the growth of these animals. The ratios of spongioblasts to oligodendrogliablasts to oligodendrogliacytes to astrocytic series decreased as compared with the previous fetus to approximately 1.8 :1.6 :0.8 :0.1. The total count of all these cellular elements was 112. In the new born rabbit at term the relative numbers of spongioblasts and oligodendrogliablasts were small as compared to young oligodendrogliacytes ; the latter were distinguished by the smaller nucleus than that of oligodendrogliablasts, but containing more chromatin than that of adult 116 ANATOLE DEKABAN oligodendrogliacytes. The density of cellular elements in the posterior funiculus has markedly increased, amounting to 240 cells per 3 mmz (the transverse diameter of one posterior funiculus at this stage is still smaller than 4 mm2, so an area of 4mm was counted and multiplied by two). The total count of oligodendroglia in one posterior funiculus of this rabbit after 23 cells of astroglia series were subtracted was 156. The transverse diameter of the majority of medium sized axis cylinders has increased to approximately 0.8 p. With the increase in thickness of the spinal cord in a oneday-old rabbit the distribution of cells in the posterior funiculus was less dense than in a new born animal. The total count of oligodendroglia cells in one posterior funiculus was 191 after subtraction of 26 cells of astrocytic series. This showed an increase in the number of oligodendroglia cells over that of the new born. The transverse diameter of the majority of axis cylinders also showed a slight increase during the same period of time. I n 2- and 4-day-old rabbits the degree of myelination in the posterior columns had almost doubled and tripled respectively as compared with that of the new born. The spongioblasts and oligodendroblasts were only occasionally seen, the bulk of cells being composed of young oligodendrogliacytes. The size of the majority of axis cylinders of 4-day-old rabbits had increased in diameter to approximately 1.1p. There were 202 oligodendroglia cells present in one posterior funiculus of a 2-day-old rabbit (after subtraction of astroglial cells). The oligodendroglia counts in one posterior funiculus of a 4-day-old rabbit amounted to 209 cells. I n 6-, 8-, and 10-day-old rabbits, the degree of myelination in the posterior funiculus continued to increase at almost the same rapid pace as in the earlier stages of postnatal life, however, the total counts of oligodendrocytes remained quite constant. The minor fluctuations in counts were dependent on individual variations and possible counting error. Spongioblasts were seen no more, the oligodendroblasts were encountered only occasionally, the predominant type of cell AXONS, OLIGODENDROGLIA A N D MYELINATION 117 being still young oligodendrogliacytes. The astrocytes counts also remained quite constant at these ages, their numbers averaging from 18 to 26 in one posterior funiculus. The total oligodendrogliacytes count in this location of a 6-day-old rabbit was 226 cells, of 8 days old 218, and of 10 days old it was 221 cells. The axis cylinders continued to increase in size progressively, the diameter of the majority of fibers had almost doubled in the 10-day-old rabbits over that of new 30 20 118 ANATOLE DEKABAN born animals. More detailed data of measurements and counts in the progressive stages of animals’ maturation may be seen in figure 2 A, B, and C. The degree of myelination in rabbits between the 12th and 36th day of age continued to increase although at a very slow rate. The oligodendrogliacytes assumed a more adult appearance, showing marked decrease in the chromatin content as revealed by lighter staining of their nuclei. The total oligodendrogliacyte counts in one posterior funiculus of these animals were quite constant and averaged from 219 to 231 cells. The axis cylinders continued to increase in size; in a 30-day-old rabbit the diameter of the majority of medium sized axis cylinders increased three times over that of the new born. From the age of 40 days onwards and in adult rabbits there was no further increase in myelin content in the same sized square area as estimated by the light absorption method, although by measurement the transverse diameters of myelin sheath (inclusive those of axis cylinders) were increasing slightly until adulthood. The average oligodendrogliacyte counts in one posterior funiculus were within the same range as in the animals of 1 2 to 36 days of age. The majority of axis cylinders in this location at the adult rabbit had further increased in size over that of a 40-day-old rabbit. The average diameter of axis cylinders of 40-day-old rabbits was 33 times larger