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 .
Bid Rev . (1979). 54. PP. 155-197
Printed in Great Britain

HYPOTHESES CONCERNED WITH

AXONAL REGENERATION IN THE MAMMALIAN
NERVOUS SYSTEM
BY J . A . KIERNAN
Department of Anatomy. The University of Western Ontario.
London. Ontario N6A ~ C ICanada
.

(Received 20 December 1978)

CONTENTS
I . Introduction . . . . . . . . . . . . . . . 156
11. Pertinent experimental data . . . . . . . . . . . . 157
(I) Regeneration in the peripheral nervous system . . . . . . . . 158
(2) Axonal regeneration in the C.N.S. . . . . . . . . . . 158
(a) Wounds of the brain and spinal cord . . . . . . . . . 158
(b) Transplantation of peripheral nervous tissue into the C.N.S. . . . . I59
(3) Neurosecretory axons . . . . . . . . . . . . 160
(4) Monoaminergic fibres . . . . . . . . . . . . 161
( 5 ) Regeneration through spinal roots . . . . . . . . . . 161
(6) Primary olfactory neurones . . . . . . . . . . . 161
(7) Regeneration of axons into non-neural tissues transplanted into the brain . . . 162
(a) Skin . . . . . . . . . . . . . . . 162
(b) Striated muscle . . . . . . . . . . . . . 162
(c) Smoothmuscle . . . . . . . . . . . . . 162
(d) Other tissues . . . . . . . . . . . . . 163
(8) Pharmacological effects onregenerating axons . . . . . . . . 163
(a) Thyroid hormones . . . . . . . . . . . . 163
(b) Pyrogens. corticotrophin and corticosteroids . . . . . . . 164
(c) Immunosuppressive drugs . . . . . . . . . . . 165
(d) Local treatments . . . . . . . . . . . . . 165
(e) Combined local and systemic treatment with enzymes . . . . . . 166
(9) Foetal and neonatal mammals . . . . . . . . . . . 167
(10) Laminar lesions . . . . . . . . . . . . . 167
(I I ) Submammalian vertebrates . . . . . . . . . . . 167
(12) Growth of neurites in culture . . . . . . . . . . . 168
( a ) Elongation of neurites . . . . . . . . . . . 168
(6) Contact guidance and chemotaxis . . . . . . . . . 168
(13) Mechanisms of axonal growth . . . . . . . . . . . 169
.
I11 Hypotheses . . . . . . . . . . . . . . . 169
( I ) Intrinsic inability of central neurones to regenerate their severed axons . . . 170
(a) Statement of hypothesis . . . . . . . . . . . 170
(b) Arguments supporting hypothesis . . . . . . . . . 170
(i) Lower vertebrates . . . . . . . . . . . 170
(ii) Mammalian neurosecretory. monoaminergic and olfactory fibres . . . 170
(iii) The immature central nervous system . . . . . . . . 170
10-2
 J .A . KIERNAN
(c) Discussion of arguments opposing the hypothesis . . . . . . 171
(i) Motor neurones. . . . . . . . . . . . 171
(ii) Transplantation experiments . . . . . . . . . 171
(2) Inadequacy of theperiaxonal environment . . . . . . . . 171
(a) Schwann cells and contact guidance . . . . . . . . . 171
(i) Development of hypothesis and supporting evidence . . . . . 171
(ii) Arguments against the hypothesis . . . . . . . . 172
(b) Compatibility between regenerating axom and the surrounding cells . . . 172
(i) Development of hypothesis . . . . . . . . . . 172
.
(ii) Discussion . . . . . . . . . . . . 173
(3) Physical barriers to regeneration . . . . . . . . . . 174
(a) Observations indicating existence of barriers to regeneration. . . . 174
(b) Evidence against blockade of regeneration by scars . . . . . * I75
(4) Inappropriate formation of synapses . . . . . . . . . . 175
(a) Bernstein's experiments and deductions . . . . . . . . I75
(b) Discussion. . . . . . . . . . . . . . 176
(5) Autoimmune inhibition of regeneration . . . . . . . . . I77
(a) The hypothesis of Berry & Riches . . . . . I . .
. 177
(b) Arguments supporting the hypothesis . . . . . . . . . I77
(c) Observations incompatible with the hypothesis . . . . . . . 178
.
(6) Necessity of periaxonal vascular permeability . . . . . . . 178
(a) Statement of hypothesis . . . . . . . . . . . 178
.
(b) Reconciliation of the hypothesis with experimental observations . . . 179
(i) Axonal regeneration and blood-brain barriers . . . . . . I79
(ii) Monoaminergic and sensory neurones . . . . . . . . 179
(iii) Foetal mammals and submammalian vertebrates . . . . . . 180
(iv) Pharmacological effects . . . . . . . . . . 180
(7) Growth factors and axonal regeneration . . . . . . . . . 181
(i) Statement of hypothesis . . . . . . . . . . 181
.
(ii) Discussion . . . . . . . . . . . . 182
IV . Conclusions . . . . . . . . . . . . . . . 182
V.Summary . . . . . . . . . . . . . . . 184
.
VI References . . . . . . . . . . . . . . . 186

.
I INTRODUCTION
The different consequences of injury to peripheral nerves and to the central nervous
system (C.N.S.) have puzzled neurologists for many years. The axons of a peripheral
nerve can be transected either by severance of the whole trunk of the nerve (neuro-
tmesis) or by blunt trauma such as crushing, which breaks the axons (axonotmesis) but
leaves the non-neuronal supporting structures in physical continuity. Localized
freezing produces effects closely similar to those of crushing (Mira. 1977). The parts
of the axons that have been separated from their cell-bodies die and are phagocytosed
by their ensheathing Schwann cells. If the cut nerve is repaired by suturing. or if the
crushed nerve is simply left alone. the damaged axons in the proximal stump elongate
and enter the distal stump. There. they continue to grow longer until eventually they
reach and re-innervate sensory and effector organs. where recovery of function ensues.
It must be emphasized that this sequence of events. known as axonal regeneration. is
one in which cells are replacing their amputated cytoplasmic processes. It does not
involve mitotic activity on the part of the neurones or the formation of new neurones
 Axonal regeneration in mammals I57
from undifferentiated precursor cells. In these respects the word ‘regeneration’ has
a meaning somewhat different from the one it has when applied to cellular replace-
ment in injured non-nervous tissues. The recovery of normal ultrastructure in the
perikarya of neurones damaged but not killed by treatment with toxic drugs has also
been called ‘regeneration’ (Rohkamm, 1977). This is, however, an incorrect and con-
fusing use of the term.
Transected axons in some invertebrates can repair themselves by fusion of the
central stumps with their surviving peripheral processes (Hoy, Bittner & Kennedy,
1967). Similar ‘regeneration per primam’ has been described in micrurgically severed
vertebrate nerve fibres growing in tissue culture (Levi & Meyer, 1945). Fusion of the
proximal and distal stumps of the cytoplasmic processes of cultured neuroblastoma
cells has also been observed, following transection by laser microbeam irradiation
(Rieske & Kreutzberg, 1978). This type of primary axonal repair almost certainly
does not occur in vivo in mammals and will not be further discussed.
Return of function can follow axonal damage in some regions of the brain and
spinc alord, but such recovery is due almost entirely to the re-organization of alter-
native, intact neuronal circuitry. This involves structural rearrangements (variously
known as ‘axonal sprouting ’, ‘collateral sprouting’ and ‘plasticity ’) of the synaptic
terminals and dendrites of uninjured neurones. For full discussion of many aspects of
plasticity the reader is referred to Barlow & Gaze (1977) and Lund (1978). Axonal
regeneration probably has no appreciable significance in the mammalian C.N.S. and
a lesion which is so situated that there are no adequate alternative pathways (e.g. a
substantial infarction of the internal capsule or a complete transection of the spinal
cord) produces permanent disability. However, central axons can regenerate and in
some experimental circumstances, which will be discussed, they may do so to a useful
extent. The clinical benefits which will follow the discovery of means of encouraging
effective regeneration of severed axons in the C.N.S. are unlikely to be seen until
it is known why, under ordinary conditions, this regenerative process is unsuccessful.

11. PERTINENT EXPERIMENTAL DATA

Several theories have been advanced which purport to explain the failure of central
axonal regeneration in mammals. In order to be acceptable, any hypothesis must
account for all the available experimental observations. The most relevant of these will
now be reviewed, though many related topics that are not directly applicable to the
hypotheses are not considered in this article. For broader coverage of the literature
the reader is referred to Cajal (1928), Windle (1955, 1956), Guth (1956, 1975),
Clemente (1964)~Sunderland (1968)~Guth & Windle (1970)~Jellinger (1976), Yeo
(1976)~Nesmeyanova (1977) and Puchala & Windle (1977). Unless otherwise stated,
the data cited below relate to the nervous systems of adult mammals. Experiments
have been conducted principally on mice, rats, rabbits, cats, dogs and monkeys.
Axonal regeneration in the C.N.S. of lower vertebrates is considered briefly in Section
11(I I) since its successful occurrence in some of these animals must be allowed for in
the evaluation of the hypotheses discussed in Section 111.
158 J. A. KIEFWAN
(I) Regeneration in the per$heral nervous system
Axons of all types can regenerate after severance within the territory of the peripheral
nervous system (P.N.S.). Such fibres, however, generally are unable to enter central
nervous tissue. For example, if a dorsal spinal root is injured between the sensory
ganglion and the spinal cord, most of the regenerating axons grow up to but not into
the tissue of the cord (Bikeles, 1907; Kimmel & Moyer, 1947), though small numbers
have been shown to penetrate into the spinal grey matter (Ikeda & Campbell, 1971 ;
Nathaniel & Nathaniel, 1973). I n some experiments (see p.161) it has been possible
to induce larger numbers of axom to grow into the C.N.S. through spinal roots.

(2) Axonal regeneration in the C.N.S.

