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Oligodendroglia and axis cylinders in rabbits before during and after myelination.

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Shaughnessy Hospital and the Department o f Medicine,
Ti. B . C . Medical School, Vancouver, B. C.
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.
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.
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.
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
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
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.
TI. sc CT. or. s?.
C O R D IN ao.nn.
a 800 boo0
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
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
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
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
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.
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
- of
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
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
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.
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-
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.
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axis, oligodendroglioma, myelination, rabbits, cylinder
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