Download by David Zimmerman The ultimate in nerve regeneration

by David Zimmerman
The ultimate in nerve regeneration—a treatment for paraplegiais still far off. But it is vo longer considered beyond question.
Following injury, the living cells of skin,
liver and most o t h e r b o d y tissues
divide rapidly, repair the damage and
restore the organ's normal functions.
Neurons—nerve cells—restore themselves far
less vigorously, least vigorously of all those
of the central nervous system—the brain and
spinal cord—of mammals.
Restoration of nerve tissue occurs in two
ways; in both, regenerative ability diminishes
the higher one goes on the phylogenetic
tree. O n e way is by mitosis—-cell division—
which creates wholly new neurons. These
nerve cells then can forge n e w connections
to old t e r m i n a l s , t h u s r e s t o r i n g n o r m a l
function. Nerve cells that can reproduce by
mitosis are found in m a n y invertebrates and
in some reptiles and amphibians. But the
rule is that neuronal mitosis is not found in
higher animals.
The second process of nerve repair is axonal
reextension, in which a nerve cell's axon—the
often lengthy, signal-carrying nerve f i b e r will regrow following injury and ultimately
reconnect to its target. Axonal reextension
occurs in the central nervous systems of
lower animals, and even of some vertebrates.
But in higher organisms including h u m a n s ,
axonal reextension commonly occurs only
in the peripheral nervous systems—motor
and tactile sensory neurons, for example. A
s p i n e - i n j u r e d goldfish will regain s o m e
f u n c t i o n s mediated by central a x o n s , for
instance, but not others. And a newt—a small
s a l a m a n d e r — w i l l r e g r o w an e n t i r e tail,
extending central nervous system axons into
it. A h u m a n may, over time, find motion
and sensation returning to a seriously cut
finger as severed peripheral axons reestablish
their connections. But despite some claims
to the c o n t r a r y , axonal r e g e n e r a t i o n a n d
functional recovery from injury to spinal
n e u r o n s h a v e y e t to be d e m o n s t r a t e d
convincingly in any higher vertebrates, including mammals.
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It once was considered to be impossible.
T o d a y , it no longer is. A n understanding of
the factors that inhibit or promote mitosis in
some organisms but not in others, or axonal
reextension in some central nervous systems
b u t not in others, has become an important
concern of many neuroscientists. And, no
matter h o w fundamental their studies, most
scientists investigating nerve regeneration
readily acknowledge w h a t one calls their
"compassionate c o n c e r n " for the paralyzed
h u m a n victims of spinal-cord injury. In the
United States, there are an estimated 300,000
paraplegics and quadriplegics. About 15,000
n e w victims of spinal-cord injury are added
to the list each year.
A decade ago, recalls neuroanatomist Lloyd
G u t h of the University of Maryland School
of Medicine, the treatment of spinal paralysis
was not a concern of basic researchers; the
possibility of successful treatment seemed
t o o r e m o t e . But a s c i e n t i f i c c o n f e r e n c e
c o n v e n e d in 1970 revealed to skeptical
neuroscientists that there was indeed cause
for t h e m to be concerned: the existence of
valid scientific clues to an understanding of
barriers to neural regeneration and to methods
that might be employed to overcome them.
Since then, leaders in the field have come to
believe that, as G u t h puts it, "experimental
studies on the molecular basis of vertebrate
regeneration . . . undoubtedly will lead to
m e t h o d s which will enable us to control the
(neuronal) regnerative process by minimizing
the effects of deleterious extrinsic factors
a n d b y e n h a n c i n g the n e u r o n ' s intrinsic
regenerative capacity."
W h y the confidence? O n e part of the the
explanation is new research; another is a
reinterpretation of old findings.
A crushed or severed axon's first response
to the injury is to die back from the wound.
This axonal degeneration ceases after a week
or so. Then, in those neurons that are capable
of it, the neuron appears to stabilize itself
metabolically and the axon to regenerate,
g r o w i n g past the injury site and restoring
function.