(a) Wounds of the brain and spinal cord
With a few exceptions, to be described presently, most axons fail to regenerate
across lesions in the brain and spinal cord, in the absence of any pharmacological
intervention. The regenerative process begins a few days after placement of the lesion
but after about 2 weeks the axonal sprouts die back (Sala, 1909; Cajal, 1928;Brown &
McCouch, 1947; Lampert & Cressman, 1964) and form ‘retraction bulbs’, many of
which eventually synapse with the dendrites and perikarya of other neurones (Bern-
stein & Bernstein, 1971) or become entangled in the cytoplasmic processes of pro-
liferated neuroglial cells (Phelps, 1969). This sequence of events has been called
‘abortive regeneration’ (Brown & McCouch, 1947).
When incised wounds of the brain are carefully examined after 8 weeks, small
numbers of axons can sometimes be seen passing from the nervous tissue into the
connective tissue that has occupied the slit made by the experimenter’s knife. On rare
occasions, an axon is found which crosses to the ’other side of the lesion. Such truly
regenerated fibres represent, however, only a tiny proportion of all those severed at
the time of operation (Fertig, Kiernan & Seyan, 1971; Heinicke, 1977). As will be
seen, the numbers of regenerating axons can be increased experimentally.
There have been a few reports of substantial axonal regeneration, with associated
functional recovery, following transection of the spinal cord in the rat and the dog.
These claims must, however, be balanced against the results of many comparable
studies in which no such recovery could be obtained (see Windle, 1956; Clemente,
1964 for original literature). It has recently been shown that surgical transections of
the rat’s spinal cord, however carefully carried out, are frequently incomplete (Feringa,
Kinning, Britten & Vahlsing, 1976). Functional reorganization of connexions in the
partially transected cord takes place mainly through the ventral funiculi (Barker &
Eayrs, 1967), which may be the parts most likely to be spared when transection is
attempted from the dorsal aspect. There have also been several claims of functional
recovery due to axonal regeneration in the transected human spinal cord. However,
there is only one thoroughly studied case in which any motor control or conscious
sensation followed unquestionably complete transection. This patient eventually
became paraplegic again, some years after surgical repair of her spinal cord (see
Windle, 1955, pp. 259-271 for critical appraisal of the clinical literature).
 Axonal regeneration in mammals I59
(b) Transplantation of perzpheral ~ O U tissueF into the C.N.S.
Tello (191I , cited from Cajal, 1928) stated that grafts of rabbits’ peripheral nerves
in the brain were penetrated by numerous cerebral axons, but his observations could
not be reproduced by Clark (1942). In the mouse, however, such grafts are penetrated
by numerous axons, which are seen to be longitudinally orientated in the transplanted
nerves (Horvat, 1966; Heinicke, 1978, 1979). When a spinal or sympathetic ganglion
is transplanted into the brain of the same or another animal, axons of the neurones in
the ganglia grow out into the surrounding connective tissue, but not into the cerebral
parenchyma. Neither do central axons regenerate into the grafted ganglia (Ranson,
1914;Tidd, 1932; Clark, 1942). The growth of axons out of ganglia transplanted into
muscle or subcutaneous connective tissue is probably more extensive than that from
intracerebrally grafted ganglia (Nageotte, 1907; Cajal, 1928; Ward, 1936). In related
experiments, the central stumps of cut motor nerves were implanted into the brain.
h o n s could be traced from the nerve into the mass of Schwann cells that formed
around its site of termination and even beyond this region into the adventitial connec-
tive tissue of cerebral blood vesseIs, but never into the proper nervous tissue of the
brain of the rabbit (Clark, 1943) or rat (Kiernan, 1969). These results were confirmed
in the cat and rat by Windle, Clemente & Chambers (1952) and Clemente (1958) but
in both the latter studies it was found that large numbers of axons grew out into the
cerebral parenchyma if the animals received post-operative courses of injections of
Piromen or corticosteroids (see p. 164).
It has been claimed that if the gap between the stumps of a transected rat’s spinal
cord is filled by a piece of peripheral nerve (from the same animal), dense scars do not
form and spinal axons enter the grafts (Kao, 1974). Similar Observations made by
Sugar & Gerard (1940), who used homografts, could not be confirmed by Barnard &
Carpenter (1950). The growth of axons into autogenous grafts was more extensive
if the latter were inserted seven days after spinal transection, when necrotic central
nervous tissue could be removed (Kao, Chmg & Bloodworth, 1977). Fragments of
autogenous cerebellum, used in a similar way, provoked scarring, but if the cerebellar
tissue had been maintained in culture for one week it was less irritant and behaved
in the same way as a fresh piece of peripheral nerve (Kao, Shimizu, Perkins & Free-
man, 1970). The functions of these grafts are far from clear, Grey matter of the adult
C.N.S. (Williams, 1953; Kiernan, 1969), unlike that of the P.N.S. and the foetal
C.N.S. (Dunn, 1917;Das & Altman, 1972; Olson & Seiger, 1972; Thuline & Bunge,
1972; Lund & Hauschka, 1976; Hoffer, Seiger, Freedman & Olson, 1977; Olson,
Freedman, Seiger & Hoffer, 1977) does not survive autotransplantation. Small pieces
of adult central nervous tissue can be maintained in culture for up to 2 weeks (Kier-
nan & Pettit, 1971; Tsiquaye & Zuckerman, 1974), though many of their cells die
(Drayton & Kiernan, 1973; Davis, Gascho & Kiernan, 1975). Central white matter
is more amenable to transplantation. Aguayo et al. (1978) have autografted pieces of
the optic nerves of adult rats and mice into the skin and into the sciatic nerve. The
grafts survived and in the latter site were penetrated by peripheral axons, a few of which
became myelinated by neuroglial cells.
I 60 J. A. KIERNAN
A different kind of transplantation into the transected spinal cord was tried by
Turbes & Freeman (1958), Jakoby, Turbes & Freeman (1960)and Perkins, Barbini &
Freeman (1964) in dogs. An intercostal nerve which took its origin above or below
the level of the lesion was anastomosed to the distal or proximal stump of the cord.
The authors stated that half of the animals attempted to stand and walk about z weeks
later and showed further improvement over the following months. Voluntary move-
ments of the hind limbs were said to be immediately abolished by cutting the anasto-
mosed nerve. Regenerated axons were demonstrated histologically, passing from
the nerve into the cord and electrophysiological evidence of conduction along the
reconstituted pathway was obtained. These results have not, unfortunately, been
reproduced in other laboratories (see Benes, 1968).

(3) Neurosecretory axons

Neurosecretory cells are distinguished from other types of neurone by their charac-
teristic tinctorial and histochemical properties. In mammals such neurones occur only
in the magnocellular nuclei of the hypothalamus. Their unmyelinated axons pass
through the median eminence and the pituitary stalk into the posterior lobe of the
pituitary gland, where they end around capillary blood vessels. The neural components
of the three last-named structures collectively form the neurohypophysis, a region of
the C.N.S. characterized by the presence of morphologically distinctive neuroglial
cells, the pituicytes (Weaver & Bucy, 1940) and by the absence of a blood-brain
barrier (Cappell, 1929; Wislocki & King, 1936).
When severed within the territory of the neurohypophysis (e.g. in the pituitary
stalk) neurosecretory axons regenerate vigorously. This phenomenon was first de-
scribed by Stutinsky, Bonvallet & Dell( 1949) in the dog and hassince beenextensively
studied in many mammalian species including man (see Dellmann, 1973 for a com-
prehensive review). If the posterior lobe of the pituitary gland becomes atrophic
post-operatively, a new neurohaemal organ is formed which, though small, is fully
functional. Should the original posterior lobe survive, it is reinnervated by neuro-
secretory fibres and becomes indistinguishable from the normal organ. These two
modes of regeneration have been termed, respectively, ‘proximal’ and ‘proximo-
distal’ regeneration of the neurohypophysis (Kiernan, 1970). Fendler et al. (1970)
state that proximal regeneration of the neurohypophysis in the rat can be prevented
by prior extirpation of the superior cervical sympathetic ganglia. This interesting
observation requires further investigation. It is a surprising finding since sympathetic
innervation of the normal neurohypophysis has never been described. Sympathectomy
would be expected to result in vasodilatation in the region and the most obvious
consequence of this would be an improvement rather than an inhibition of the re-
generative activity.
Neurosecretory axons probably do not regenerate following severance within the
hypothalamus in mammals (Kiernan, 1969) though they may grow into intrahypo-
thalamic grafts of neurohypophysial tissue (Desclin & Flament-Durand, 1963).
Neurosecretory fibres do not regenerate into autotransplanted pieces of peripheral
nerve and the surgically re-directed proximal stumps of transected nerves are unable
 Axonal regeneration in mummuls 161
to regenerate into neurohypophysial tissue. Neither can ordinary central axons grow
into the posterior pituitary lobe following its transplantation into the brain (Kiernan,
1971).
(4)Monoaminergic j i b e s
Certain unmyelinated axons containing noradrenaline, dopamine and serotonin
can be demonstrated in the brain and spinal cord by fluorescence histochemical
methods for these amines. It has been shown by Nygren, Olson & Seiger (1971)that
monoaminergic fibres can regenerate across sites of transection of the rat's spinal
cord and by Nygren et al. (1971), Bjorklund, Nobin & Stenevi (1973) and Nobin,
Baumgarten, Bjorklund & Lachenmayer (1973) that the same axons can regenerate
after severance produced chemically, by the application of toxic analogues of meta-
bolic precursors of the natural monoamines. Bowen, Karpiak, Demirjian & Katzman
(I975) have observed similar phenomena in the cryosurgically injured cerebral cortex
of the rabbit. After compression of the spinal cord, Bjorklund, Katzman, Steveni &
West ( I 971) observed copious regenerative sprouting of noradrenaline- and serotonin-
containing s o n s into the necrotic tissue present at the site of injury. This axonal
growth started on the third post-operative day and attained its maximum extent on
the fourteenth day. Regeneration of monoaminergic axons all the way through the
site of compression was not found by Bjorklund et al. (1971), but specimens were not
examined later than 3 weeks after placement of the lesions.
Nygren, Olson & Seiger (1977) have demonstrated regeneration of monoaminergic
axons out of pieces of foetal medulla grafted into the spinal cords of adult rats. Other
studies of the regeneration of central monoaminergic axons have made use of intra-
cerebrally transplanted non-neural tissue and will be discussed, along with other such
transplantation experiments, in Section I1 (7).

( 5 ) Regeneration through spinal roots

Although axonal regeneration within dorsal roots is generally arrested at the
Schwann cell-neuroglia junction (see above), it has been shown (Ikeda & Campbell,
1971; Nathaniel & Nathaniel, 1973, 1977a, b) that some axons can penetrate into the
spinal cord. It is not known whether the axons that grow across the junctional zone
are of some special type or whether axons of all types are occasionally successful in
their regenerative efforts.
Barnes & Worrall (1968) anastomosed ventral roots to the central stumps of dorsal
roots (having removed the ganglia) and demonstrated histochemically the growth of
acetylcholinesterase-containing axons into the dorsal horn of the spinal grey matter.
The animals they used were, however, all treated post-operatively with cortisol (see
Section I1 (8(b)).
(6) Primary olfactory neurones
The cell-bodies of the primary olfactory neurones are receptor cells in the nasal
epithelium. Their unmyelinated axons are at first invested by Schwann cells in the
olfactory nerves and then by neuroglia within the olfactory bulb. The olfactory recep-
tors can be destroyed by application of a zinc sulphate solution, which does not
162 J. A. KIEFWAN
damage the ordinary respiratory epithelium of the nose. The treatment results
in stripping of the entire olfactory epithelium from its basement membrane and
is followed by complete degeneration of the olfactory axons. The epithelium is
then regenerated, probably from cells of the ducts of Bowman's glands in the
underlying lamina propria. Receptor cells differentiate in the new epithelium in
the frog (Smith, I~SI), rabbit (Mulvaney & Heist, 1971)and mouse (Matulionis,
1975),but not in the rat (Smith, 1938), though in the last-named species infection
may have interfered with the regenerative process (Takagi, 1971).
Transection of the olfactory nerves is invariably followed by death of the neuronal
somata in the epithelium, though the non-neuronal supporting cells survive. Graziadei
& DeHan (1973)and Harding, Graziadei, Graziadei & Margolis (1977)have shown in
the mouse that this retrograde degeneration is followed by replacement of the recep-
tors, probably as a result of mitosis and differentiation of primitive cells within the
olfactory epithelium. The axons of the regenerated receptors grew through the
olfactory nerves and into the olfactory bulb. This is the only known instance of
regenerative replacement of mammalian neurones. The new axons must regenerate
through tissues of both the P.N.S. and the C.N.S. in order to reach their destinations.