than that of the new born, whereas the average diameter of axis cylinders of an adult rabbit was 3% times larger. DISCUSSION The pregnancy cycle of rabbits is short (31 days), and at birth these animals are still very immature; their eyes, for example, are not open until they attain their 12th day of life. For this reason they proved to be a suitable experimental animal for the study of myelination, maturation of cellular elements, and for investigation of possible interrelationship among these components. AXONS, OLICODENDROGLIA A N D MYELINATION 119 The first evidence of deposition of myelin in the posterior funiculi of rabbits occurred at the stage of about two days before birth. At birth the myelin content was still very slight. Subsequently, until about the 10th day of life, the myelin content in the posterior funiculi advanced at a very rapid pace, increasing its amount by 4 times over that at birth. From the 12th day onwards until about the 40th day there was very little increase of myelin content as estimated in an area of the same size. Beyond the 40th day up to adulthood there was no change in the myelin content as estimated by the method of light absorption in the same sized area. However, the transverse diameter of the myelin sheath continued to increase slightly in size beyond the stage of 40 days as revealed by measurements. This discrepancy can be explained by the fact that the transverse diameter of the spinal cord continued to increase in size, whereas the number of its cellular elements remained constant, although they became larger and less densely distributed. I n consequence, the photovolt readings of the transmitted light through the same sized area of the 'section stained for myelin were practically unchanged between the ages of 40 days and adulthood. I n spite of this, the method had to be adopted because the measurement of the transverse diameters of single myelin sheaths in very early stages is impossible, as its deposition occurred in an irregular fashion around the axis cylinders. Clearly, for the purpose of this study the stage of early growth was of chief importance. The number of oligodendrogliacytes in the posterior funiculus from the second day of life onwards remained quite constant, showing only minimal fluctuations which can be explained by individual variation in the size of animal and by possible counting error. By means of the staining methods used it was possible to distinguish 4 stages in the development of oligodendroglia. The first stage consisted of a precursor cell: unipolar, bipolar and apolar spongioblasts ; second -of oligodendrogliablast with large hyperchromatic nucleus ; third 120 - of ANATOLE DEKABAN young oligodendrogliacyte with small nucleus but still abundant chromatin; finally, the 4th stage - of adult oligodendrogliacytes possessing smaller nucleus and scanty chromatin. The spongioblasts in the posterior columns of rabbits have largely disappeared by the second day of life. Occasional oligodendroblasts were still seen in animals 4 days old. The change of characteristics of young oligodendrogliacytes t o adult form occurred between the 13th and 16th day after birth. The proliferating activity of oligodendroglia of new born rabbits was not an isolated phenomenon, as there were also mitotic figures present in other cell series. It was felt, therefore, that the appearance of myelination in a rabbit of 29 days' gestation age and the presence of active proliferation of oligodendroglia at this stage may be coincidental and should not necessarily be interpreted as evidence of their fuiictional interrelationship which was inferred by some investigators (Penfield, '28). Certainly, between the 2nd and 10th day of rabbit life the myelination in the posterior funiculi progressed at a very rapid pace although the number of oligodendroglia in the same region remained quite constant. Also, the large and hyperchromatic nucleus of young oligodendrogliacytes were the common characteristics of all maturing cells and cannot be either accepted as evidence of their specific activity such as the production of myelin. The transverse diameter of axis cylinders in the posterior funiculi u7as observed t o continue increasing in size from the early stage of development t o adulthood as may be seen in figure 2 C. This growth was somewhat more rapid in a very young animal than in the stage of approaching adulthood. The analysis of data derived from this study did not reveal the presence of a relationship between the progressive deposition of myelin and the number of oligodendroglia cells during maturation of rabbits. Unfortunately, there are no methods available which would allow estimation of