But not all axons can regenerate. As first
described by the Spanish Nobelist Santiago
R a m o n y Cajal at the turn of the century,
damaged cells in the central nervous systems
of higher animals first die back, as do other
n e u r o n s . T h e n new nerve sprouts, which
m a y come either from the damaged axons or
from undamaged collateral fibers nearby,
begin to appear. But the sprouts grow only
briefly; they soon stop growing and die.
M a n y neuroscientists have interpreted this
p h e n o m e n o n as a d e m o n s t r a t i o n of the
impossibility of regeneration of neurons in
the central nervous system. But according
to G u t h , it was Cajal's view—which G u t h
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MOSAIC September/October 1980
n o w shares—that s p r o u t i n g d e m o n s t r a t e s
instead the central n e u r o n s ' " i n t r i n s i c
regenerative capacity." In this more sanguine
view, the task n o w is to define whatever
limitations on regrowth are present in order
to overcome them, and to understand h o w
lower life forms manage to accomplish this
task with the hope of applying these findings to repair of h u m a n injury.
Tracing the pathways
One way to approach this intrinsic capacity,
if it indeed is there, is through study of
pathways by which some neurons in central
nervous systems of some animals do regenerate, tracing them so that their sources,
routes and targets can be k n o w n . Frank
Scalia and Dan Matsumoto of the Downstate
Medical Center in N e w York, among others,
are trying to do this through a study of the
o p t i c n e r v e s a n d n e r v e p a t h w a y s in t h e
leopard frog which, unlike those of most
vertebrates, appear capable of regeneration
following surgical interruption.
T h e tracer they are using is the enzyme
h o r s e r a d i s h peroxidase. It has a useful
property: W h e n injected into a n e u r o n or
taken up by severed axons, it diffuses, Scalia
says, or is transported actively t h r o u g h o u t
the cell or affected axons, yet does not leak
out through the cell membrane into adjacent
a r e a s . T h u s , it m a k e s p o s s i b l e a clear
delineation for microscopic study of the nerve
cell and its axonal pathways.
Using this technique, Scalia has been able
to pick out individual optic nerve fibers as
fine as 0.2 micrometer (micron) in diameter
and to trace their paths from the eye to their
t e r m i n a l s in t h e b r a i n . By i n t r o d u c i n g
horseradish peroxidase into regenerated optic
nerves, he and Matsumoto are trying to trace
the pathways of axons to see which—and
h o w many—of the nerve cells in the retina
are responsible for regeneration. Specifically,
they hope to learn w h e t h e r the information
that dictates the route of an optic-nerve
axon's return to its target is distributed all
along the p a t h w a y or is available only at or
near the target.
T h a t there is such a m a p p i n g system is
suggested to them by the fact that frogs
regain effective vision following optic nerve
regeneration. It is believed that this results
from precise, point-for-point connections
present between the retina and the visual
c e n t e r s in the b r a i n . D u r i n g a f r o g ' s
embryonic development, these connections
are k n o w n to be mediated by embryonic
precursors of the optic nerve. A n earlier
finding by Scalia and M a t s u m o t o suggests
the presence in the adult of what may be a
similar system during regeneration.
Their studies have s h o w n that, following
injury, optic fibers leading to different targets
in the brain degenerate at different rates.
M o r e i m p o r t a n t , s o m e of t h e s e a x o n s
degenerate very slowly; they may remain
present and intact d u r i n g recovery. These
residual fibers, Scalia speculates, may serve
to g u i d e the o t h e r r e t u r n i n g axons back
toward their targets. T h e y thus may be an
important factor in the u n u s u a l regnerative
ability of the frog's optic nerve. If so, finding
out how these fibers resist degeneration, as
Scalia and Matsumoto now hope to do, could
point toward ways to assist regrowth in other
injured central n e r v o u s system p a t h w a y s .
Crippled fish
Another extraordinary model of central
nervous system injury, which a principal
investigator calls the "paraplegic goldfish,"
offers s t r i k i n g l y different insights into
regeneration. The researcher is neurobiologist
S t e v e n J. Zottoli of W i l l i a m s College in
Massachusetts. The model is valuable because
the fish's dramatic and highly visible ability
to perform a sudden tail flip that displaces it
sideways out of d a n g e r ' s way is effected via
a single pair of h u g e , identifiable axons,
called M - a x o n s . W h e n the M-axons are cut,
the tail flip d i s a p p e a r s . T h i s m e a n s that
Zottoli, unlike Scalia, has the advantage of
working with accessible axons that specify
easily detectable behavior.
