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Fine structure of regenerated ependyma and spinal cord in Sternarchus albifrons.

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THE ANATOMICAL RECORD 205:73-83 (1983)
Fine Structure of Regenerated Ependyma and
Spinal Cord in Sternarchus albifrons
MARILYN J. ANDERSON, STEPHEN G.WAXMAN, AND MICHAEL LAUFER
Department of Neurology, Veterans Administration Medical Center and Stanford
University School of Medicine. Pa10 Alto, CA 94304
ABSTRACT
The caudal-most regenerated spinal cord in Sternarchus albifrons consists solely of an ependymal tube. Ependymal cells are enlarged radially
and are more numerous than in unregenerated cord. Projections of ependymal
cell cytoplasm and Reissner’s fiber fill most of the central canal. Small groups of
neurites and cell processes filled with dense-cored vesicles lie between abluminal
processes of ependymal cells.
Rostra1to this, additional cells appear dorsal and lateral to the inner ependymal
layer. Some cell bodies contain numerous dense-cored vesicles. Larger bundles of
neurites, some with synapses, are present. Invaginations of the peripheral edge
of the cord create enclosed spaces lined with basal lamina. In the peripheral
region, longitudinally oriented neurites extend through extracellular spaces or
channels. The ventral portion a t some levels of regenerated cord is completely
filled with neurites, processes containing dense-cored vesicles, and capillaries.
Similar masses of neurites and processes containing dense-cored vesicles lie outside the cord proper, in or near the meningeal layer.
In rostral-most sections, the organization of regenerated spinal cord approaches
that of normal cord, with the regenerated cord exhibiting groups of myelinated
axons, differentiated fibrous astrocytes and oligodendroglia,cell bodies containing
dense-cored vesicles, and differentiated electromotor neurons. These observations
indicate a degree of pluripotency in some of the ependymal cells in adult Sternarchus. Moreover, they are consistent with a role of ependymal cells in the
guidance of regenerating neurites.
It has long been recognized that the vertebrate central nervous system does possess some
regenerative capability after injury. In mammals, limited functional recovery and regrowth of axons may occur after injury to the
spinal cord (Bernstein and Bernstein, 1973;
Guth, 1975; Puchala and Windle, 1977). Various experimental manipulations are reported
to enhance regeneration of mammalian spinal
axons: Application of an electromagnetic field
(Wilson and Jagadeesh, 1976), grafting peripheral nerve into the injured cord (Kao et al.,
1977; David and Aguayo, 1981), wrapping the
cut end of the cord with millipore filter (Scott
and Liu, 19641, and adding various chemical
agents (McMasters,1962;Bernstein et al., 1978;
Feringa et al., 1979).Despite these provocative
approaches, regeneration in mammalian spinal
cord is limited and the mechanism underlying
it appears to be largely the regrowth of cut
axons, rather than the generation of new nerve
cell bodies.
More complete regeneration of the spinal cord,
in some cases including the generation of new
neurons, occurs in lower vertebrates: teleosts,
urodele amphibians, and lizards (Kirsche, 1951;
Butler and Ward, 1967; Simpson, 1968; Bernstein and Gelderd, 1973). In lizards, the regenerated spinal cord is comprised of an ependymal tube and descending nerve fibers (Egar
et al., 1970). In addition, in newt spinal cord
new neuronal cell bodies are regenerated after
amputation of the tail (Egar and Singer, 1972).
In regenerated spinal cord of the teleost Sternarchus a1bifrons, new electromotor neurons
are produced in even greater numbers than are
present normally (Anderson and Waxman,
1981).
One important problem concerns the differences between the inframammalian spinal cord,
which readily regenerates, and mammalian
Received April 29,1982;accepted September 28,1982.
0003-276X/83/2051-0073$03.500 1983 ALAN R. LISS, INC.
74
M.J. ANDERSON, S.G. WAXMAN, AND M. LAUFER
cord, which does not, The structure and role of
the ependyma are undoubtedly important in
the ability of inframammalian spinal cord to
regenerate. Enlargement and proliferation of
ependymal cells has been noted as one of the
first steps in regeneration after amputation of
the tail in amphibians and lizards (Simpson,
1968; Nordlander and Singer, 1978) and also
after transection of the spinal cord in larval
lampreys (Wood and Cohen, 1981). The sequence of generation of new cells from the
ependymal layer has been described in detail
in amphibians (Egar and Singer, 1972; Nordlander and Singer, 1978) and lizards (Egar et
al., 1970). In addition, the presence of ependyma is necessary for regeneration of spinal
cord and other structures of the tail in salamander (Goldfarb, 1909) and lizard (Kamrin
and Singer, 1955; Simpson, 1964).
