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On the occurrence of microtubules within mature astrocytes.

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On the Occurrence of Microtubules within
Mature Astrocytes
CEDRIC S. RAINE AND HENRYK WISNIEWSKI
Department of Pathology (Neuropathology ),
Albert Einstein College of Medicine,
Bronx, N e w York 10461
Microtubules have been described to occur frequently within the subABSTRACT
pial fibrous astrocytes of the adult spinal cord of three species. This finding is related
directly to perfusion fixation by glutaraldehyde. Immersion fixation produced no or
fewer tubules in this area. It is suggested that the presence of microtubules in these
cells might reflect a specific skeletal function. Astrocytes from other locations within
the central nervous system displayed fewer microtubules within their cytoplasm.
During a recent study on experimental
demyelination and remyelination (WiSniewski et al., '69; Raine et al., '69; Prineas et
al., '69) it was observed that within the spinal cord, the subpial astrocytes and those
penetrating the base of each spinal nerve
root contained an abundance of microtubules in addition to bundles of filaments
(Prineas et al., '69). The existence of microtubules within fibrous astrocytes has never
been emphasized as a diagnostic criterion
for the identification of this cell type (Farquhar and Hartman, '57; Schultz et al., '57;
Palay et al., '62; Mugnaini and Walberg,
'64; Wendell-Smith et al., '66; Kruger and
Maxwell, '67). The validity of this observation forms the subject of the present
report in which the ultrastructure of fibrous astrocytes from several locations
within the central nervous system (CNS)
of three species has been examined.
hours, dehydrated through a graded series
of ethyl alcohol, passed through propylene
oxide and epon, and embedded flat in capsules and silicone rubber molds. Slices of
cord fured by simple immersion in buffered
glutaraldehyde alone, in buffered glutaraldehyde followed by post-osmication, and
Dalton's chrome osmium alone, were taken
from one rabbit and processed identically
for comparison with glutaraldehyde perfused material. Sections 1 thick, stained
with 1 % toluidine blue were examined
light microscopically prior to sectioning
for grids. Ultramicrotomy was carried out
on Porter-Blum MT1 and Reichert OMU2
microtomes; thin sections were stained
with lead citrate and uranyl acetate (Venable and Coggeshall, '65), and scanned in
a Siemens Elmiskop 1A.
RESULTS
The subpial fibrous astrocytes of the
spinal cord were intimately bounded along
Twenty-four adult animals, 22 New their leptomeningeal surfaces by a 600 A
Zealand albino rabbits, one cat, and one thick basement membrane (fig. 1). The
dog were used for this study. Animals were cell surface beneath this membrane conanesthetized with intraperitoneal sodium tained periodic osmiophilic regions ("halfpentobarbitone prior to sacrifice. Fixation desmosomes" - Hirano et al., '69), and
was achieved by arterial perfusion through between these superficial cells and their
the left ventricle with 100 ml of 4% para- processes, frequent desmosome-like juncformaldehyde followed by 5 1 of 5% gluta- tions could be seen. Astrocytes in these reraldehyde in 0.1 m phosphate buffer at gions provided a tight continuous covering
pH 7.3 (WiSniewski et al., '69). Following over the white matter, varying in thickness
fixation, coronal sections of the brain from a single layer of processes to several
1-2 mm thick, and transverse sections of whole cells. Close examination revealed
spinal cord at C2 and L6, were post-fixed that each of the astrocytic cell processes
in Dalton's chrome-osmium at 4°C for 1-2
Received Nov. 7 , '69. Accepted Jan. 28, '70.
MATERIALS AND METHODS
ANAT. REC., 167: 303-308.
303
304
CEDRIC
s.
RAINE AND HENRYK W I ~ N I E W S K I
Fig. 1 Rabbit spinal cord; subpial layers. Superficial astrocytic processes are covered by a basement membrane ( B M ) . Superficial cell membranes show periodic densifications and some processes
have small desmosome-like junctions (arrows). Microtubules ( T ) can be discerned within astrocytic
processes. Nonmyelinated fibers occur below. x 22,000.
(less obviously the somata) contained compact bundles of filaments 90 A in diameter,
each associated with accompanying discrete collections of 250 A microtubules
(fig. 2). The latter were absent when osmium immersion fixation was used (fig.
3 ) . Microtubules were found within the
subpial astrocytes of all three species studied and many displayed a central osmiophilic core with a diameter of approximately 40 A (figs. 2, 4 ) . Essentially, the
dimensions of these microtubules were
identical to those described in developing
astrocytes (Peters and Vaughn, '67), and
were ultrastructurally indistinguishable
from normal neurotubules.
The presence of the accompanying fibrillar bundles and desmosome-like junctions
between cell processes facilitated the differentiation of small glial processes from
non-myelinated nerve fibers (fig, 1 ) . In
transverse section these filaments could be
seen to contain a 20-25 A lumen (fig. 2).
Microtubules, under the prevailing perfusion fixation conditions proved to be a
common constituent of astrocytes throughout all the areas of the CNS studied. However, nowhere were microtubules as common and precisely arranged as within the
subpial layers of the spinal cord. Those
astrocytic processes seen to penetrate the
spinal nerve root entry zones also contained bundles of both microtubules and
filaments (Prineas et al., '69). Astrocytes
situated within deeper layers of the spinal
white matter, the anterior horns, and sub-
MICROTUBULES WITHIN ASTROCYTES
305
Fig. 2 Rabbit spinal cord; subpial layers. A n astrocytic process is seen i n transverse section to
contain filaments (some displaying a lumen), and several microtubules to the right. A central density can be seen i n one tubule (arrow). Desmosome-like contact at top. Glutaraldehyde perfusion.
x 102,000.