T h e M - a x o n s are named for their 19th
century discoverer, Bohemian ophthalmologist Ludwig M a u t h n e r . In the goldfish,
they run the length of the spinal cord and
are large enough to be identified with the aid
of a low-power microscope. Each M - a x o n is
100 micrometers in diameter, at least twice
the size of the next largest axon in the
goldfish's central nervous system.
Large axons transmit impulses more rapidly
than small ones. And since the huge M-axon
transmits an impulse that can be readily
identified at the point where it enters the
M-cell body in the brainstem, Zottoli is able
to distinguish its input in the brain from
that of other axons.
W h e n its spinal cord is cut, a goldfish will
lie on its side; it cannot swim upright and
has lost its tail-flip ability. After several
weeks' convalescence, the fish may regain
the ability to swim upright, a behavior that
is believed to be controlled by nearby spinal
a x o n s . But its tail flip, mediated by the
M-axons, does not return. Zottoli's next round
of experiments will be an effort to find out
why. He will attempt to determine whether
M-axon regeneration is limited by an extrinsic
factor—a mechanical barrier or a chemical
i n h i b i t o r s e c r e t e d b y n e a r b y cells, for
example—or whether it is inhibited by an
intrinsic factor, perhaps a metabolite produced
within the M - a x o n or the cell body itself.
Several hypotheses have been advanced
to explain w h y mammalian central nervous
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in regenerating than is the goldfish M-cell.
Scar tissue, which may include glial cells
that normally provide structural and metabolic
support for the axons, might block regrowth.
In a very recent experiment, G u t h and his
University of Maryland co-worker, pharmacologist Edson X. Albuquerque, eliminated
scar formation as a variable in their work
with another animal, the thirteen-lined ground
s q u i r r e l . T h e s e s q u i r r e l s are l o n g a n d
profound hibernators. T h e y are so damped
d o w n metabolically w h e n hibernating that
they do not synthesize scar tissue in response
to injury. In the experiment, spinal axons
were cut while the rodents were hibernating
so that no scar tissue would appear. T h e
axons nevertheless regenerated only up to
the point of injury. There they " s t o p p e d
dead in their tracks," G u t h says, indicating
that something other than scar tissue inhibited
their regrowth.
Another possible hypothesis for the failure
of c e n t r a l n e r v o u s s y s t e m n e u r o n s to
regenerate is that the embryonic stem cells
that originally produce central nervous system
neurons may no longer be present or are
only selectively present in an adult animal.
Another is that regrowth may be frustrated
by the a b s e n c e of the s u p p o r t cells t h a t
originally laid out the pathway and provided
the directional cues during embryonic neural
development.
A l t e r n a t i v e l y , the m y e l i n s h e a t h t h a t
surrounds and encloses the M-axon and other
central nervous system axons may become
impenetrably fused following injury. Or these
nerves may be prevented from regenerating
by the very slow rate at which the materials
required for elongation can be transported
d o w n the axon. These latter possibilities,
Zottoli suspects, are the most promising;
they are the ones he is n o w pursuing.
In his current experiments, Zottoli is trying
to cut just the M-axon, leaving other, nearby
axons intact. T h e ability to do this, he says,
will eliminate m a n y of the extrinsic f a c t o r s such as mechanical barriers and vascular
interruptions—that have complicated previous
studies of this kind.
Biochemical cues
Some hard data already are available on
one of Zottoli's questions: the rate at which
cut axons regrow. Axonal reextension closely
reflects the rate at which actin, one of the
principal cellular building blocks of nerve
fibers, is transported from a nerve's cell body
d o w n to its axon's growing tip.