The ependymal cells may also influence or
guide the outgrowth of regenerating neurites
through regenerating spinal cord. Ependymal
cell processes are in close contact with outgrowing axons in regenerating Xenopus spinal
cord (Michel and Reier, 1979) and may influence their growth by direct contact. In regenerating and embryonic spinal cords of some
species, longitudinal channels, formed by spaces
between ependymal cell processes, have been
observed to anticipate the appearance of neurites. It has been proposed that such channels
may guide neurite outgrowth through the cord
(Singer et al., 1979; Nordlander and Singer,
1978, 1982).
Inframammalian systems provide important
models for the study of spinal cord injury and
regeneration in several major ways. First, regeneration can initially be studied more easily
in a system where it unequivocally occurs; results may then be extrapolated to systems (such
as mammalian spinal cord) where regeneration occurs incompletelyif at all. Secondly, some
of the basic processes of regeneration, e.g. regrowth of nerve fibers, are common to both
mammals and inframammalian vertebrates.
Thus, information gained from the study of
spinal cord regeneration in lower vertebrates
may be in some cases applicable to the mammalian (nonregenerative) nervous system.
The teleost Sternarchus albifrons represents
an especially advantageous system for study
of both spinal cord and peripheral nerve regeneration. First, spinal cord regeneration, including the generation and differentiation of
new neurons, occurs readily after amputation
of the tail (Anderson and Waxman, 1981). Second, the spinal cord of Sternarchus contains a
class of easily identified neuronal perikarya,
whose axons peripherally comprise the electric
organ of this weakly electric fish (Waxman et
al., 1972; Pappas et al., 1975). The electrocyte
axons run a highly distinctive and stereotyped
course and have a very specific pattern of myelination which varies along the length of the
axon. They also contain two types of nodes of
Ranvier: normal, excitable nodes (Type I) and
large, inexcitable (Type 11) nodes. Thus, the
electrocyte axons provide a good system for
studying the factors which guide nerve outgrowth and influence myelination. Moreover,
the electrocyte axons are asynaptic, ending
blindly in the electric organ. Thus, these cells
are amenable to the investigation of the dependence of certain properties of neurons on
synaptic connections, e.g. chromatolysis (Waxman and Anderson, 1982) and cell death (Anderson et al., 1982). Lastly, the production in
regenerated spinal cord of significantly more
electrocytes than normal suggests this system
as a possible model in which to study the stimulation and control of cell division in response
to injury. In light of the potential usefulness
of this system of identifiable regenerating neurons, we have undertaken this description of
the ultrastructure and relationship of neuronal and ependymal elements in the regenerating spinal cord of Sternarchus albifrons.
MATERIALS AND METHODS
To initiate regeneration, tails were amputated from Sternarchus anesthetized in a
1:15,000 solution of tricaine methanesulfonate
(MS 2221, and the fish were returned to their
tanks. After a regeneration period of 3 weeks,
4 months, or 1 year a t 26" C, the regenerated
spinal cord was fixed either by perfusion or
direct immersion in a solution of 2.5%glutaraldehyde in 0.1M sodium phosphate buffer with
2.5%dextrose and 0.01%CaC12, pH 7.35, 350
mOsm. The spinal cord was dissected in fixative, then immersed in a fresh vial of fixative,
for a total of 3 hours fixation (at room temperature). The tissue was washed in 4 changes
of the phosphate buffer over 45 minutes, and
postfixed for 2 hours in 2% OsOlin the phosphate buffer, a t room temperature. The tissue
was then washed for 2 hours in 0.2M acetateacetic acid buffer (pH 5) and stained en bloc
with uranyl acetate. Tissue was embedded in
Epon-araldite and thin sections were stained
with lead citrate and uranyl acetate prior to
observation on a JEOL 100 CX electron microscope.