(7) Regeneration of axom into non-neural tissues transplanted into the brain
(a) Skin
When small pieces of skin are auto- or homografted into the cerebral hemispheres
of rabbits (Shirai, 1935;Glees & Erikson, 1954),rats (Nathaniel & Clemente, 1959;
Heinicke & Kiernan, 1978) or mice (Horvat, 1966,19673;Heinicke, 1978), they are
invaded by nerve fibres from both grey and white matter. These axons ramify in the
dermal and epidermal tissue, but do not form characteristic cutaneous end-formations.

(b) Striated muscle

Nathaniel & Clemente (1959)demonstrated that fragments of striated muscle
autotransplanted into the brains of rats were invaded by axons of central origin.
Motor end-plates were not formed, but the density of the regenerated innervation
was thought to be somewhat greater than that observed in cutaneous implants. Similar
experiments were carried out in mice by Horvat (1967a)and Heinicke (1978),who
found that the grafts contained fewer nerve fibres than comparable grafts of skin.

( c ) Smooth muscle
The re-innervation of smooth muscle autotransplanted into the C.N.S. has been
the subject of several investigations. The iris, when placed in the midbrain or spinal
cord, is rapidly invaded by histochemically demonstrable monoamine-containing
axons. The distribution of the regenerated fibres within the grafts resembles the nor-
mal pattern of noradrenergic innervation of the dilator pupillae muscle. The smooth
muscle of the uterus, which does not normally receive sympathetic fibres, does not
acquire a monoaminergic innervation when transplanted into the C.N.S. (Bjorklund
et al. 1971;Bjorklund & Stenevi, 1971).The regenerative central innervation of
 Axonal regeneration in mammals '63
transplanted smooth muscle by monoamifiergic axons is enhanced by intraventricular
administration of nerve growth factor (N.G.F.) (Bjerre, Bjorklund & Stenevi, 1973).
N.G.F. is a protein which has positive trophic effects on developing noradrenergic
sympathetic neurones in the P.N.S. and which accelerates the elongation of axons in
tissue cultures of sensory and sympathetic ganglia. It is conceivable that regenerated
axons detected in the dorsal spinocerebellar tracts and dorsal funiculi of the spinal
cords of cats treated with N.G.F. (Liu & Scott, 1958; Scott & Liu, 1964) may also
have been monoaminergic. N.G.F. may be necessary for the regeneration of central
monoaminergic fibres since the innervation of transplanted smooth muscle described
above can be inhibited by administration of anti-N.G.F. serum (Bjerre, Bjorklund
& Stenevi, 1974). It has been shown by Saunders (1972) that N.G.F. has no effect on
the rates of axonal regeneration in peripheral nerves of rats and rabbits.
The intracerebrally transplanted iris is also invaded by acetylcholinesterase-
containing axons and these form close contacts with the muscle fibres of the constric-
tor pupillae (Svengaard, Bjorklund & Stenevi, 1976), which normally receives a
cholinergic innervation.

( d ) Other tissues
Horvat (1966, 1967a, b, 1969) has homotransplanted cartilage, lung, liver, kidney,
pancreas and submandibular salivary gland, from neonatal mice into the cerebral
hemispheres, cerebella and spinal cords of adult mice. All except the cartilage were
invaded by central axons, though the grafts of lung, liver and pancreas were largely
replaced by connective tissue. The most profusely innervated tissue was that of the
salivary gland. Using autografts into the cerebral hemispheres of adult mice, Heinicke
(1978, 1979) found only slight axonal growth into salivary gland tissue, but fairly
abundant innervation of pieces of thyroid gland. The latter author also observed
axonal regeneration into vascularized regions of fragments of tendon transplanted
into the brain. Avascular regions of the tendon grafts contained healthy collagenous
tissue but did not accommodate any axons.

(8) Pharmacological effects on regenerating axons

Exogenous substances affecting axonal regeneration can be considered under four
headings.

(a) Thyroid hormones

Thyroxine (T,) and triiodothyronine (T3)are the natural secretions of the thyroid
gland, the proper functioning of which is necessary for the normal development of
the C.N.S. in foetal and post-natal life. In the developing C.N.S., these hormones
act to increase the rate of synthesis of protein, but this effect can no longer be elicited
when the nervous system becomes mature (see Sokoloff, 1971).
In adult animals, axonal regeneration is retarded in hypothyroidism (Marinesco
& Minea, 1910) but can be restored to its normal rate by replacement therapy
with T, (Isenschmid, 1932). Experimental hyperthyroidism results in accelerated
164 J. A. KIERNAN
functional recovery from peripheral nerve injuries (McIsaac & Kiernan, 1975a ;
McQuarrie, 1975; Berenberg et al., 1977). This effect may be due to faster axonal
growth (Cockett & Kiernan, 1973; Kiernan & Heinicke, 1977) or to more rapid re-
innervation of motor end-plates (McIsaac & Kiernan, 1975b). In some experiments
it has not been possible to demonstrate accelerated axonal regeneration in T,-treated
animals (Forman & Berenberg, 1977; Stelmack & Kiernan, 1977). Treatment with
T, also results in the formation of thicker myelin sheaths around both normal and
regenerated axons (Stelmack & Kiernan, 1977). This effect is probably due to
stimulation by the hormone of the metabolic activity of Schwann cells (Valcana
et al., 1975). The degenerative as well as the regenerative sequelae of injuries to
peripheral nerves are accelerated in hyperthyroid animals (Hines & Knowlton,
1934; Diaz-Guerrero, Thomson & Hines, 1947; McIsaac & Kiernan, 1975b).
Neuronal degeneration occurring in the normal ontogeny of the nervous system
is also thyroid-dependent (Race, 1961; Tata, 1966).
The mode of action of T, and T, in accelerating peripheral axonal regeneration is
not fully understood. I suggested (1972) that the injured neurone in an adult animal
might, while regenerating its axon, revert to an immature metabolic state and again
become responsive to thyroid hormones. This suggestion receives support from the
observation that the rate of protein synthesis in the ganglion cells of sensory axons
regenerating in the rat’s vagus nerve is increased by treatment with T, (Cook &
Kiernan, 1976).
Treatment with T, encourages the regeneration of axons across sites of crushing
of the spinal cord of the rat (Harvey & Srebnik, 1967) and T3has a similar effect on
wounds of the cerebral hemisphere (Fertig et al., 1971). The latter effect, however, is
less extensive than was at first thought (Heinicke, 1977). T, does not affect the pro-
liferation of neuroglia (Flint & Berry, 1972, 1973) and has only a slight suppressive
effect on the formation of connective tissue (Heinicke, 1977) at sites of injury to the
brain. It may be presumed, therefore, that enhancement of central axonal regener-
ation is, as in the P.N.S., due to an action of the hormone upon the neurones them-
selves. Other drugs that stimulate RNA and protein synthesis have also been tested
for enhancement of axonal regeneration in the C.N.S. Some success was achieved
with dinitryl malonic acid, but the toxicity of this compound severely limited its
therapeutic value (Nesmeyanova, 1977).
(b) Pyrogens, corticotrophin and corticosteroids
The first substance shown to promote central axonal regeneration was Piromen, a
pyrogenic bacterial polysaccharide. Animals with transected spinal cords sometimes
regained the use of their hind limbs following treatment with Piromen, and histo-
logical and physiological studies showed that the recovery was due to the regeneration
of axons across the sites of transection. The lesions were occupied at this stage by
loose connective tissue, through which the axons passed. Unfortunately, the improve-
ment was rarely sustained for more than a few months. The animals became paralysed
again, owing to compression of their cords by constricting fibrous scars. These studies
have been reviewed by Windle (1956) and Clemente (1964). More recently these
 A x m l regeneration in mammals 165
observations have been confirmed by Russian investigators using Pyrogenal, which,
like Piromen, is obtained from Pseudomonas bacteria (see Nesmeyanova, 1977).
Evidently, pyrogens derived from other types of bacteria are able to reduce the
density of scarring in the injured spinal cord but do not enhance the regeneration of
axons (Arteta, 1956a).
Pyrogens stimulate the secretion of corticotrophin and growth hormone by the
adenohypophysis (Hawley & Bowers, 1967; Jenkins, 1968). This endocrine effect
may be responsible for their actions on the injured C.N.S., though direct actions on
connective tissue have been suggested by Nesmeyanova (1977). Corticotrophin and
adrenal corticosteroids are able to reduce the density of the connective tissue scar
forming at the site of a spinal transection (Clemente, 1955) or of an injury to the brain
(Fertig et al., 1971). The former hormone was found by McMasters (1962) to be
somewhat more efficacious than Piromen in promoting axonal regeneration in the
spinal cords of rats. Treatment with corticotrophin does not affect the numbers of
neuroglial cells either in regions of axonal degeneration (Cavanagh & Joseph, 1958)
or around the sites of lesions (Flint & Berry, 1972, 1973). Corticosteroids may
influence axonal regeneration by actions other than those upon the formation of
connective tissue. The hormones influence the activities of several enzymes in the
brain (Shimada, Piddington & Moscona, 1967; DeVellis & Inglish, 1968) and probably
also increase the rate of synthesis of neuronal proteins (Vernadakis & Timiras, 1967;
Semiginovsky & Jakoubek, 1971).

( c ) Immunosuppressive drugs
Since one current theory (to be discussed later) invokes autoimmunity as a cause
of unsuccessful axonal regeneration in the C.N.S., it is interesting to see that the
effects of immunosuppressive treatments on the consequences of spinal transection
have been examined. The treatments tried have included the administration of
various antimitotic drugs and of antilymphocytic serum (Palladini & Alfei, 1965;
Feringa et al., 1973; Feringa, Wendt & Johnson, 1974) and the induction of im-
munological tolerance (Nelson, Feringa & Vahlsing, 1977). Although evidence for
conduction of impulses across the sites of transection was sometimes obtained, this
was shown to be associated in both experimental and control animals with incomplete
transection of the cord (Feringa et al., 1976). The promotion of axonal regeneration
in the C.N.S. by immunosuppression has not, therefore, been demonstrated.
( d ) Local treatments
In peripheral nerves, scarring at sites of injury is reduced by treatment with
pyrogens (Arteta, 19563), allantoin (Loots, Loots, Joubert & Kruger, 1976) or cis-
hydroxyproline, an inhibitor of collagen biosynthesis (Pleasure, Bora, Lane &
Prockop, I 974). While treatment with allantoin and cis-hydroxyproline may accelerate
axonal regeneration, pyrogens do not do SO (Arteta, 1956b). Systemic corticosteroids
may even delay the return of function (Lytton & Murray, 1954).
Axonal regeneration in the transected spinal cord may be enhanced by various
techniques intended to prevent the formation of dense collagenous scars. These
I 66 J. A. KIERNAN
procedures include enclosure of the approximated stumps in Millipore, a material
permeable to serum but not to cells (Bassett, Campbell & Husby, 1959; Campbell &
Windle, 1960) and enzymatic removal of necrotic debris with trypsin (Freeman,
McDougall, Turbes & Bowman, 1960) or with a mixture of deoxyribonuclease and
fibrinolysin (Perkins, Solow & Freeman, 1970). Exposure to a pulsed magnetic field
may have similar effects (Wilson & Jagadeesh, 1976). The functional usefulness of
these procedures is rather doubtful, and it seems likely that most of the observed
regenerating axons are derived from the dorsal nerve roots rather than from the
spinal cord itself (Perkins et al., 1970). Recently it has been claimed that substantial
axonal regeneration can be obtained in the spinal cord following combined local and
systemic treatment with a variety of enzymes. These experiments will be discussed in
Section (e) below. Puromycin, an inhibitor of ribosomal protein synthesis, has also
been applied to the injured spinal cord. Bernstein, Wells & Bernstein (1978) placed
pieces of gelatin foam soaked in a solution of this drug at sites of hemisection of the
spinal cords of rats. During the first 30 post-operative days, axons grew into the
cicatrices that formed at the sites of the lesions in the treated animals, but were not
seen in controls with saline-impregnated gelatin foam. These axons later degenerated
and none were left by the 60th post-operative day.