These findings come from the Case Western
Reserve University School of Medicine, where
neurobiologist R a y m o n d J. Lasek and his
colleagues have s h o w n that repair materials,
packaged in discrete intracellular structures,
move d o w n the axon at varying rates of up
to 400 millimeters a day. Actin, however, is
carried by one of the slower of these packets,
which Lasek calls slow component b. It travels
at a b o u t two millimeters per day. T h i s
neatly—but not surprisingly—turns out to
be almost identical to the rate of axonal
r e g r o w t h . A n d Irv M c Q u a r r i e , a n e u r o surgeon working with Lasek, has shown
that twice-injured axons appear to regrow
faster than axons injured but once, suggesting
that whatever the repair mechanism turns
out to be, it can be stimulated.
According to another of Lasek's associates,
neurobiologist Scott Brady, these findings
u n d e r s c o r e the p o i n t t h a t nerve g r o w t h
depends on biochemical cues as well as on
p h y s i c a l stimuli and c o n s t r a i n t s . C o n s e quently, it will require new approaches to a
field that heretofore has been dominated by
morphological, physiological and behavioral
studies. Already, for example, Lasek and his
colleagues have found biochemical differences
in the structure of uninjured axons and those
re-elongated following injury. They are now
at w o r k t r y i n g to s p e c i f y w h a t t h e s e
differences are.
Sleeves and channels
If growth—elongation—is one fundamental
element in nerve regeneration, a second is
direction. A n elongating axonal tip must
travel toward its severed peripheral or central
connections if it is to contribute to functional
r e s t o r a t i o n . Since the axon d e g e n e r a t e s
MOSAIC September/October 1980
13
backward from both sides of a nerve wound,
regenerative repair from one side only may
be a long process, even for a fiber that has
been injured close to its target endpoint.
A pioneer investigator of this problem
has been L a s e k ' s chief at C a s e W e s t e r n
Reserve, neuroanatomist Marcus Singer, who
says he n o w may have part of the answer to
h o w the nerve reaches its destination. He
rejects the notion that the target tissue secretes
a specific substance that the regrowing axon
can sniff and h o m e in on like a beagle dog.
" N e r v e fibers are not smart e n o u g h to reach
their targets by themselves/ 7 Singer says.
R a t h e r , he a n d d e v e l o p m e n t a l n e u r o biologist Ruth H . Nordlander suggest, on
the basis of a decade's research, the axons
are guided from the injury site toward their
original connections by a system of "canals"
c o m p o s e d of e p e n d y m a l cells, a type of
epithelial support cells. These canals develop
ahead of—and so guide—the regenerating
axon tip. This guidance may be mediated by
tactile cues or by substances secreted by the
canal walls.
T h e existence of an ependymal guidance
system first was inferred over a decade ago
from electron m i c r o g r a p h s of n e w t tails
regenerating after amputation. N e w t s are a
principal model for Singer, who long has
been a leader in the experimental study of
limb regeneration.
Cross-sectional electron micrographs of
Mr, Zimmerman,
author of "Rh, The Intimate History of a Disease and its Conquest"
and "To Save a Bird in Peril," has also
written articles for Smithsonian, A u d u b o n ,
Natural History and T h e N e w York Times
Magazine.
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MOSAIC September/October 1980
n e w t - t a i l s p i n a l c o r d , m a d e s o o n after
a m p u t a t i o n , s h o w e d m a n y light areas—
a p p a r e n t l y e m p t y spaces—between cells.
Initially, these were thought to be artifacts
of the fixation techniques. However, slides
taken days or weeks later showed axons
growing within these spaces. "It made us
t h i n k , " N o r l a n d e r recalls, " t h a t the pattern
of vertical fiber pathways was being directed
by the e p e n d y m a l cells. I think there are
channels there . . . toward the target."
This i n t e r p r e t a t i o n was considerably
strengthened in recent years when Nordlander
discovered strikingly similar channelization
in the n e u r o n a l development of embryonic
newts. In n o r m a l development, as in regeneration after injury, it appears that epithelial
channels guide the growing axon tips toward
the a p p r o p r i a t e targets. The a p p a r e n t
similarity between the two events strongly
s u g g e s t s to Singer and N o r d l a n d e r that
regeneration is a replay of embryogenesis.
Injury, in the newt at least, reactivates the
developmental process, and the mystery of
h o w regenerating axons find their way back
thus ties to the question of h o w nerves grow
and establish connections in the first place.