REGENERATED SPINAL CORD
75
Fig. 1. Transverse section of caudal regenerated ependyma, 8.5 mm from the site of transection in a 4-month
regenerated fish. Central canal is largely filled with Reissner's fiber (R) and protrusions of ependymal cell cyto-
plasm (arrows). N, ependymal cell nucleus; M, meningeal
layer. Inset shows abluminal cell processes filled with densecored vesicles. x 5,400; inset, x 11,400.
RESULTS
radial axis, and are more numerous than in
the intact spinal cord. We have counted up to
86 cells bordering the central canal in a regenerated spinal cord, as opposed to an average
of 36 cells in normal, unregenerated ependyma. Lateral borders of the caudal regenerated ependymal cells are generally straight.
Ependymal cell cytoplasm contains many ribosomes and polyribosomes, rough endoplasmic reticulum, mitochondria, Golgi apparatus, clear vesicles, and occasionally a
multivesicular body. The Golgi apparatus is
always located in the apical (luminal) half of
the cell. The nuclei are large, usually oval, and
have dispersed nucleoplasm or small patches
of condensed chromatin. The regenerated
ependymal cells are of one uniform cytoplasmic
density.
Large irregularly shaped projections of the
apical border of caudal ependymal cells (Fig.
1) extend for distances up to 3 km into the
central canal, where they fill much of the space.
Reissner's fiber is present in the central canal,
We have examined regenerated spinal cord
in 15 fish, which had regenerated for periods
ranging from 3 weeks to 1 year. This paper
primarily presents results from one typical
specimen, a 4-month regenerate. Sections a t
different levels along the rostro-caudal axis of
the regenerated spinal cord from this animal
will be presented, as well as sections from two
other fish, which regenerated €or different periods of time. Regeneration of the tail in Sternurchus occurs a t an initial rate of 0.6-1.7 mm
per week at 26" C. After 3-4 months, regeneration is slower. Regenerated spinal cord close
to the site of transection is of normal diameter,
and decreases in diameter as the spinal cord
progresses caudad.
The caudal region of regenerated spinal cord
in a fish with a four month regeneration period
is presented in Figure 1.At this level (Fig. l),
the cord consists solely of a tube of enlarged
ependymal cells which are elongated along the
76
M.J. ANDERSON, S.G. WAXMAN. AND M. LAUFER
along with many cilia extending from the
ependymal cells. The cilia contain the typical
9 x 2 arrangement of doublet microtubules.
Cross-striated rootlets with dense bands a t a
periodicity of 600-700 A, are sometimes seen
extending from the basal bodies of the cilia for
several pm into the apical cytoplasm. Adjacent
ependymal cells are joined a t their luminal
edges by junctional complexes, followedby desmosomes along the lateral edges. Long desmosomes and series of 3-5 closely spaced desmosomes are common. Except for the terminal
bar, tight junctions or gap junctions between
ependymal cells are not seen in these caudal
sections.
Large cell processes packed with dense-cored
vesicles occur in the abluminal region between
some caudal ependymal cells (inset, Fig. 1).
The membrane-boundvesicles are 800-1400 A
in diameter. Some of the vesicles appear less
dense than others. A few mitochondria, microtubules, and 100 A filaments are seen amidst
the dense-coredvesicles in such processes. While
caudal-most sections of regenerated ependyma
have only a few of these vesicle-filledprocesses,
they are more frequent as one progresses rostrally in the regenerated cord. Dense-cored
vesicles are also frequently found in typical
neurites within the regenerating ependyma,
and in the meningeal layer adjacent to the
ependyma.
Individual fibers and small bundles of neurites are seen in the abluminal region of caudal
ependyma (Fig. 2, inset). The neurites are in
close proximity to adjacent ependymal cell processes. Large empty spaces between ependyma1 cells were not observed. Neurites in this
region of regenerated cord are unmyelinated
and are oriented parallel to the longitudinal
axis of the ependyma. No synapses are seen
between neurites in the caudal-most sections.
Neurites can occur close to the central canal
(within 1.5 pm), although they are more usually found near the periphery. Neurites are
also frequently located outside the regenerating spinal cord in the meningeal layer and beyond it. Such extra-spinal neurites are always
surrounded by glial processes, and are often
grouped together inside a ring of fibroblastic
processes and collagen. In sequential sections
progressing caudally from the section shown
in Figure 1,a large myelinated axon was seen
first external to the meningeal layer, then inside the regenerating ependyma, lying between the abluminal portions of ependymal
cells.