the functional activity of these cells ; the histological characteristics of developing oligodendroglia as described above mere of AXONS, OLIGODENDROGLIA A N D M Y E L I N A T I O N 121 similar order as those of other developing cell series. The question then, as to whether or not the oligodendroglia participate in the deposition or maintenance of myelin, cannot be answered definitely at the present time. This is in spite of the suggestive finding that oligodendrogliacytes largely disappear from the demyelinated areas. There does exist a parallelism between the progressive increase in the transverse diameter of myelin sheaths and that of the corresponding axis cylinders. This fact, in addition to the findings in Wallerian degeneration, may indicate a degree of metabolic interdependence between the axis cylinders and myelin sheaths enveloping them. SUMMARY Applying quantitative and qualitative methods, the oligodendroglia and axis cylinders were studied during the process of myelination in rabbits. It was found that the onset of myelination in the posterior funiculi of this animal occurred on the 29th day of gestation time. Up to about the 10th day of life myelination progressed at a very rapid pace, increasing its amount 4 times over that of a new born animal. From the 12th day of age onward there was only a slight increase in myelin. The increase of the transverse diameter of the majority of medium sized axis cylinders in the posterior funiculi progressed slowly but steadily up to adulthood. I n a new born rabbit there were 156 oligodendroglia cells in a transverse section of one posterior funiculus, 5 mm below the obex. These cells were increasing in number only slightly up to the age of about 4 days. From the 6th day onward the number of oligodendroglia cells in this location was quite constant, averaging from 218 to 231 cells. Histological characteristics of the developing oligodendroglia were considered to be of a similar nature as those seen in other cell series. No relationship could be demonstrated between the myelination and the number of oligodendroglia during maturation of rabbits. The parallelism between the progressive increase in the transverse diameter of the axis cylinders and of the myelin sheaths enveloping them may suggest a degree of meta- 1.22 ANATOLE DEKABAN bolic interdependence between these two structures. This is further strengthened by the findings in Wallerian degeneration. The author wishes to acknowledge his gratitude to Miss Marie Kendall for efficient technical assistance and f o r the preparation of the histological sections. LITERATURE CITED BRAIN,W. R., AND J. G. GREENFIELD 1950 Late infantile metachromatic encephalopathy with primary degeneration of the interfascicular oligodendroglia. Brain, Y3 : 291-317. CAJAL,S. R. 1913 Contribucione a1 conocimiento de la neuroglia del cerebro humano. Trab. del lab. de inv. biol., 11: 255-315. CONTI, R. G., J. 0. BLANDAND D. 5. RUSSELL 1937 Tissue culture of gliomata. Cinematograph demonstration. A. R. N. M. D., 2 6 : 1-24. CRANER,F., AND B. J. ALPERS 1932 The function of the glia in secondary degeneration of the spinal cord. Arch. Path., 13: 23-55. CREUTZFELD'P,H. G., AND A. METZ 1926 Ueber Gestalt und Tatigkeit der Hortegazellen bei pathologischen Vorgangen. Ztschr. f . d. ges. Neurol. u. Phychiat., 106: 18-53. DEL RIO-HORTEGA, P. 1921 La glia, de escasas, radraciones (oligodendroglia) . Bol. SOC.espan. de hist. nat., 21: 63-92. 1928 Tercera aportacion a1 conocimiento morf ologico e interpretacion functional de la oligodendroglia. Men. de la R. Soe. ESP. de Hist., 2 4 : 5-122. Madrid, Museo Nacional de Ciencias Naturales. FERRARO, A., AND L. M. DAVIDOFF1928 Reaction of oligodendroglia to injury of brain. Arch. Path., 6: 1030-1053. GLOBUS, J. 1928 Glia response in chronic vascular disease of brain. Arch. Neurol. and Psychiat., 20: 14-33. GREENFIELD,J. G. 1933 A form of progressive cerebral sclerosis in infants associated with primary degeneration of interfascicular glia. Roy. SOC. Med. Proc., 2 6 : 690-697. JAKOB, A. 1927 Normale und pathologische Anatomie und Histologie des Grosshirns. Leipzig. Vol. 1. LUMSDEN,D. E. 1951 Fundamental problems i n the pathology of multiple sclerosis and allied demyelinating diseases. Brit. Med. J., 1 : 1035-1043. MEDUNA, L. 1937 Beitrage zur Histopathologie der Mikroglia. Arch. f. Psychiat., 82: 123-193. PENFIELD, W., IN COWDRY, E. V. 1928 Special Cytology. New York, Paul Hoeber, I : 1032-1068. POMERAT, C. M. 1951 Pulsatile activity of cells from the human brain in tissue culture. J. Nerv. and Ment. Dis., 114: 430-449. I(. 1926 Ueber die Hortega Microglia. Ztschr. f. Anat. u. EntwickSCH~FFER, lungs., 81: 715-719.