Blueprint hypothesis
Since repair may represent reactivation of
a developmental blueprint, Singer, Nordlander,
and M a r g a r e t Egar w h o demonstrated the
same phenomenon in lizard-tail regeneration,
call their theory " t h e blueprint h y p o t h e s i s . "
They say the ependymal cells in these animals
have "retained the capacity to unroll a blueprint that the nerve fibers have to follow to
reach their destination. . . . In addition to
providing specific highways for regenerating axons, the blueprint hypothesis implies
that the individual axons 'recognize' and
follow p a r t i c u l a r itineraries, even w h e n
challenged by m u l t i p l e h i g h w a y s . " T h i s
guidance system, Singer adds, is probably
not target-specific. It brings the axon to the
ppropriate area, b u t some other,still-to-beelucidated cue then must tell it to stop elongating and make a connection.
T o check both of these components of the
blueprint hypothesis-—to induce a severed
neuron to extend its axon beyond the wound
and seek reconnections—a bridge across the
wound has been sought. The possibility that a
surgically implanted graft of healthy nerve
tissue could serve as such a bridge is one
approach being tried.
N e u r o l o g i s t A l b e r t A g u a y o , of McGill
University, and Carl C. Kao, a Georgetown
University neurosurgeon, both have used
i m p l a n t s of e m b r y o n i c nerve tissue and
reported some success in inducing central
n e r v o u s system axons to regrow into such
" c a b l e g r a f t s " in d o g s . S i n g e r a n d his
Cleveland colleagues have introduced a couple
of wrinkles into the cable-graft option.
Reasoning that the regenerative spinal cord
from lower v e r t e b r a t e s may h a v e useful
properties that are lacking in the nonregenerative mammalian cord, anatomist Keith
Alley, an associate of Singer's, transplanted
a "bologna slice" of lizard tail. The slice,
with is e p e n d y m a l s u p p o r t - c e l l c h a n n e l s
correctly aligned, was inserted into a notch
cut into the spinal cord of a nude mouse, a
mutant chosen because it lacks immunologic
competence and so cannot reject the graft.
He later found axons, which he believes are
from the mouse, in the cable graft. But, he
says, there were disappointingly few of them.
He plans horseradish peroxidase studies to
see If these axons will reenter the mouse
tissue on the far side of the bridge.
Peripheral neurons
T h e guidance role that support cells play
in axonal r e e x t e n s i o n has been further
suggested by experiments that University
of Utah neurophysiologist Kenneth W. Horch
conducts with cats. He focuses his attention
on sensory nerves and their receptors in the
skin of the cat's hind leg, a peripheral nervous
system model.
Horch stimulates raised pressure-receptor
domes in the shaved skin of a cat's leg in
order to delineate the skin area served by a
nerve he has exposed surgically. The borders
of the region and the domes themselves are
marked to produce a m a p of the receptors
activated by the nerve. T h e nerve then is
either cut or crushed, and the wound is closed.
After the nerve has had several months to
regenerate, Horch repeats the procedure and
compares before-and-after maps.
With crush, the before-and-after maps
are essentially the same, leading Horch to
conclude that reinnervation proceeds along
preexisting channels that survive the injury.
With cut, which disrupts the channels, the
picture is far different; more than half the
receptor domes will have disappeared from
the skin by the time of the reexamination;
they a p p a r e n t l y a t r o p h y for w a n t of a
functional axonal contact. Those that remain
appear to have been innervated by other
than their original axons. This scrambling
of the wires, as it were, may well compromise
the organization of sensory receptors and
their neuronal connections to the brain.
Compensating adaptation
But functioning may be restored even if
circuits remain hopelessly scrambled.
There is a current belief, Horch says, that
in mammals everything is "hard-wired," that
i n a p p r o p r i a t e r e c o n n e c t i o n s are n o t selfrectified. T h e alternative that some investigators are coming to accept, and that Horch
n o w is exploring, suggests that everything
MOSAIC September/October 1980
15
is not hard-wired, that there is some innate
plasticity in the mammalian nervous system.