Rostra1 to the section in Figure 1, regenerated ependyma has several layers of cells surrounding the central canal (Fig. 2). There is
no clear demarcation separating the cells immediately lining the central canal and those
more peripherally located. One of the peripheral cells in Figure 2 (asterisk) has a dispersed
nucleoplasm and a cytoplasm distinctly different from that of the ependymal cells, thus representing one of the first differentiating cells
in the regenerating spinal cord. This cell also
has dense-cored vesicles in its cytoplasm.
Ependymal cells in this section are joined, as
they are more caudally, by a terminal bar and
series of desmosomes. Gap junctions are not
seen between ependymal cells at this level of
regenerating cord. Projections of ependymal cell
cytoplasm into the central canal are smaller
than in the more caudal section, while the large
Reissner’s fiber occupies much of the space in
the central canal.
The peripheral edge of the regenerating
ependyma (Fig. 3) is bounded by an external
lamina. Outside the external lamina, a layer
of longitudinally oriented collagen, 1-5 pm
thick, lies between the ependyma and the surrounding meningeal cells. The basal portions
of ependymal cells are convoluted, interdigitating among each other. Desmosomesjoin adjacent cell processes, even a t the very edge of
the ependyma (Figs. 3, 4). Invaginations and
infoldings of the extra-ependymal space,
bounded by the external lamina, frequently occur at the edge of the cord (Fig. 3). In some
cases the basal lamina appears attenuated in
these invaginations. The spaces lined with basal
lamina can be relatively large, up to 10 pm
along their major axis. In more rostra1 sections, such lamina-lined spaces also occur more
internally in the regenerated cord, and neurites have occasionally been seen in them.
A large bundle of neurites from regenerated
spinal cord is shown in Figure 4. Progressing
rostrally in the regenerated cord, neurite bundles increase in size and may have some distinctly larger fibers at the edge of the bundle.
A synapse, characterized by clear vesicles and
a density a t the pre- and post-synaptic membrane, can be seen in the upper part of this
figure. Dense-cored vesicles can occur in both
pre- and post-synaptic processes. A second specialized contact, seen in the lower portion of
the micrograph, may be an axo-glial synaptoid
relationship. Clear vesicles joining with the
plasma membrane, reminiscent of micropinocytosis, are seen at borders between cells and
at the peripheral edge of the cord. As in the
more caudal sections, neurites lie close to the
surrounding ependymal cell processes.
A section of regenerated spinal cord from a
different fish (with a 3-week regeneration period) shows many layers of cell bodies lateral
Fig. 2. Regenerated spinal cord 1.5 mm rostra1 to the
section in Figure 1. Additional cells are present lateral to
the immediate ependymal layer. One of these cells (asterisk)
is differentiated morphologically from Lie ependymal cells,
and contains dense-cored vesicles in its cytoplasm. Central
canal with Reissner’s fiber at lower left; invaginations of
peripheral border of cord (arrowhead, and Fig. 3); neurite
bundle (N, and Fig. 4). Inset shows neurites extending in
close contact with ependymal cell borders, located 1.6 pm
from the central canal in a second regenerated spinal cord.
X 5,000; inset, x 13,800.
Fig. 3. Edge of regenerated spinal cord from Figure 2.
An external lamina lines the periphery of the cord and the
enclosed spaces (arrows). Outside this lamina lie longitudinally-oriented collagen fibrils (C). Cell processes at the
edge of the cord are interdigitated and are frequently joined
by desmosomes. x 30,100.
78
M.J. ANDERSON, S.G. WAXMAN, AND M. LAUFER
Fig. 4. Neurite bundle from a section of regenerated spinal
cord close to Figure ?,. Large neuritea surround the distal
edge of the bundle. A synapse (wide arrow) is present near
top. A second synaptoid profile in lower half (thin arrow)
may represent an axo-glial contact. Note desmosomes near
outer edge of spinal cord (top), and basal lamina (B) in enclosed space. Micropinocytotic-like vesicles join with plasma
membranes a t the periphery (inset and arrowheads) and a t
lateral cell borders (arrowhead). X 17,800;inset, x 49,400.