(e) Combined local and systemic treatment with enzymes

Matinian & Andreasian ( I 976) demonstrated axonal regeneration, associated with
substantial functional recovery, after complete transection of the spinal cords of
rats which were treated post-operatively with mucolytic and proteolytic enzymes.
Similar claims had already been made (see preceding Section), but the experiments
of the Armenian investigators involved much larger numbers of animals and were more
thoroughly documented than any of the earlier studies. The most effective regimes
were : ( a ) testicular hyaluronidase (injected subcutaneously at the site of operation
for the first I 5 post-operative days) followed by systemic (intramuscular) trypsin for
10 weeks, and (b) trypsin (injected at the site of operation) combined with elastase
(intramuscularly, 30 min after the trypsin), for 15 days. Several other regimes were
less effective. The best results were always obtained after combinations of local and
systemic injections of enzymes. With the most effective treatments nearly half of the
animals recovered almost completely and only 7-10% showed no evidence of func-
tional recovery. Extensive axonal regeneration was demonstrated histologically and
electrophysiological evidence of conduction of impulses across the sites of transection
was obtained. The control rats (injected only with the solvents for the enzymes)
showed no functional or microscopic signs of axons having grown across the lesions.
Two recent investigations have yielded results apparently incompatible with
those of Matinian & Andreasian (1976). Guth, Bright & Donati (1978) used an en-
zymatic treatment similar to one of the regimes of Matinian & Andreasian in rats
with hemisections of the upper cervical region of the spinal cord. The expected effect
of axonal regeneration (recovery of respiratory movement in the ipsilateral hemi-
diaphragm) did not occur and no histological evidence of regeneration could be found.
Knowles & Berry (1978) used two types of treatment (trypsin and elastase; hyalu-
 Axonal regeneration in mammals 167
ronidase followed by trypsin) in rats with incised cerebral wounds. No histological
differences between the experimental animals and their controls were detected.
Neither of these studies was an exact repetition of the work of Matinian & An-
dreasian (1976), though it was certainly reasonable to expect enhancement of axonal
regeneration by the treatments that were tried. The enzymes used in the later studies
were not obtained from the same sources as those used by Matinian & Andreasian
(1976)~so it is possible that the presence of unknown contaminants in some of the
preparations might account for the discrepant results.

(9) Foetal and neonatal mammals

Foetal and neonatal rodents exhibit considerably more functional recovery than
do adult animals, after injury to the brain or spinal cord. Thus, Gerard & Koppanyi
(1926) found that when the spinal cords of foetal rats were transected 8 days before
birth, the animals could walk at the same time as their unoperated controls. With
neonatal rats, much poorer results were obtained, but Ranson (1903)~Dunn (1917)
and Reinis (1965) have observed axons crossing lesions in the brains of newborn and
5-day-old rats. However, it has not yet been determined whether the apparently
regenerated axons in these cases arise from pre-existing neurones or from nerve cells
that differentiate after placement of the lesions (Sechzer, 1974). Furthermore, it has
been shown that even when the transected spinal cord of the newborn rat does not
reunite, appreciable recovery can ensue as a result of autonomous activity in the
isolated distal part of the cord (Stelzner, Ershler & Weber, 1975).
Axonal sprouting of neurones spared by lesions of the C.N.S. occurs more vigor-
ously and over greater distances in foetal than in adult animals (Miller & Lund, 1975;
Lund, 1978). Such sprouting may have been responsible for some of the functional
recovery attributed in earlier experiments to axonal regeneration.

(10) Laminar lesions

When the brain is suitably irradiated with monoenergetic a-particles, a thin layer of
the cerebral cortex, parallel to its surface;, is damaged. Neuronal somata are destroyed
and axons and dendrites are transected by such laminar lesions. Axons were seen
entering the lesions several weeks after irradiation (Rose, Malis, Kruger & Baker,
1960)~but these may not have been regenerated axons. Examination using silver
methods (Estable-Puig, de Estable, Tobias & Haymaker, 1964) and electron
microscopy (Kruger & Hamori, 1970) has revealed that not all neuronal processes
are destroyed within laminar lesions. One must be sceptical of any claims of
axonal regeneration in the mammalian C.N.S. when it has not been shown that all
axons passing through the sites of the lesions have been severed.

(I I) Submammalian vertebrates
The injured central nervous system has a greater capacity for regeneration of
axons in some lower vertebrates than is seen in mammals.
Full recovery from spinal transections nearly always occurs in adult cyclostomes
(Hibbard, 1963) and teleost fishes (Hooker, 1932; Tuge & Hanzawa, 1937; Koppanyi,
I 68 J. A. KIERNAN
1955;Phelps, 1969). The axons of the Mauthner and Miiller neurones of these animals
do not regenerate in adults but do so, albeit aberrantly, in the ammocoete larvae of
cyclostomes (Rovainen, 1976). In fishes extensive regeneration follows injury to the
telencephalon (Bernstein, 1967) and the optic nerve can also regenerate readily
@Perry, ‘945).
Axons can regenerate across sites of transection of the brain stem and spinal cord
in adult urodele amphibia (Sperry, 1948; Piatt, 1955), but in the adult anuran cord,
the regenerating fibres, though numerous, appear to be deflectedto useless destinations
by transversely oriented connective tissue (Schonheit, 1968). The optic nerve,
however, regenerates efficiently in both adult and larval anura (Sperry, 1944 ;
Gaze, 1960; Hibbard & Ornberg, 1976). The regeneration can be made to occur
more rapidly by crushing the nerve for a second time, between the retina and the site
of the first lesion, 4 or I I days after the initial injury (Brock, 1978). The optic axons
regenerate to their appropriate places in the tectum, even though the arrangement of
the fibres is considerably disorganized at the site of the lesion (Udin, 1978). The
neurosecretory axons of the hypothalamo-neurohypophysial tract, which are among
the few groups of central fibres capable of regeneration in mammals (see above),
regenerate rapidly in both amphibia (Dierickx, 1965) and fishes (Sathyanesan, 1965).
In adult reptiles and birds (Hamburger, 1955), regenerative phenomena have not
been detected in the central nervous system.

(12) Growth of neurites in culture

( a ) Elongation of neurites
The study of nervous tissue in vitro has provided valuable insights into the
mechanics of axonal growth and is relevant to the problems associated with the
regeneration of injured nerve fibres. Harrison (1907) first described the tips of fibres
growing out from nerve cells in explants of the larval amphibian neural tube. In this
and later investigations, it has usually been difficult to determine whether the cyto-
plasmic processes of neurones in cultures of ganglia and parts of the C.N.S. are axons
or dendrites, so the conveniently vague term ‘neurites’ is widely used. The continued
protrusion and retraction of minute filopodia by the growing tip is generally thought
to be an exploratory activity in which the neurite seeks out those chemical and
physical domains in the substrate which are the most suitable for continued growth
(Weiss & Taylor, 1944). The advancing tips of neurites in vitro are called ‘growth
cones’. Similar structures in vivo are difficult to detect, but have been described in
the skin (Roberts, 1976) and at the tips of regenerating axons in peripheral nerves
(Toh, Cragg, Singh & Koh, 1978).

(b) Contact guidance and chemotaxis

Nerve fibres in vitro grow along interfaces between liquid and solid or between
liquid and gelled colloid phases (Weiss, 1934). Their courses over long distances are
probably determined largely by the disposition of such interfacial planes. Changes of
direction and branching can occur when the tip of a fibre meets a physical obstacle.
 A x o m l regeneration in mammals 169
Over short distances (perhaps up to 500 pm), the neurites may follow chemical con-
centration gradients (Harrison, 1935; Crick, 1969) so it is possible that a chemotactic
mechanism is involved in the development of specific synaptic connexions between
functionally associated neurones.

( I 3) Mechanisms of axonal growth

The elongation of the neurite involves proximo-distal transport of neuronal cyto-
plasm. This is necessary for addition to the volume and surface-area of the growing
nerve fibre. The orthograde flowing of cytoplasmic material (see Weiss, 1961), as
well as the retrograde transport of particulate matter (Burdwood, 1965), has been
observed by cinemicrographic methods. New membrane is probably manufactured
in the perikaryon and transported distally as vesicles of agranular reticulum, which
then fuse with the plasmalemma at the growing tip of the axon (see Peters, Palay &
Webster, 1976).
Cultured neurones have also been shown to be able to absorb identifiable proteins
added to the nutrient media (Klatzo & Miquel, 1960; Holtzmann & Peterson, 1969).
Ultrastructural evidence of pinocytosis is seen between the bases of filopodia at the
tips of neurites (Yamada, Spooner & Wessels, 1971; Bunge, 1977) and the endo-
cytosed vesicles may be involved in the conservation and re-cycling of the plasmalemma
(Bray, 1973) as well as in the introduction of protein molecules from the medium into
the cell (Tischner, 1977). Intact protein molecules are probably essential nutrients for
neurones in vitro, since these cells cannot be cultured in the absence of some kind
of serum or tissue extract. Two individual proteins shown to improve the survival of
neurones in vitro are insulin, which may be beneficial to cells from all regions of the
nervous system (see Murray, 1971; Kiernan, 1974) and nerve growth factor (N.G.F.),
which acts principally (see Levi-Montalcini & Angeletti, 1968) but not exclusively
(Hillman & Sheikh, 1968) on the cells of sensory and sympathetic ganglia. Insulin
and N.G.F. have similar chemical structures and evoke similar metabolic changes in
cells responsive to their actions (Frazier, Angeletti & Bradshaw, 1972).
It is well known that in vivo, exogenous tracer proteins can be absorbed from pre-
synaptic terminals in muscle (Kristensson, Olsson & Sjostrand, 1971; Kristensson &
Olsson, 1973) and in the C.N.S. (LaVail, Winston & Tish, 1973; LaVail, 1974) and
from axons injured by cutting or crushing peripheral nerves (Furstman, Saporter
& Kruger, 1975; Kristensson & Olsson, 1974, 1975, 1976). The absorbed proteins are
transported retrogradely to the perikarya, where they can be detected by histological
and histochemical methods. Similar absorption occurs at the tips of actively regener-
ating peripheral axons (Sparrow, 1978; T. Olsson, Forsberg & Kristensson, 1978;
Sparrow & Kiernan, 1979).

111. HYPOTHESES
The various theories purporting to explain the differences between the regenerative
properties of axons in the C.N.S. and P.N.S. will now be discussed and attempts will
be made to reconcile them with the experimental data outlined above.

I1 B R E 54
170 J. A. KIERNAN

(I) Intrinsic inability of central neurones to regenerate their severed axons

( a ) Statement of hypothesis
Severed axons regenerate readily in all types of peripheral nerves, indicating that
the non-neuronal components (principally Schwann cells) of the P.N.S. form a
favourable environment for growing axons. However, when pieces of peripheral
nerve are transplanted into the brain of the rabbit, axons of the C.N.S. do not grow
into them. Clark (1942, 1943) deduced from this observation that central neurones
had little or no capacity to regenerate their axons. Such an intrinsic lack of regener-
ative capability would explain the lifelong disabilities caused by spinal transection and
by lesions in other parts of the C.N.S. that cannot be functionally bypassed by
reorganization of surviving neuronal connexions.