Such plasticity would enable a cat, or a human,
to use higher centers in the brain to learn
other methods for processing information
delivered through an altered p a t h w a y . (See
"The Neural Net: Change in a Fixed System/ 7
Mosaic, Volume 9, N u m b e r 2.)
Vanderbilt University neurobiologist Jon
H. Kaas is pursuing similar possibilities in
experiments with a primate on which the
mapping is being performed higher in the
central nervous system—in the brain. The
mapped target is an area of the cerebral
cortex that is activated by sensory impulses
carried from the palm of the h a n d via the
median nerve. T h e owl m o n k e y was the
p r i m a t e c h o s e n for t h e s e e x p e r i m e n t s ,
because, in this South American species, the
cortical area activated b y the palm is a large
area on the surface of the brain; it can be
easily mapped before and after the nerve is
damaged at the level of the wrist.
W h e n the median nerve is crushed, says
Kaas, it regenerates correctly; points on the
palm reactivate their former target points in
the cortex. With cut injuries, however, he
finds, as did Horch, " m a n y mislocations"
on the map; sensory points on the palm now
target different points in the cortex.
Kaas now is looking for changes in cortical
organization in a follow-up experiment in
which the nerve serving the palm is cut and
then prevented from regenerating. He has
found that in such a case the cortical area
previously served by the palm via the median
nerve becomes reactivated by another
nerve—either the ulnar or the radial—which
carries sensory impulses from the back of
the hand to its own parts of the brain.
To learn the effects of such anomalous
connections, Kaas has conditioned his owl
monkeys to reach u p w a r d in response to
stimulation on the back of the h a n d and
downward when the palm is stimulated. The
q u e s t i o n is w h e t h e r a m o n k e y , after the
severing of the median nerve serving the
palm and after takeover of the palmar part
of the cortex by either the radial or ulnar
nerve, will continue to move the h a n d up, as
it has been taught, w h e n the back of its hand
is stimulated.
If it doesn't, if it reaches d o w n in the
palmar direction in response to back-ofthe-hand stimulation, the implication is that
the reactivated palmar or median area of the
cortex—which, because it is as m u c h as 20
times the size of the area served by the radial
or u l n a r nerves—overrides a n d p r o d u c e s
sensations on the palm no matter h o w it got
its message. T h e inference is not conclusive,
because the radial or ulnar nerves would
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MOSAIC September/October 1980
still be performing their normal function as
well and might not be overridden. But the
implication w o u l d be t h a t the b r a i n is
hard-wired rather than plastic. T h e palmar
region remains the palmar region however it
is stimulated.
But if such a monkey later correctly reaches
u p w a r d in r e s p o n s e to b a c k - o f - t h e - h a n d
stimulation, despite the fact that the brain's
dominant palmar area is being stimulated,
Kaas adds, then the absence of hard-wiring
is suggested; there is plasticity in the cortex
that permits its palmar area—or presumably
any newly i n n e r v a t e d region—to serve
whatever neuron brings messages to it. T h e
brain would be adapting itself to respond
a p p r o p r i a t e l y e v e n to s c r a m b l e d i n p u t
pathways. "This would be very exciting from
the point of view of recovery from nerve
d a m a g e , " Kaas says. His experiment has not
yet been completed; his hypotheses are still
hypotheses. Though training has begun and
the necessary surgery has been scheduled,
several months m u s t elapse after surgery for
the animals to heal before testing.
An exciting exception
In these experiments, Kaas, like many of
his fellow investigators of n e u r o n a l regeneration, is s t u d y i n g a x o n a l r e g r o w t h of
peripheral n e u r o n s . But from the medical
viewpoint, and perhaps scientifically too,
the u l t i m a t e c h a l l e n g e is t h e m a m m a l i a n
central nervous system, where injured axons
sprout and die back without ever reconnecting
and where the power of mitotic division
appears to have been lost; mitotic division
and recovery of function for the most part
simply do not appear to occur in the mammalian central n e r v o u s system.