79
REGENERATED SPINAL CORD
to the immediate ependymal layer (Fig. 5).Cells
appear to progress radially away from the
ependyma. The cells close to the ependymal
layer resemble the ependymal cells in shape
and scant cytoplasmic content. More peripheral cells are polygonal in shape. Some peripheral cells have an electron dense cytoplasm
and resemble oligodendroglia; other cells contain prominent dense bodies or lysosomes. As
in the 4-month regenerated ependyma (Fig. 2),
there is no demarcation separating the immediate ependymal layer from the more peripheral cells. Near the edge of the cord in this
specimen, groups of unmyelinated neurites extend through extracellular spaces (inset, Fig.
5 ) which resemble the channels described by
Singer et al. (1979).A light flocculant material
is also present in some of these channels. The
channels are located in the rostral portion of
this 3 week-regenerated tail, 2 mm from the
end of the tail.
In the 4-monthregenerated spinal cord, many
cell bodies and cell processes containing densecored vesicles are present. In some regions, virtually the entire ventral half of the cord is filled
with neurites and processes containing densecored vesicles, interspersed with capillaries and
epithelial cell processes (Fig. 6). Many of the
processes are totally filled with clear and densecored vesicles, and abut directly upon the basal
lamina of a capillary. However, no signs of vesicle depletion or secretion into the capillaries
were observed. Dense-cored vesicles have not
been observed in myelinated axons, nor in the
central canal.
The cell bodies containing dense-cored vesicles are 10-20 pm in diameter and very irregular in shape, often having bulbous or long
thin projections which also contain the densecored vesicles. The vesicles are sometimes
closely associated with the Golgi apparatus.
Caudally, the cells containing dense-coredvesicles are always located near the periphery of
the regenerated cord. In more rostral sections,
they are found in many locations, including the
area just lateral to the ependymal cells. The
cells containing dense-cored vesicles receive
multiple synapses from unmyelinated axons.
The frequency of dense-cored vesicles in cells
and processes in the regenerated spinal cord is
especially striking in comparison to their infrequent occurrence in unregenerated spinal
cord at the same spinal level and in cord anterior to the transection in the operated fish.
In the latter two cases, one or two dense-cored
vesicles are found in a few neurites, and no cell
bodies containing dense-cored vesicles are seen
(see Anderson and Waxman, 1982, pp. 85-92,
this volume).
Rostra1 sections of the 4-month regenerated
spinal cord (1-5 mm from the site of transection) are characterized by the presence of many
myelinated axons, large masses of unmyelinated neurites near the periphery, and by differentiated oligodendroglia,fibrous astrocytes,
motor neurons and the differentiated electromotor neurons (Anderson and Waxman, 1981).
The differentiated electrocytes are identified
by their size, spherical shape, lack of dendrites,
and their cytoplasm packed with mitochondria
and ribosomes. Fibrous astrocytes are plentiful, and a well-defined glia limitans is present.
Cells with electron-dense cytoplasm, presumably oligodendroglia, are frequently seen partially surrounding small-diameter myelinated
axons. The ependymal layer is clearly distinct
from adjacent cells and axons. Some ependyma1 cells have a long, fiber-filled process which
extends deeply into the neuropil. Gapjunctions
are seen between lateral borders of ependymal
cells.
In contrast to the more central position of
cell bodies in normal, unregenerated cord, differentiated cell bodies of both the electrocytes
and the cells containing dense-cored vesicles
can occur a t the very edge of the regenerated
cord. Although the number of cell bodies containing dense-cored vesicles decreases in rostral regenerated cord, degeneration or death
of these cells has not been observed. Cell processes filled with both clear vesicles and densecored vesicles are seen in rostral regenerated
cord, but are more frequent in the caudal regenerated cord.
We have found no limit to the number of
times the spinal cord will regenerate in one
fish, nor to the distance rostrally that the cord
will regenerate. Tails have been re-amputated
from several fish 4 times each, over a period
of one year, and regeneration proceeds as rapidly the fourth time as the first. The spinal cord
and other structures of the tail still regenerate
when a tail is amputated as far rostral as 5.5
cm from its caudal end, which can be virtually
half the length of the fish. Although we have
not amputated the spinal cord rostral to this
level, it seems likely that it too would regenerate.