(b) Arguments supporting hypothesis

(i) Lower vertebrates. The successful axonal regeneration seen in the spinal cords
of cyclostomes, fishes and some amphibia might indicate that the ability to regenerate
has been unfortunately lost during the course of the evolution of the more complex
central nervous systems of reptiles, birds and mammals. The failure of regeneration
of the Mauthner axons in the spinal cords of fishes (see p. 168) could be a result
of excessive morphological specialization. The organization of the P.N.S. has not
changed nearly as much as that of the C.N.S. with phylogenetic development. Since
the morphological plan of the P.N.S. is essentially the same in all vertebrates, it is
not surprising that axonal regeneration in peripheral nerves occurs in mammals, just
as in the lower forms.
(ii) Mammalian neurosecretory, monoaminergic and olfactory jibes. The neuro-
secretory neurones of the hypothalamus, whose axons are able to regenerate in
mammals, are cells of a phylogenetically very old type. Neurones with identical
histochemical properties and closely similar ultrastructural features occur in the
nervous systems of most classes of invertebrates (see Heller & Clark, 1962; Gabe,
1966). These cells are all thought to secrete peptide neurotransmitter substances.
Willmer ( I974) has suggested that the vertebrate hypothalamo-neurohypophysial
system evolved from the peptidergic neurones supplying the retractor muscle of the
proboscis in certain nemertine worms, which may have been the ancestors of the
vertebrates and other chordate animals. The primitive nature of the neurosecretory
cells may account for the retention of regenerative propensities which have been lost
by most of the other neurones of the mammalian C.N.S. Monoaminergic neurones
are also phylogenetically old (see Gershon, 1977)and can also regenerate following
severance in the mammalian C.N.S. The primary olfactory neurones, which have
been shown to be capable of regeneration (see p. 161) are located in an epithelium and
are perhaps comparable to similarly situated receptors in many invertebrates.
(iii) The immature central nervous system. The regenerative capacity of neurones in
the C.N.S. of foetal and possibly of neonatal rodents might be explained in terms of
ontogeny repeating phylogeny, with the cells losing the ability to re-grow amputated
 Axonal regeneration in mammals 171
axons once they have matured. However, as explained earlier, it is by no means cer-
tain that severed axons do regenerate in the C.N.S. of foetal mammals.
(c) Discussion of arguments opposing the hypothesis
(i) Motor neurones. The idea that the mammalian C.N.S. contains neurones so
specialized as to be unable to support axonal regeneration fails to explain some
experimental observations. Thus, the neurones supplying striated muscles have their
cell bodies in the brain-stem and spinal cord, yet their axons are fully capable of
regeneration following injuries to peripheral nerves. The same is true of the pre-
ganglionic autonomic neurones, whose axons go to sympathetic and parasympathetic
ganglia (Langley & Anderson, 1904). These efferent neurones are, like the neuro-
secretory and monoaminergic ones, phylogenetically old, being present in all chordate
animals (see Papez, 1929)~so it would be of great interest to know whether their
axons would regenerate if they were selectively transected within the C.N.S. This
experiment does not appear to have been attempted per se, though Cajal (1928)
commented that when the intraspinal portions of ventral roots were involved in
lesions transecting the cord, axonal regeneration did not occur. Tower (1943)
observed some regeneration into ventral roots that were avulsed from the spinal cord
and re-implanted, but in this case there is uncertainty as to whether the neurotmesis
occurred in an environment of Schwann cells or of neuroglia. If it were shown that
these efferent axons could regenerate within the C.N.S., the theory presently under
discussion would be supported.
(ii) Transplantation experiments. It is difficult to reconcile the regenerative inner-
vation of intracerebrally transplanted non-neural tissues with a postulated intrinsic
inability of central axons to regenerate. Indeed, the observed growth of central fibres
into such grafts provides important evidence of the ability of central neurones to
regenerate their axons.
(2) Inadequacy of the periaxonal environment
( a ) Schwann cells and contact guidance
(i) Development of hypothesis and supporting evidence. Within the degenerated distal
segment of an injured peripheral nerve, the Schwann cells are arranged in long cords,
being held in this orientation by a rigid framework of basement membranes. The
latter form tubes, just as they did in the intact nerve. A regenerating axon grows
along the cleft between the plasmalemmae of the Schwann cells and the inside of the
tube formed by the encompassing basement membrane (see Ohmi, 1962; Wettstein &
Sotelo, 1963; Satinsky, Pepe & Liu, 1964). The arrangement of Schwann cells and
their basement membranes obviously provides a favourable substrate for the reception
of regenerating axons and for their guidance to peripheral end-organs. No such
organized channels exist in the C.N.S. There, Schwann cells are absent and the
neuroglial cells, with their numerous cytoplasmic extensions, may largely fill the
available space. The only basement membranes present are those of capillary blood
vessels (Palay & Palade, 1955). The differences between the arrangements of support-
ing cells and basement membranes in the P.N.S. and C.N.S., and especially the
11-2
172 J. A. KIERNAN
absence of Schwann cells in the latter, have been thought to account for the different
regenerative phenomena that follow injury to the two divisions of the nervous system
(Cajal, 1928).
The growth of central axons into non-neural tissues transplanted into the C.N.S.
may be compatible with Cajal’s hypothesis, since Schwann cells of small nerves
originally present in the tissues may proliferate and migrate to the peripheral zones
of the grafts (Horvat, 1969).
(ii) Arguments against the hypothesis. There are several serious objections to Cajal’s
simple explanation of the failure of regeneration of injured central axons.
First, the cellular architecture of the adult C.N.S. is essentially the same in all
classes of vertebrate animal, yet axonal regeneration takes place efficiently only in
the brains and spinal cords of fishes and some amphibia. While it is possible that
subtle structural substrates for the guidance of regenerating nerve fibres might be
present in the central nervous systems of lower vertebrates, there is, as yet, no evidence
of their existence. Secondly, only small numbers of central axons grow into pieces of
peripheral nerve grafted into the adult mammalian C.N.S. Such grafts retain their
linear arrangements of Schwann cells and there is no obvious reason why they should
not accommodate abundant regenerating central fibres. Thirdly, some axons do
regenerate following severance within the C.N.S., despite the absence of Schwann
cells. Notable among these are the neurohypophysial neurosecretory fibres, which
can regenerate amongst pituicytes, but not amongst Schwann cells. Fourthly, axonal
regeneration in the C.N.S. can be enhanced by treatment with some hormones,
enzymes and other drugs. Some of these agents modify the micro-environment of
regenerating axons, but none induce structural organization similar to that occurring
in peripheral nerves.

(b) Compatibility between regenerating axons and the surrounding cells

(i) Development of hypothesis. In the adult mammal, some types of axon can
regenerate amongst some types of non-neuronal supporting cell. With other combi-
nations of axons and cells, regeneration does not occur. Table I summarizes various
combinations of axonal types and the kinds of cells amongst which they have been
required to regenerate in experiments involving autotransplantation of different
tissues.
Although some experimental results are still in dispute and not all the possible
combinations have been tested, it can be seen that all the axonal types shown in Table
I are capable of regeneration in some circumstances. It is notable that ordinary
central axons grow more readily into non-neural tissues than amongst neuroglial
cells, but it is not known how long the regenerated portions of such axons might
eventually become, since the grafts are necessarily only I or 2 mm in diameter. Nerve
fibres of other types are evidently capable of regeneration amongst the cells that
normally accompany them, or into tissues that are normally innervated by physio-
logically similar axons elsewhere in the body.
From these considerations, it is evident that for regeneration to occur there must
be a favourable interaction between the axons and the cellular or intercellular matrix
 Axonal regeneration in mammals I73
Table I. Occurrence and failure of axonal regeneration into dzFt?rent tissues or
cellular environments in mammals
Type of axon

Central Central Other

Peri- Neuro- Mono- AChE- Primary central
Cell-type or tissue pheral secretory aminergic containing olfactory fibres
Schwann cells + - 0 0 +* kt
Ordinary neuroglia 21 (A) +I 0 f* - II
Pituicytes - + 0 0s 0 -
Skin + 0 0 +** 0 +**
Smooth muscle (iris) + 0 + + 0 0
Striated muscle + 0 0 ott 0 + tt
AChE = Acetylcholinesterase; + means that regeneration occurs; - means that regeneration does
not occur; o means that information is not available; ( ) indicates doubt or disputed results;
+ indicates discrepant observations.
-
* These are the axons of neurones newly differentiated in the injured olfactory epithelium (see
p. 162).
+
t in mouse; - in rabbit and rat.
1 Peripheral axons regenerate into grafts of optic nerve, but not into the brain (except in Piromen-
treated animals). Some axons can regenerate through nerve roots into the spinal cord.
Perhaps not always (see p. 161).
I/ Regeneration occurs to a very limited extent and can be accentuated by some drugs (see pp.163-167).
9 Neurosecretory fibres in some mammals contain AChE (Koelle & Geesey, 1961 ; Kiernan, 1964).
** Of all the AChE-containing cerebral axons abutting against an implanted piece of skin, only a
small minority grow into the graft (Heinicke & Kiernan, 1978). Most of the axons in such transplant8
do not contain AChE.
tt It is possible that some or all of the central axons entering striated muscle grafts in the brain
might contain AChE.

into which the axons are required to grow. I suggested (1971)that non-neuronal cells
of different types selectively permitted or encouraged the regenerative growth of
particular kinds of nerve fibre, and that regeneration failed to occur when there was
no such compatibility between the axons and their surrounding cells. This hypoth-
esis, though rather vague, accounted for the failure of peripheral axons to grow into
the neurohypophysis and of neurohypophysial neurosecretory axons to grow amongst
Schwann cells. More recent observations of the regenerative innervation of trans-
planted smooth muscle by central monoaminergic and acetylcholinesterase-containing
fibres are easily explained in similar terms.
(ii) Discussion. Axonal growth is dependent upon synthesis of structural proteins
in the perikaryon, so any modulating factors operative at the elongating tip of the
nerve fibres must transmit their influences in some way to the cell-body of the neurone.
Retrograde axoplasmic transport would provide a suitable mechanism and will be
discussed later in connexion with other hypotheses.
While it seems that specific relationships must exist between axons and non-
neuronal elements, the nature of the affinity is unknown. Chemotactic mechanisms,
suggested by Cajal (1928), could operate only at very short range (Crick, 1969),
if indeed they exist at all. Electric fields may influence the direction of growth of
I74 J. A. KIERNAN
neurites in tissue culture (Marsh & Beams, 1946) but no evidence of ‘galvanotropism’
in vivo has ever been obtained. Since nerve fibres in vitro can grow on various sub-
strates, it is unlikely that the purely physical properties of the intercellular matrix
or of the surfaces of cells affect axonal regeneration in a specific manner. The
chemical constituents of the structures into which axons attempt to regenerate may,
however, exert selective influences comparable with the ‘neurotropic’ effects of
Schwann cells first postulated by Cajal. Berry (1979) and Lund (1978) have speculated
that specific growth factors akin to N.G.F. may exist for all types of neurones. Investi-
gation of the interactions of different extracellular materials and of components of
the external surfaces of cells with regenerating axons is clearly needed.