Recently, h o w e v e r , a s u r p r i s i n g and
provocative exception has come to light:
neurons of the olfactory nerve. These are
part of the central nervous system and are
often called an extension of the brain outside
the cranial cavity. U n d e r w h o l l y n o r m a l
conditions, these n e u r o n s c o n t i n u o u s l y die
and are replaced t h r o u g h mitotic division of
neuroepithelial stem cells. W h e n olfactory
n e u r o n s are d e s t r o y e d , t h e y n o t o n l y
regenerate, b u t they avidly search out and
forge n e w connections. T h e y will seek out
the olfactory bulb in the brain, their normal
t e r m i n a l , or they will seek deeper b r a i n
structures if the bulb has been surgically
ablated.
These remarkable regenerative powers are
present in mature as well as immature animals,
in the "morphologically identical" olfactory
systems of mice, rats, cats and m o n k e y s ,
and very likely also in h u m a n s . T h e y are
present as well in the sensory apparatus of
lower animals, including the octopus in which
they were first elucidated.
T h e researchers who n o w have confirmed
the admittedly wild hypothesis that the prim a r y olfactory neurons continuously turn
over, doing something no other mammalian
n e u r o n appears able to do, are neuroanatomists Pasquale P. C. Graziadei and Giuseppina
A. M o n t i Graziadei—husband and wife—of
Florida State University. A colleague, biochemist Frank L. Margolis of the Roche
Institute of Molecular Biology, has isolated
and characterized a low-molecular-weight
protein that occurs in the olfactory neurons
of all vertebrate species studied, but apparently not in other central nervous system neurons.
This olfactory marker protein, Pasquale
Graziadei says, is unique in that "it is the
only protein that has been found that appears
to be specific to a class of n e u r o n s . " This
suggests, he adds, that " m a y b e we have a
golden egg inside." T h e possibility, which
he and other researchers now are avidly
p u r s u i n g , is t h a t t h i s s i n g u l a r p r o t e i n
stimulates or de-represses mitotic regeneration
in olfactory neurons.
So far, efforts to crack the "golden e g g "
have been hindered by the scant supply of
the marker protein. The marker, says Pasquale
Graziadei, has thus far been " v e r y difficult"
to isolate and purify. O n e of the Graziadeis'
first experimental efforts, using an antibody to
the olfactory marker protein supplied by
Margolis, is to try to block the protein in
living olfactory neurons. They hope thereby
to see if such blocking compromises the
ability of the cells to r e g e n e r a t e . " W e ' r e
working, and we do have leads," is all that
Pasquale Graziadei will say at this time of
this provocative work in progress.
The olfactory neuron's unique regenerative
properties reflect its unique—and Pasquale
G r a z i a d e i believes quite primitive—traits.
He first explored these more than two decades
ago in the analogous neuronal elements of
the octopus, an animal with olfactory-like
sentivity a thousand times that of h u m a n s .
He located sensory receptors on the octopus's
lips and on the rims of the suckers on each
of its arms. He found that in octopuses, as in
the higher animals that he has studied since,
c h e m o s e n s o r y n e r v e cell b o d i e s — s o m e
animals may have as many as 50 million of
them—are spread t h r o u g h o u t the sensory
tissue, or n e u r o e p i t h e l i u m , a n d are not
collected in groups or ganglia as is the case
with other types of sensory n e u r o n s . A n d
unlike other central nervous system sensory
neurons, the dendrites and ciliary appendages
of these cells penetrate the epithelial surface
and are in direct contact with the environment.
T h e regenerative capacity of vertebrate
olfactory neurons had been suspected by
some 1 9 t h c e n t u r y a n a t o m i s t s , b u t this
possibility was not fully appreciated by their
20th century successors, Pasquale Graziadei
says, until he and his wife demonstrated its
existence. They showed that the nucleoside
thymidine, which is incorporated by cells
only during mitotic division, is present in
the cytoplasm and nucleus of olfactory stem
cells. It was then seen in the olfactory neurons'
d a u g h t e r cells, after the s t e m cells were
incubated with thymidine. "For the first time,"
Pasquale Graziadei exclaims, " w e have a
neurogenic matrix that replaces the neurons
that die out—and the new n e u r o n s regrow
their axons. W e have a neural system that
recovers from injury."