DISCUSSION
The caudal-most region of regenerated spinal
cord in Sternarchus consists solely of the ependymal tube, as in regenerated spinal cord of
lizard and newt (Egar et al., 1970; Egar and
80
M.J. ANDERSON, S.G. WAXMAN, AND M. LAUFER
Fig. 5. Regenerated ependyma from a specimen with a
3-week regeneration period. Many cells extend radially from
the central canal ( 0 , which contains cell processes and secreted material. Inset shows neurites in a channel between
peripheral cell processes. Neurites are separated by cell processes with a darker cytoplasm. Basal lamina a t leR of inset
pertains to adjacent space internal in the cord (not the cord
periphery). x 2,700; inset, x 15,550.
Fig. 6. Ventral region of a regenerated spinal cord, filled
with neurites (wide arrows) and cell processes containing
dense-cored vesicles (thin arrows). A capillary (Cp) with
nucleated red blood cell is present in lower right comer. x
10.900.
REGENERATED SPINAL CORD
Singer, 1972). Rostra1 to this, ependymal cell
mitoses generate numerous cells peripherally
which differentiate into the various neuronal
and glial cells of the spinal cord. This process
occurs with some rapidity, producing new spinal
cord segments a t a rate of about 1 mm per
week. A similar sequence of spinal cord regeneration has been described in regenerating amphibian cord (Nordlander and Singer, 1978).
The results presented here indicate that cells
of the spinal cord s t a r t differentiating very early
in the process of tail regeneration. Differentiating cells are present in the earliest sections
where cells are noted peripheral to the ependyma. These cells, which contain dense-cored
vesicles, are probably the cells of origin of the
vesicle-packed processes seen in caudal sections of regenerated cord. In somewhat more
rostral regenerated cord, oligodendroglia and
fibrous astrocytes have differentiated. Even
more rostrally, the large electromotor neurons
characteristic of Sternarchus spinal cord differentiate and recapitulate their normal, distinctive morphology (Anderson and Waxman,
1981).
The first stages of differentiation of the electromotor neurons have not yet been identified.
Fully differentiated electrocytes are present in
rostral regenerated cord within 3 months after
amputation, but are not yet generated in the
3 week-regenerate. It is unclear whether some
of the cells which initially contain dense-cored
vesicles can later differentiate into electromotor neurons or any other cell type. We know
that the cells containing dense-cored vesicles
are among the first to differentiate, and are
prevalent in more caudal sections of regenerated cord. In more rostral sections, the electrocytes are the prominent neuronal cell type,
with fewer cells containing dense-cored vesicles in evidence. Since differentiation procedes
in a rostro-caudal direction in regenerating
spinal cord, with the rostral-most cord having
regenerated the longest amount of time, and
caudal-most cord the least, we are led to ask
what happens to the cells containing densecored vesicles as the regenerating cord “matures.” If these cells do not represent an initial
stage in the differentiation of some other cell
type, then their absence in mature regenerated
cord may be due to cell death or migration.
No evidence of death or degeneration of the
cells containing dense-cored vesicles was observed in this study. However, we cannot rule
out this possibility, especially if the region of
cell death is relatively localized. The second
possibility, that the cells containing dense-cored vesicles are later converted to a different
81
cell type, also remains open. There is evidence
for the conversion of adrenergic cells (exhibiting synapses characterized by dense-cored
vesicles) to cholinergic cells (with synapses
characterized by clear vesicles) both in vitro
and in vivo (Patterson, 1978). Cell processes
filled with both clear and dense-cored vesicles,
which might be indicative of an intermediate
stage in differentiation, do occur in the regenerated spinal cord in Sternarchus. However,
these processes were not necessarily associated
with other synaptic specializations. In addition, these processes are most frequently found
in the caudal region of regenerated cord (where
the cells and processes containing dense-cored
vesicles are also most prevalent).
It is also noteworthy that the cells containing dense-cored vesicles regenerate, after amputation of the tail, from rostral locations where
normally none of these cells would be found.
If regenerated spinal cord is generated from
the extant ependyma alone, these results argue for the pluripotency of at least some of the
rostral, “mature” ependymal cells in Sternarchus.