(3) Physical barriers to regeneration

( a ) Observations indicating existence of barriers to regeneration
The growth of regenerating axons in the C.N.S. may be hindered as a result of
structural changes at the site of injury. The simplest type of central nervous lesion
is a clean incised wound of the brain. The events involved in the healing of such a
wound have been described in detail by Penfield (1927). The space separating the
intact tissues on either side of the lesion is at first occupied by clotted blood and
necrotic nervous tissue. These are later organized to form connective tissue, the
collagenous fibres of which blend with the leptomeninges. The thickness and density
of the connective tissue are variable. Thick aggregations of tightly packed collagen
fibres are occasionally seen, but a looser, reticular arrangement is more usual, es-
pecially in the brain. In the deeper parts of a cerebral wound the connective tissue
scar may be represented only by a thin lamina, resembling a basement membrane
(Willis, Berry & Riches, 1975). In places, this connective tissue is absent (Heinicke,
1977) or at least apparently so in optical microscopy. Regenerating axons are never
seen traversing dense collagenous scars, but are most frequently observed in places
where the collagen forms loose networks. The axons are sometimes seen in close
association with fine strands of connective tissue (Fertig et al., 1971). It must be
emphasized, however, that most of the axons severed at the time of making the lesions
do not even enter the zone of connective tissue.
On either side of the incised wound neuroglial cells, mostly astrocytes, proliferate
and their cytoplasmic processes intermingle to form an aggregation of what neuro-
pathologists call ‘glial fibres’. The thickest and most conspicuous of the astrocytic
processes are aligned at right angles to the plane of the wound, with one end of each
cell attached to the collagenous scar and the other end to a capillary blood vessel in
the undamaged nervous tissue. Penfield (1927) held that this orientation of the
astrocytes resulted from the slow contraction of the collagenous scar. Other cells
appearing in the vicinity of the lesion are macrophages. While some of these may be
derived from the microglia, the majority are probably of haematogenous origin (Flint
& Berry, 1973; Wakefield & Eidelberg, 1975; Ling, 1978). Since it is within the zone
of glial fibres that most of the transected axons are found to terminate, Cajal(1928),
Chambers (1955) and many others (see Windle, 1955, 1956) have proposed that the
 Axonal regeneration in mammals I75
neuroglial component of the scar forming at sites of injury to the C.N.S. constitutes
a major physical barrier to axonal regeneration.
Support for such a view comes from observations of enhanced regeneration in
animals treated with Piromen, corticotrophin, corticosteroids, and proteolytic
enzymes, agents which certainly suppress the development of collagenous connective
tissue and may also reduce the obstructive powers of the proliferated neuroglial cells
(Windle & Chambers, 1950; Windle et aZ., 1952). The proliferation of neuroglial
cells and the influx of macrophages around lesions are unaffected by treatment with
corticotrophin (Flint & Berry, 1973), but these observations do not preclude an affect
of the hormone on the cytoplasmic processes of the reactive astrocytes. The concept
of blockade of regeneration by scarring may also be consistent with the successful
regeneration of central axons severed in immature animals, in which less dense scars
are formed (Chambers, 1955). It is, however, by no means proven that axonal regener-
ation does take place in young mammals (see p. 167).

(b) Evidence against blockade of regeneration by scars

Axonal regeneration in the piscine C.N.S. occurs vigorously despite the presence
of a scar which looks just as potentially obstructive as that found in mammals. I n
fishes, the axons grow through the substance of the scar (Tuge & Hanzawa, 1937;
Koppanyi, 1955; Bernstein & Bernstein, 1967). In the transected spinal cords of
adult anuran amphibia, axons regenerate through the neuroglial component of the
scar and are deflected laterally by dense collagenous connective tissue (Schonheit,
I 968).
The postulated existence of physical barriers to axonal regeneration in the mam-
malian C.N.S. fails completely to explain the regeneration of monoaminergic and
neurosecretory fibres, the innervation of intracerebrally transplanted non-neural
tissues and the effects of thyroid hormones. While it is entirely reasonable to suppose
that axons cannot grow through densely packed collagenous connective tissue,
there is little experimental support for the view that axonal regeneration in the C.N.S.
is obstructed by proliferated neuroglia.

(4)Inappropriate formation of synapses

( a ) Bernstein’s experiments and deductions
Experiments performed by Bernstein & Bernstein (1967, 1971) may provide an
explanation for the different sequelae of injuries to the spinal cords of fishes and mam-
mals. These investigations will now be briefly described.
Axonal regeneration and the associated functional recovery were usually well
advanced 30 days after transecting the spinal cord of the goldfish, but could be
prevented if, at the time of operation, a piece of Teflon (polytetrafluoroethylene) was
inserted between the stumps of the cord. Subsequent removal of this barrier was not
followed by regeneration of axons across the site of transection and electron micro-
scopic examination revealed unusually large numbers of axodendritic and axoaxonal
synapses in these regions. It was shown that many of the boutons at these synapses
belonged to the axons of the long tracts of the cord. If, at the time of removing the
176 J. A. KIERNAN
Teflon barrier, the cord was transected again at a more rostra1 level, axons now
regenerated across the sites of both lesions. The substantial glial-ependymal scar
that had formed at the site of the earlier transection did not obstruct the regeneration
of the axons of the long tracts and functional recovery was observed in the caudal
parts of the body. These results indicate that when axonal regeneration in the teleo-
stean spinal cord is made to fail, the nerve fibres in the region of the lesion make
synaptic connexions which are inappropriate to their normal functions. Once these
synapses are established, further regeneration does not occur unless the axons are
cut for a second time. In the latter eventuality, regenerative growth proceeds as if the
original lesion had never been made.
Comparable experiments in mammals were not possible, but it was shown (Bern-
stein & Bernstein, 1971) that large numbers of axodendritic synapses developed
around sites of hemisection of the rat’s spinal cord and that some of these involved the
axons of long tracts. It was suggested that axons in the injured spinal cord of the rat
regenerated only for very short distances before forming inappropriate synapses. The
stimulus to regenerative growth was presumed to be lost as a consequence of the
establishment of such connexions. Bernstein et al. (1978) suggested that the transient
axonal regeneration that followed application of puromycin to the injured spinal cord
(see Section II(8d)) was due to prevention of the development of the dendritic
varicosities which received the inappropriate synapses.

( b ) Discussion
Axons in the spinal cords of fishes evidently exhibit weaker tendencies to synapse
with the wrong neurones than do their mammalian counterparts. Expressed in this
way, Bernstein’s concept recalls hypotheses invoking the evolutional ‘ over-maturity ’
of mammalian central neurones. Instead of considering the axon in the mammalian
C.N.S. as being incapable of regenerating, perhaps we should regard it as being less
‘choosey’ in its selection of a synaptic partner than its piscine counterpart.
The success of axonal regeneration in the mammalian P.N.S. is easily justified in
terms of the ideas discussed above. There is nothing in a peripheral nerve with which
an axon can synapse until an end-organ is reached. The regeneration of the
hypothalamo-neurohypophysial tract within the pituitary stalk might be similarly
explained, though capillary blood vessels, upon which the fibres of this tract terminate,
would surely be present throughout the course followed by the regenerating
neurosecretory axons. If the latter were to stop growing as soon as they encountered
any capillaries, proximo-distal regeneration of the neurohypophysis (see p. I 60)
would never occur. The failure of axonal regeneration in the mammalian optic
nerve, despite the preservation of retinal ganglion cells (Eayrs, 1952) is not explained
by Bernstein’s hypothesis, since this part of the C.N.S. contains no neurones with
which axons could form synapses.
Further experimentation is needed in order to determine whether inappropriate
synaptogenesis is a cause or an effect of the failure of axonal growth in the injured
spinal cord.
 Axonal regeneration in mammals '77
( 5 ) Autoimmune inhibition of regeneration
(a) The hypothesis of Berry €Y Riches
The neurones and neuroglial cells of the C.N.S. and the tissues within the endo-
neurium in the P.N.S. are sequestered from the circulation by brain-blood and
nerve-blood barriers. If the lymphoid tissues are deliberately exposed to suitably
compounded homogenates of brain or peripheral nerve, antibodies (humoral and cell-
borne) are formed and their reactions with the auto-antigens result in autoimmune
diseases, experimental allergic encephalomyelitis (E.A.E.), or peripheral neuritis
(E.A.N.). These are both demyelinating diseases, the antigens principally responsible
for their causation being proteins of the myelin sheaths.
Any damage to the brain or spinal cord may be expected to release potentially
antigenic material into the blood, but simple injuries of the C.N.S. do not induce
E.A.E. The involvement of an autoimmune process in preventing the regeneration
of axons severed within the C.N.S. was suggested by Feringa, Wendt & Johnson
(1g74), but was first expounded in detail by Berry & Riches (1974). The hypothesis
advanced by the latter authors can account for many of the phenomena associated
with injury to the nervous system.
Berry & Riches suggested that antibodies and antibody-bearing cells circulating
in the blood as a result of injuring the C.N.S. could only attack at the site of the lesion.
The blood-brain barrier, which prevents the passage of proteins and cells from the
blood into the C.N.S., is known to be defective for 2-3 weeks at and around sites of
trauma to the brain and spinal cord. It was postulated that while the severed axons
were in the earliest stages of regeneration their tips imbibed antibodies by endocytosis
and transported them to their neuronal perikarya. Here, the antibodies were thought
to interfere with the synthesis of structural protein, thereby arresting the regenerative
process. The success of axonal regeneration in peripheral nerves was attributed to
rapid phagocytosis and subsequent denaturation of antigens by the Schwann cells
surrounding the degenerated axons and their myelin sheaths.

(b) Arguments supporting the hypothesis

The following pieces of evidence are congruent with the hypothesis of Berry &
Riches (1974).
(i) The growth-cones of recently injured and actively regenerating axons are able
to imbibe proteins and transport them retrogradely to their perikarya (see p. 169).
(ii) Neurosecretory axons normally exist in a region where there is no blood-brain
barrier to proteins and would not, therefore, be auto-antigenic.
(iii) Monoaminergic axons may secrete at their advancing tips amines that can
block the reaction between sensitized lymphocytes and myelin antigens (Carnegie,
Caspary, Smythies & Field, 1972). More regeneration of these axons is seen after
degeneration due to administration of toxic analogues of the metabolic amine pre-
cursors than after surgical transection (Nygren, Fuxe, Jonsson & Olson, 1974) and the
drug-induced lesions may not be associated with opening of the blood-brain barrier
(Sidman & Wessels, 1975).
178 J. A. KIERNAN
(iv) The enhancement of axonal regeneration in the C.N.S. by corticosteroids
and by agents that increase their endogenous secretion may be due to the immuno-
suppressive rather than to the anti-inflammatory actions of these hormones.