T h e Graziadeis have c o n f i r m e d these
f i n d i n g s m o r p h o l o g i c a l l y , by d i s c o v e r i n g
regenerated cells that reestablished synaptic
c o n n e c t i o n s in m o u s e b r a i n s . T h e y h a v e
confirmed morphological recovery following
surgical injury to the olfactory nerve even in
monkeys. Functional and behavioral studies
are continuing in the Graziadeis' Tallahassee
laboratory and in several others. " W e have
found a neuron that is exceptional," Pasquale
Graziadei says. " W e must n o w try to find
the parameters of its exceptionality."
Postulates for nerve research
M u c h current research is directed to the
elaboration of models that may point to factors
t h a t r e p r e s s or p r o m o t e n e r v e g r o w t h .
Experimental rigor is s o u g h t because m a n y
of these experiments are difficult to conduct
and interpret, given the n u m b e r of neurons
a n d t h e w e l t e r of t h e i r a x o n s in m o s t
physiologically significant nerve tracts. T h e
frog optic nerve, to cite one such model,
carries almost half a million axons emanating
from a like number of nerve cell bodies.
Experimental rigor is sought also because
claims have been made that several experimental surgical and pharmacologic therapies
h a v e successfully p r o m o t e d s p i n a l cord
regrowth in higher animals, even h u m a n s .
" N o n e of these therapies has produced
regeneration," says neuroanatomist Lloyd
G u t h . But "these misleading claims have
had the unfortunate effect of confounding
the scientific literature, promoting wasteful
duplication of scientific effort and d i s a p pointing the paraplegic c o m m u n i t y . "
T o limit m i s l e a d i n g claims, G u t h , a n d
associates in the Stroke and Trauma Program
of the National Institute of Neurological
and Communicative Disorders and S t r o k e ,
recently published a set of "Koch's postulates"
or rules for proving regeneration, w h i c h ,
G u t h says, have thus far not been w h o l l y
satisfied in any single experiment. (Robert
Koch, the t u r n - o f - t h e - c e n t u r y G e r m a n
bacteriologist and Nobelist, established a set
of rules—Koch's postulates—for confirming
the connection between a particular m i c r o organism and a particular disease. T h e t e r m
has been generalized to signify rules of proof
in any scientific inquiry.)
To conclude that functional regeneration
of spinal neurons has occurred, Guth's postulates stipulate, a researcher must demonstrate:
• T h a t the e x p e r i m e n t a l lesion c a u s e d
disconnection of the nerve processes.
• That the new central nervous s y s t e m
fibers bridge the level of the injury and make
j u n c t i o n a l contacts as d e m o n s t r a t e d b y
anatomical studies.
• T h a t the regenerated fibers also can be
shown to generate nerve responses across
the injury.
• That some functional change—preferably
for the better—can be s h o w n to result from
new connections.
These postulates thus become, in a sense,
an agenda as well as a challenge a n d a
c o n c e p t u a l f r a m e w o r k for r e g e n e r a t i o n
research in the 1980s. T h e propriety of s u c h
a f r a m e w o r k is u n d e r l i n e d by d e c i s i o n s
reached, in the spring of 1980, at a conference
in B e r m u d a u n d e r t h e aegis of D u k e
University and the Paralysis Cure Research
Foundation. Just a decade after the earlier
conference, which Guth recalls as the turning
point of the field, participants in the Bermuda
meeting identified central nervous s y s t e m
r e g e n e r a t i o n r a t h e r t h a n , for i n s t a n c e ,
improved acute care of spinal injury, as a
reasonable research target for the future.
Such a target is b o u n d to breed u n d e r s t a n d a b l e b u t unrealistic e x p e c t a t i o n s in
persons afflicted by nerve injuries a n d in
those who care about them. T h e " c o m p a s sionate concern" that investigators of neural
regeneration express for the victims of spinal
cord injury requires that the scientists avoid
to the extent possible the e n c o u r a g e m e n t of
such premature or u n w a r r a n t e d o p t i m i s m .
Such strictures as Koch's postulates for nerve
research may well be an appropriate screen. •
The National Science Foundation
contributes
to the support of the research discussed in
this article through its Neurobiology
and
Sensory Physiology and Perception Programs,
MOSAIC September/October 1980
17