The morphology of the cells containing densecored vesicles very closely resembles that of
the neurosecretory, or “Dahlgren,” cells of the
caudal neurosecretory system normally found
in teleosts (Bern and Takasugi, 1962; and see
Anderson and Waxman, 1982, pp. 85-92, this
volume). Regeneration of caudal neurosecretory cells from the remaining ependyma after
amputation of the tail has been reported in
another teleost, Tilapia mossambica (Fridberg
et al., 1966). The question arises whether the
presence of neurosecretory cells in regenerated
spinal cord is functionally significant. In Sternarchus, it is not yet known if the secretory
products (dense-cored vesicles) or the physiological function are the same as those of caudal
neurosecretory systems of other fish (Anderson
and Waxman, 1982, pp. 85-92, this volume).
No signs of depletion or secretion of dense-cored vesicles (as are seen in Tilapia) were seen
in the neuro-hemal areas of regenerated Sternarchus spinal cord. It is possible that the neurosecretory cells (cells containing dense-cored
vesicles) are regenerated simply because their
normal location is at the end of the tail and
that is where regeneration is occurring. The
means of encoding and recognizing such positional information would be of great interest.
What signals spinal cord in the middle of the
fish to differentiate with characteristics appropriate to the end of the fish, after amputation of the tail? It may be that amputation,
formation of the blastema, or multiplication of
82
M.J. ANDERSON, S.G. WAXMAN, AND M. LAUFER
the ependymal cells triggers a recapitulation
of embryological development,which results in
regeneration of the caudal neurosecretory system.
The grouping of longitudinally oriented neurites in extracellular spaces near the periphery
of regenerating Sternarchus spinal cord corresponds to the outgrowth of neurites in channels between ependymal cell processes in regenerating amphibian and reptile cords (Egar
et al., 1970; Simpson, 1968). The outgrowth of
regenerated spinal neurites in Sternarchus differs from that proposed in the “blueprint hypothesis” of Singer et al. (1979) in that no evidence was found for channels present in advance
of neurite outgrowth. In the caudal sections of
regenerating Sternarchus spinal cord, individual neurites and groups of neurites extend in
close contact t o ependymal cell borders. Only
in more rostral sections of regenerating cord
were large extracellular spaces with neurites
in them observed. Axonal regeneration without preexisting ependymal channels has also
been observed in transected and r e d spinal
cord of Xenopus tadpoles (Michel and Fkier,
1979).
The unmyelinated neuritic processes observed in early regenerating ependymal tube
are probably axonal rather than dendritic, since
they are usually found in groups and are parallel to each other. These processes presumably
extend posteriorly from cell bodies located in
the unregenerated part of the spinal cord rostral to the transection, or from differentiated
cells in the rostral part of the regenerated cord.
The fact that individual neurites do also grow
through the ependyma indicates that the
grouping of neurites is not necessary for their
outgrowth. Likewise, axons growing outside of
the regenerating cord are often in groups, but
single axons (surrounded by glial processes)
are also common.
The possible role of astrocytes in retarding
or encouraging regeneration has been commented upon by a number of workers. The
presence of numerous fibrous astrocytes in regenerating Sternarchus spinal cord is noted as
evidence that fibrous astrocytes do not, per se,
inhibit regeneration. Also addressing this point,
b i e r (1979)has shown that regenerating optic
axom can penetrate astrocytic scars.
While the regeneration of spinal cord in Sternarchus is in large part similar to spinal cord
regeneration previously reported in other animals (Simpson, 1968; Nordlander and Singer,
19781, Sternarchus has several outstanding
differences which may be functionally important to an interpretation of the process of spinal
cord regeneration. These include the lack of
obvious channels in advance of neurite outgrowth, the frequent invaginations of adluminal ependymal cell surface, the presence of
numerous cells and processes containing densecored vesicles, and the (previously noted) generation of more electrocyte cell bodies than are
normally present.
ACKNOWLEDGMENTS
This work was supported in part by the Medical Service, Veterans Administration, and by
grants from the National Institutes of Health
and the Paralyzed Veterans of America.
We are grateful to Mary E. Smith and Marsha s.Kantor for their excellent technical support.
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albifrons, spina, structure, ependymal, cord, regenerated, fine, sternarchus
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