(c) Observations incompatible with the hypothesis

The concept of autoimmune inhibition of axonal regeneration in the C.N.S. has
been tested in three experiments, the results of which render the hypothesis untenable.
(i) Although even minor injuries to the C.N.S. evoke the production of humoral
antibodies to some cerebral lipids (see Berry & Riches, 1974; Berry, 1979), there is
no evidence of cell-mediated immunity to myelin or to whole-brain tissue even after
severe trauma (Willenborg, Staten & Eidelberg, 1977). Immunosuppression probably
does not promote axonal regeneration in the transected spinal cord (see Section I1
(W-
(ii) It has been shown (Mervart & Kiernan, 1978) that, in the rat, severed axons in
the sciatic nerve regenerate at the same rate in control animals as in animals with
E.A.N. The explanation given by Berry & Riches (1974) for peripheral axonal
regeneration cannot, therefore, be entirely correct.
(iii) Axons regenerate into intracerebral skin grafts in rats, despite the absence of
a blood-tissue barrier to a fluorescent-labelled protein tracer introduced into the
circulation (Heinicke & Kiernan, 1978). It is likely that humoral antibodies would
have had access to the tips of regenerating axons within the transplanted skin. From
the presence of extravascular leukocytes in the dermis it was inferred that sensitized
lymphocytes could probably also have infiltrated the grafts. Similar observations of
a variety of transplanted non-neural tissues in mice (Heinicke, 1978, 1979) are in
agreement with these findings.

(6) Necessity of periaxonal vascular permeability

(a) Statement of hypothesis
This hypothesis (Heinicke & Kiernan, 1978;Kiernan, 1978), first advanced in order
to account for the regeneration of axons into intracerebrally transplanted skin, will
now be discussed in greater detail.
It is suggested that axons of any type can only regenerate when their growing tips
are bathed in extracellular fluid containing proteins derived from the circulating
blood. Such proteins (whose identities are unknown) may be imbibed by the regener-
ating axonal sprouts and transported to the neuronal perikarya, there to exert an
effect that causes stimulation of the re-growth of the axons. Pre-requisites for the
operation of this mechanism are ( a )the breakdown of blood-tissue barriers in regions
into which axons are growing and (b) the capability on the part of regenerating axons
to assimilate proteins derived from the extracellular fluid. These conditions are also
necessary for the autoimmune inhibition of regeneration as discussed above, but the
present theory does not invoke a function for any antibodies that may be produced
as a consequence of injuring any part of the nervous system.
 Axonal regeneration in mammals I79
(b) Reconciliation of the hypothesis with experimental observations
(i) Axonal regenneration and blood-tissue barriers. Protein molecules can permeate
into the endoneurium distal to a site of axonotmesis in a peripheral nerve for a time
greatly in excess of that required for the completion of axonal regeneration (Mellick &
Cavanagh, 1968; Olsson, 1972; Olsson & Kristensson, 1973; Motte & Allt, 1976).
Plasma-derived proteins would, therefore, be available to the tips of the growing nerve
fibres. In the injured C.N.S., on the other hand, the blood-brain barrier to proteins
is breached for only 2 or 3 weeks and then only in the immediate vicinity of the lesion
(Lee & Olszewski, 1959; Klatzo, 1967; Bakay, 1972; Persson, Hansson & Sourander,
1976). The reconstitution of the blood-brain barrier occurs at the time when the
regeneration of axons ceases (see p. 158). It is suggested that these axons stop growing
because they are no longer surrounded by an exudate derived from the blood. The
formation of inappropriate synapses described earlier may occur only after the axonal
elongation has been arrested. The exact sequence of events at the site of an injury to
the C.N.S. would be clarified by electron microscopy of tissue taken from animals
injected intravenously before death with a suitable tracer protein, at various intervals
after placement of lesions.
The blood-brain barrier is normally absent in the neurohypophysis (see p. I ~ o ) ,
so axons regenerating in the pituitary stalk would always have access to plasma pro-
teins. Thus, the hypothesis presently under discussion can account for the effective
regeneration of these fibres. The failure of ordinary central axons to grow into the
transplanted neurohypophysis is difficult to account for in terms of the present theory.
It is possibIe that a blood-tissue barrier develops after the neurohypophysis is placed
in the brain. Such a barrier is not present in intracerebrally transplanted skin
(Heinicke & Kiernan, 1978) and other tissues (Heinicke, 1979), into which central
axons do regenerate. Similar reasoning would account for the growth of mono-
aminergic and acetylcholinesterase-containingaxons into grafts of smooth muscle.
When the proximal stump of a transected motor nerve is implanted into the cerebral
hemisphere, axons probably do not grow amongst the neurones and neuroglial cells
(where the breach in the blood-brain barrier is soon healed) though some fibres
regenerate into regions occupied by Schwann cells that have migrated into the brain
(see p. 159). Probably, the capillary blood vessels associated with these proliferated
endoneurial elements remain permeable to large molecules for a much longer time
than do the corresponding vessels of the true parenchyma of the brain. Again, further
experimental evidence is needed.
(ii) Monoaminergic and sensory neurones. The regeneration of severed mono-
aminergic fibres within the substance of the C.N.S., as distinct from their growth
into smooth muscle grafts, may be due to effects of leakage of the neuro-
transmitter substances. The secretion of vasoactive substances, especially serotonin
(Westergaard, 1975) from the tips of monoaminergic axons might cause a localized
transudation of plasma proteins sufficient to maintain the regenerative growth of
these fibres. Blunt injury to the spinal cord (the trauma being insufficient to transect
all the axons) is followed by effusion of amines, notably noradrenaline, into the
I 80 J. A. KIERNAN
injured nervous tissue. The amines may come from the traumatized mono-
aminergic axons of the cord (see Osterholm, 1974). The vasoconstriction, oedema
and haemorrhagic necrosis resulting from this type of injury can be reduced in
severity by the preoperative administration of catecholamine-blocking drugs
(Hedeman & Sil, 1974). This therapy does not, however, promote recovery from
traumatic paraplegia (Gurden & Feringa, 1974), which is hardly surprising if the
presence of oedema fluid is necessary for the occurrence of axonal regeneration. The
role of noradrenaline in causing oedema and necrosis in the injured spinal cord is,
however, still controversial (see Rawe et al., 1977a, 6, for some evidence against the
hypothesis, and discussion).
A potent vasoactive peptide, substance P, is present in some of the first-order
sensory neurones of the spinal ganglia and may function as a neurotransmitter in
the dorsal horn of the spinal grey matter (see Iverson, Nicoll & Vale, 1978). A high
concentration of substance P has been demonstrated immunocytochemically in the
substantia gelatinosa (Pelletier, Leclerc & Dupont, 1977). Local vascular exudation
caused by substance P could be responsible for the limited amount of axonal regener-
ation observed to occur from injured dorsal roots into the spinal cord.
(iii) Foetal mammals and submammalian vertebrates. Regeneration of axons severed
in the central nervous systems of foetal rodents may be due to immaturity of the
blood-brain barrier to proteins at early stages of development (see Lee, 1 9 7 1 ;
Klatzo, 1977). The barrier is functional in neonatal rodents, but it has not been
convincingly shown that axonal regeneration in the C.N.S. at this stage of develop-
ment is any more effective than it is later in life (see p. 167). In fishes and amphibia,
the blood-brain barrier is similar to that of mammals (see Bakay, 1956). The conten-
tion that axonal regeneration occurs only when the barrier is defective would
be strongly supported if it were shown that the blood vessels of the piscine and am-
phibian nervous systems remained permeable to proteins for long periods following
injury.
(iv) Pharmacological efects. The beneficial actions of certain drugs and local
therapeutic measures on axonal regeneration may eventually be explained in the
terms of the present hypothesis when more data become available concerning the
effects of the treatments on vascular permeability in the injured C.N.S. I t may be
significant that the administration of bacterial endotoxins can result in breakdown of
the blood-brain barrier in the absence of any physical trauma (Bakay, 1956; Clawson,
Hartmann & Vernier, 1966; Lee, 1971).Thyroid hormones probably accelerate the
regeneration of peripheral axons by stimulating protein synthesis in axotomized
neurones (see p. 164). Their slight effects in the injured C.N.S. may be similarly
mediated, though the possibility of an independent action on the vasculature of the
C.N.S. might be worth investigating.
The promotion of axonal regeneration in the spinal cord by mucolytic and pro-
teolytic enzymes may also be explicable in the terms of this hypothesis. The normal
C.N.S. contains acid mucopolysaccharides (Young & Abood, 1960) which are
probably located in the extracellular space (Kiernan, 1965; Bondareff, 1967; Tani &
Ametani, 1971)and in the cytoplasm of astrocytes (Lewis & Lampert, 1977). Matinian
 Axonal regeneration in mammals 181
& Andreasian ( I 976) showed that the histochemically detected increase in concen-
trationof such substances in regionsof the cord adjacent to a transection was prevented
by treatment with hyaluronidase. It is possible that depolymerization of extracellular
acid mucopolysaccharides may accentuate the penetration of plasma-derived proteins
into the nervous tissue. Proteolytic enzymes may act similarly, but it is also pertinent
that trypsin and other proteinases are potent kininogenases and can cause the
liberation of vasoactive peptides (kinins) from protein precursors (kininogens) present
in the blood plasma and extracellular fluid (Rocha E Silva, Reis & Ferreira, 1967;
Habermann, I 970 ; Greenbaum, I 971). Kinins produce vasodilatation and increased
permeability of capillaries and venules to proteins and cells. They are important as
chemical mediators of inflammation (Rocha E Silva, 1970; Wilhelm, 1973) and may
also be involved in lesser degrees of vasodilatation and oedema following quite trivial
injuries (Chapman, Ramos, Goodell & Wolff, 1961; Kiernan, 1976, 1977). If the
kinin-forming action of trypsin manifests itself in the injured C.N.S., the enzyme
would cause exudation of plasma proteins around the tips of the regenerating nerve
fibres. The effects of mucolytic and proteolytic enzymes upon the blood-brain barrier
are in need of investigation.
This hypothesis would be supported if it were shown that greatly enhanced
regeneration of severed axons followed experimental abolition of the blood-brain
barrier to proteins for a prolonged period of time following injury to the C.N.S.
Unfortunately, most of the procedures known to increase vascular permeability in
the brain involve either the placement of destructive lesions or the systemic adminis-
tration of toxic substances (see Lee, 1971; Steinwall, 1977). The therapeutic value of
such treatments might therefore be doubtful.