Fig. 3 Rabbit spinal cord; subpial layers. A n astrocytic process is seen to contain n o microtubules, only filaments. Osmium immersion. X 80,000.
Fig. 4 Cat spinal cord; subpial layers. A n astrocytic process close to the soma contains many
tubules, note central density (arrow), interspersed between filaments and mitochondria, Glutaraldehyde perfusion. x 40,000.
306
CEDRIC
s.
RAINE AND HENRYK
WISNIEWSKI
Fig. 5 Rabbit spinal cord; subependymal layem. A fibrous astrocyte contains scattered microtubules (arrows). Note tubules in adjacent astrocytic cell process above. x 30,000.
ependymal layers contained scattered microtubules between bundles of filaments
(fig. 5). The subpial astrocytic layer around
the cerebral hemispheres was observed to
comprise layers of fibrous astrocytic processes in which microtubules, though usually present, were not numerous. The cytoplasm of these cells was more electronlucent than their spinal cord counterparts,
and contained glycogen granules. Oligodendrocytes with their denser cytoplasm and
containing only microtubules, i.e., no filaments (Mugnaini and Walberg, ’64; Kruger and Maxwell, ’66; Raine and Bornstein,
in press), were common and readily identifiable.
Spinal cord tissue fixed by immersion
in glutaraldehyde with or without subsequent post-osmication, while displaying
many neurotubules, contained only scattered astrocytic microtubules between bundles of filaments. On the other hand, however, tissue fixed by osmic immersion
revealed no astrocytic microtubules whatsoever (fig. 3). As a consequence of immersion fixation, general preservation was
poorer and the astrocytes contained a
“watery” cytoplasm, not evident after perfusion.
DISCUSSION
Under conditions of glutaraldehyde perfusion, microtubules are invariably found
within fibrous astrocytes. There was, however, a marked variance in the microtubule
populations encountered. Spinal cord subpial astrocytes contained more microtubules than astrocytes located elsewhere in
the CNS. The higher frequency of microtubules within these subpial astrocytes
compared with those from deeper layers of
the cord, suggested either that there was a
gradient of good fixation in these areas
(i.e., the quality of good fixation decreased
towards deeper layers), or more probably,
that the subpial astrocyte is inherently endowed with a more elaborate system of
microtubules. Such a system may serve
some specific skeletal or physiological
function in this area, more likely the former in view of the peripheral location of
the cell and the fact that microtubules
are known in many other instances to be
an essential element in the maintenance
MICROTUBULES WITHIN ASTROCYTES
of form (Tilney and Byers, '69). Ultrastructurally, this cell type possessed both
astrocytic (filaments) and oligodendrocytic (tubules) features. However, its proliferation activity in disease (Prineas et
al., '69; Bunge et al., 'Sl), its fibrillar content, and its location, identified it as astrocytic. The "transitional" macroglial cell
recognized by Bunge et al. ('61) and Bunge
and Glass ('65), during their study on
experimental remyelination, because of its
subpial location and proliferative activity,
was probably the same cell as that under
discussion here. Its ability to partake in the
process of remyelination was postulated
by Bunge et al. ('61). In a more recent
study on experimental remyelination in
this region of the CNS, this cell type appeared not to be involved in the process
of remyelination (Prineas et al., '69), and
that function was carried out by reactive
oligodendrocytes.
The majority of reports on the ultrastructure of the subpial astrocytes of the
CNS (using osmium or aldehyde fixation
by immersion and perfusion) have failed
to demonstrate the existence of microtubules in these cells (Lin et al., '60; Nelson
et al., '61; Ramsey, '65; Waggener and
Beggs, '67), although similar findings regarding the presence of a covering basement membrane and intercellular junctions were reported. Their occurrence, however, was briefly reported by Hirano et al.
('69), during a recent study on remyelination. Brightman and Reese ('69), have also
demonstrated microtubules within astrocytes quite convincingly (fig. 4, loc. cit.)
without drawing attention to the fact. Astrocytic microtubules are known to occur
in the developing optic nerve (Peters and
Vaughn, '67; Vaughn and Peters, '67) although the authors preferred to consider
this organelle a rarity in the adult cell
owing to its postulated transformation during development into filaments, and consequently de-emphasized its presence in
the mature cell. The present study shows
that within the mature CNS of the three
species studied, microtubules were always
evident, and were more common within
subpial astrocytes than those from other
sites within the CNS.
307
Microtubules have not been considered
a normal component of astrocytic cytoplasm by previous workers who have contributed to macroglial cell classification
(Farquhar and Hartman, '57; Schultz et
al., '57; Palay et al., '62; Mugnaini and
Walberg, '64; Wendell-Smith et al., '66;
Kruger and Maxwell, '66). The present
study demonstrates that like most mammalian cells, the astrocyte also contains
microtubules. In spite of their paucity (or
absence, in the case of osmium immersion) when perfusion fixation was not
used, axoplasmic microtubules (neurotubules) could usually be found. It would
appear therefore that astrocytic microtubules are more sensitive to the type of fixation employed and might indeed be different chemically from neurotubules.
ACKNOWLEDGMENTS
The authors thank Dr. Robert D. Terry
for his advice throughout this study. The
technical assistance of Karen Berkman,
Cyrilla Ho, Larry Gonzalez and Sidney
Gravney is gratefully acknowledged.
This work was supported by grants
NS 02255 and NS 03356 from the National Institutes of Health, and grant 600A-2 from the National Multiple Sclerosis
Society.
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