(7) Growth factors and axonal regeneration

(i) Statement of hypothesis. Another hypothesis purporting to explain the failure of
axonal regeneration in the C.N.S. was advanced, but not discussed in detail, by
Berry (1979). It was proposed that: " . ..all central axons would regenerate if they
had available to them their specific growth promoting factors (Kiernan, 1978).
Such factors may be proteins or polypeptides which mobilise neuronal protein
synthesis through cyclic AMP as a secondary messenger, but fail to do so in the
adult either because autoantibodies neutralise their potency or because they normally
disappear from the C.N.S. during ontogeny. " Lund (1978) has made similar
speculations.
The only growth factor yet identified is nerve growth factor (N.G.F.). This protein
is essential for the normal development of monoaminergic neurones, especially those
in sympathetic ganglia. The regeneration of the severed axons of monoaminergic
neurones in the C.N.S. into grafts of smooth muscle is enhanced by N.G.F. and
prevented by administration of antiserum to N.G.F. (Bjerre et al., 1973, 1974).
Berry (1979) suggests that hitherto undiscovered growth factors, comparable to
N.G.F., exist for all types of neurones. He suggests that the growth factors are
absorbed by the growing tips of axons and retrogradely transported to the perikarya,
where their actions on protein synthesis are effected. N.G.F. has been shown to enter
I 82 J. A. KIERNAN
noradrenergic sympathetic neurones in this manner (Hendry, Stockel, Thoenen &
Iverson, 1974; Stockel, Paravicini & Thoenen, 1974).
(ii) DiscussiOn. The sites of origin of the postulated growth-promoting factors are
not known. If these substances were present in the plasma of the blood, the ‘growth
factor hypothesis’ would become identical with the ‘vascular permeability hypothesis ’
discussed above. Alternatively, specific growth factors could be secreted by neuroglia
and neurones of the embryonic and foetal, but not of the adult C.N.S. The observed
formation of synaptic connexions with fragments of foetal superior colliculi implanted
into the visual systems of newborn rats (Lund & Hauschka, 1976) may be consistent
with such an origin.
By assuming the existence or non-existence of specific growth factors it is possible
(see Table 2) to account for many of the instances of success and failure of axonal
regeneration cited in Section I1 of this review. The arguments will not be pursued
here, however, since they are entirely speculative. With a little imagination the theory
could probably also be extended to account for the phenomena of synaptic plasticity
and of inappropriate synaptogenesis in the transected spinal cord. This hypothesis
cannot, however, account in any obvious way for the beneficial effects of enzymes and
hormones on axonal regeneration or for the outgrowth of axons from fragments of the
foetal but not the adult C.N.S. in vitro.
Unfortunately no evidence is yet available for the existence of growth factors other
than N.G.F. Should substances with the appropriate biological properties be isolated,
it will be necessary to determine their cellular localizations in mature and immature
animals in order to obtain the facts required to support or refute the hypothesis of
Berry (1979). A search for growth factors in fishes and urodele amphibia, in which
central axons regenerate efficiently, might be more rewarding than comparable
examination of extracts of adult mammalian tissues.

IV. CONCLUSIONS
Eight theories concerned with axonal regeneration in the mammalian nervous
system have been discussed. Some of these provide reasonable explanations for
greater numbers of the experimental observations reviewed in Section I1 of this
article than do others. Some of the pertinent data and their compatibilities (and
otherwise) with the different hypotheses are summarized in Table 2.
The ‘total scores’ at the foot of Table 2 cannot be taken very seriously, since equal
weight is given to each observation and no account is taken of all the zeros indicative
of experiments not yet performed. Nevertheless, four hypotheses stand out as being
quite inadequate. The phenomena of axonal regeneration in the P.N.S. and C.N.S.
cannot be explained solely on the basis of an absence of Schwann cells in the latter or
in terms only of the formation of impenetrable scars or of inappropriate synapses.
Neither can it be maintained that central axons are intrinsically incapable of re-
generating in adult mammals. The hypotheses represented in the third (2b)and in the
last three ( 5 , 6, 7) columns of Table z can all account for much of the available in-
formation, though further investigations will be necessary if the justifications proposed
for some experimental results are to be upheld.
 Axonal regeneration in mammals

Table 2 . Reconciliation of hypotheses with experimental observations

Hypothesis

Experimental observation 1 2 a 2 b 3 4 5 6 7

Successful regeneration of axom in the P.N.S. + + + + + - + o

Failure of peripheral axons to regenerate
into: Brain - + + + - + + +
Neurohypophysis - + + - - - 0 0
Abortive regeneration of most axons severed
in C.N.S.
+ + + + + + + +
Successful regeneration of neurosecretory + - + - o + + +
axons severed in the neurohypophysis
Probable failure of regeneration of neuro- - + o + + + + o
secretory axons severed in the hypothalamus
Failure of neurohypophysial neurosecretory
axons to grow into transplanted peripheral
- - + - - o o +

nerve
Failure of central axons to regenerate + + + o o + o o
into transplanted neurohypophysis
Regeneration of central monoaminergic axons + - 0 - o + + +
Regeneration of primary olfactory neurones + - 0 0 0 0 0 0
Regeneration of central axons into
transplanted skin,muscle, glands, etc.
- + + - - - + +
Effects of thyroid hormones on axonal o - - - 0 0 0 0
regeneration (C.N.S. and P.N.S.)
Effects of Piromen, corticosteroidsand 0 - o + o + o o
corticotrophin
Effects of local treatments 0 - o + o o o o
Effects of combined local and systemic o o o + o o + o
proteolytic and mucolytic enzymes
Axonal regeneration in foetal C.N.S.
(extent questionable)
+ - o + o + o +

Successful axonal regeneration in C.N.S. + - 0 - + o o o

of lower vertebrates
Growth of neurites in culture - - o + + + + o
Total score for each hypothesis +2 -3 +7 +Z +I +6 + 9 +7
+
(counting as + I , - as - I and o as zero)
Hypotheses (numbering as in Section I11 of this article): (I) Intrinsic inability of central neuron- to
regenerate their severed axons. (2a) Schwann cells and contact-guidance. (2b) Compatibility between
regenerating axons and the surrounding cells. (3) Physical barriers to regeneration. (4) Inappropriate
formation of synapses. ( 5 ) Autoimmune inhibition of regeneration. (6) Necessity of periaxonal vaacula
permeability. (7) Necessity of specific ‘growth factors’. Symbols: (+) The observation is reasonably
explained by the hypothesis, though further investigation may be desirable. (-) The observation and
the hypothesis cannot be reconciled. ( 0 ) No judgement can be made without further investigation.

The theory of autoimmune inhibition of axonal regeneration in the C.N.S. is not

acceptable in its original form, for reasons already discussed. However, studies of
the effects of auto-antibodies on injured neurones may lead to the development of a
modified version of this hypothesis.
The proposed necessary presence of exuded plasma proteins around the tips of
regenerating axons explains more observations than any of the other hypotheses
184 J. A. KIERNAN
under scrutiny. There is no experimental evidence directly opposed to this concept,
but a glance at Table 2 shows that several phenomena (9 of the 18 considered) are
not understood sufficiently to attempt their reconciliation with the hypothesis. The
most recent theory, that invoking specific neuronal growth factors, is also in need of
experimental support. Investigations specificallydesigned to test individual hypotheses
are required: it can be seen in Section I11 of this article that very few such studies
have been carried out.
The proposed explanations for the diverse regenerative responses of axons have
been considered individually, but there is no reason to believe that the various
hypotheses are mutually exclusive. They may all be partially correct. For example, the
presence of a dense scar can certainly obstruct the purposeful growth’ of axons, even
though it does not always do so. It has been observed that axons form synapses
inappropriately when they fail to grow across sites of spinal transection and the
establishment of such abnormal connexions may well result in the cessation of the
regenerative process. Allowing for possible inherent differences in the capacities of
different neurones to re-grow their axons, it is possible to account\for several pheno-
mena associated with injury to the C.N.S. in terms of inappropriate synaptogenesis.
However, many observations, especially of situations in which central axons do
regenerate, seem to require different explanations. Thus, the regeneration of central
axons into some non-neural tissues transplanted into the brain and the lack of com-
parable regeneration amongst neuroglial cells appear to involve factors extrinsic to
the growing nerve fibres. Although there is little support for the idea that neurones
of the mammalian C.N.S. are too ‘advanced’ to be able to regenerate their axons,
phylogenetic considerations should not be ignored. The process of evolution must
have resulted in changes in the functions of the neuroglia and other supporting ele-
ments of the nervous system.
The favourable and unfavourable interactions between an elongating axon and its
surroundings almost certainly depend upon the properties of nearby non-neuronal
cells as well as upon the presence of plasma-derived proteins in the extracellular
fluid. Specific ‘growth factors’, if they exist, might arise from either source. Extra-
vasated cells, antibody-bearing and otherwise, could also affect the regenerative
process. The ability of the tips of growing axons to imbibe macromolecules and
transport them to the neuronal perikarya provides the most plausible mechanism for
the mediation of extra-axonal effects of all kinds. The influence of the periaxonal
microenvironment is probably the most important and least adequately understood
aspect of the subject of axonal regeneration in the central and peripheral nervous
systems.
V. SUMMARY
I. Axons severed within the peripheral nervous systems (P.N.S.) of all vertebrate
animals regenerate vigorously and re-innervate motor and sensory end-organs.
2. Axons cut within the central nervous system (C.N.S.) fail to regenerate effectively
in mammals, except in certain circumstances. The great majority of severed centraJ
axons begin to grow into the wound but the regenerative activity ceases after about
 Axonal regeneration in mammals ‘85
2 weeks. The regenerated axons end as inappropriate synaptic contacts, proximal to
the lesion.
3. The axons of monoaminergic neurones in many parts of the C.N.S. and of
neurosecretory fibres in the neurohypophysis regenerate to a much greater extent
than do central axons of other types in mammals. No axons grow in either direction
across an artificially produced junction between peripheral nerve and neurohypo-
physial tissue. Primary olfactory neurones are also unusual, in that their axons can
regenerate through the olfactory nerves (P.N.S.) and into the olfactory bulb
(C.N.S.).
4. Some non-neural tissues, notably skin and muscle, are infiltrated by axons of
central origin following autotransplantation into the brain, but axons from peripheral
nerves cannot grow into the C.N.S. except in the case of the olfactory nerves and, to
a limited extent, at the junction of dorsal roots with the spinal cord and into fragments
of optic nerve transplanted into a peripheral nerve.
5. Axonal regeneration across wounds in the brain and spinal cord is enhanced to
a limited extent by systemic treatment with thyroid hormones, pyrogens, cortico-
trophin, or corticosteroids and by combined local and systemic administration of
mucolytic and proteolytic enzymes. Thyroid hormones probably act by stimulating
neuronal protein synthesis. The other agents reduce the formation of obstructing
collagenous scars but this may not be their sole action.
6 . In the C.N.S. of foetal rodents, severed axons probably regenerate in greater
numbers than in adults, but it is not always possible to distinguish between true
axonal regeneration and the continuation of the developmental process.
7. Effective axonal regeneration in the C.N.S., with restoration of function, occurs
in adult fishes and urodeles, and in larval anurans, but not in adult anurans, reptiles
or birds.
8. In tissue culture, there is extensive outgrowth of neuronal cytoplasmic processes
from explants of foetal central nervous tissue. Outgrowth has not been obtained from
explanted fragments of the adult C.N.S.
9. Eight hypotheses purporting to account for the failure of axonal regeneration in
the adult mammalian C.N.S. have been discussed and attempts have been made to
reconcile them with the experimental facts.
10.Five of the theories are rejected as being incompatible with many of the
experimental data. Thus, the failure of axonal regeneration in the mammalian C.N.S.
cannot be due solely to intrinsic inability of central neurones to regenerate, to the
absence of Schwann cells in the C.N.S., to the formation of impenetrable scars, to
the inappropriate development of new synapses or to the operation of an autoimmune
process. All of these factors may, however, contribute partially to unsuccessful
regeneration in some circumstances.
I I. The three remaining hypotheses can account for most of the observations, but
none have yet been specifically tested by experiment. Successful regeneration may be
dependent upon the existence of (u ) specific compatibility of unknown nature between
the axons and the non-neuronal cells amongst which they are required to regenerate,
(b) the presence of plasma proteins in the extracellular space around the growing tips
12 B R E I2
I 86 J. A. KIERNAN
of the axons, or (c) the availability of specific ‘growth factors’, normally present during
development but either absent or inactive in the adult C.N.S.
12.The hypotheses that accord most closely with the available facts are not mutually
incompatible. For all three proposed mechanisms, the retrograde axoplasmic trans-
port of proteins absorbed by endocytosis at the growing tip of the axon may be
essential for the occurrence of regeneration.

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