Download axon reaction, which may be displayed in

Four
The Human Nefvous Syslemt An Anqlomical ViewoinL
Sixlh Edition, Mvray L. Barr and John A. Kieman. J.B,
Lippincou Company, Philadelphia, O 1993.
e
a
I
cJ
Most of the cytoplasm of a neuron is removedwhen the axon is transected.
The segment that has been isolated from the cell body degenerates together with its myelin sheath, and the fragments are phagocytosed.
The neuronal cell body typically reacts to axotomy with greatly increased
protein synthesis, accompanied by structural changes known as the axon
reaction, or chromatolysis.
Axonal regeneration occurs when an axon is transected within the peripheral nervous system, but functional recovery is commonly imperfect because not all axons reach the correct destinations.
In mammals, axons transected within the central nlrvous system fail to
regenerate effectively. Synaptic rearrangements, however, can occur in
partly denervated regions of gray matter, and some recovery of function
occurs as a result of recruitment of alternative neuronal circuitry.
The distribution of fragments of degenerating axons can provide evidence
for the former existence of neuronal connections in the injured or diseased
brain or spinal cord. Investigation of neuronal activities, such as axonal
transport and glucose or oxygen metabolism, is now more widely used in
the study of connectMty and function in the central nervous system.
Neurons may be injured by physical trauma or
by involvement in pathological processes,
such as infarction caused by vascular occlu-
sion. Small interneurons are likely to suffer
total destruction, whereas injury to large neurons mav result either in destruction of the cell
,2
body or in transection of the axon, with preservation of the cell body, The best known change
proximal to the site of axonal transection is the
axon reaction, which may be displayed in
the cell body. When the cell body of a neuron is
destroyed, the axon is isolated from the syn-
Chapter 4: Response of Nerve Cells to Injury; Nerve Fiber Regeneration; Neuroanatomical
lontical Viewoint,
r A, Kiernan, J.B,
)
Methods 53
1991.
thetic machinery of the cell and soon breaks up
Nissl method), first shows signs of reaction24
to 48 hours after interruption of the axon. The
into fragments, which are eventually pha-
gocytosed, Similar changes occur distal to the
are changed
site ofan axonal injury. The degeneration ofan
axon that has been detached from the remain-
this change,
urs first be-
der of the cell is called wallerian degeneration, The process affects not only the axon, but
tween the
also its myelin sheath, even though the latter is
(Fig.
uarly
not part of the injured neuron.
spre
a-l).
j.TS
eltric
position away from the axon hillock, and the
whole cell body swells. This aspect of the reac_
Changes in the cell body after axonal transectionvary according to the type of neuron. Cells
in some locations undergo progressive degeneration and, ultimately, disappear. This happens to most neurons,when the injury occurs
before or soon after birth. Converbely, the
proximal portions of some adult neurons are
not significantly altered by cutting the axon.
The cytological details of the classical axon
reaction are best seen in large neurons, such as
those supplying skeletal muscle, which contain coarse clumps of Nissl material, The fol-
lowing account includes the more typical aspects of the response to cutting the axon of a
motor neuron,
The nerve fiber between the cell body and
the lesion is not altered appreciably. The cell
body, in sections stained by a cationic dye (the
'vith
preserrwn change
ection iri the
Figure 4-1, Motor neuron, show-
lisplayed in
with cresyl violet, x 800). Compare
Ia neuronis
rm the syn-
ing changes in the cell body 6 days
after transection of its axon. (Stained
this with the normal neuron
Figure 2-2.
in
tion reaches a maximum t0 to 20 days afrer
axonal transection, and the closer the injury is
to the cell body, the severer the swelling. Electron microscopy shows disorder of the granu_
lar endoplasmic reticulum and an increase in
.
the number of polyribosomes in the cytoplasmic matrix.
There are signs of recovery from the early
effects of trauma to the axon even while these
changes are occurring, The nucleolus enlarges,
and dense basophilic caps are often seen on
the cytoplasmic side of the nuclearmembrane.
Both the nucleolar enlargement and the.nu_
clear caps are evidence of accelerated ribo-
nucleic acid and protein synthesis, which
would favor regeneration of the axon when
conditions make regeneration possible, Recovery commonly takes several months, and the
cell body is eventually smaller than normal if
the axon does not regenerate,
54
Introduction and Neurohistology
In
cells confined to the central nervous
walls of endoneurial blood vessels. The re-
system, the axon reaction is conspicuous only
mains of the axon and its myelin sheath (or the
axon onJ.y in the case of unmyelinated fibers)
are phagocytosed. Thus the distal stump of a
degenerated nerve is filled with tubular formations, known as the bands of von Btingner,
in
some large neurons, Changes in smaller
neurons that have inconspicuous Nissl substance are not detectable by light microscopy,
and large cells may exhibit no axon reaction
when collateral axonal branches that arise
close to the cell body are spared, Some central
neurons normally have eccentric nuclei, thus
giving a false impression of an axon reaction.r
The axon reaction has been exploited in the
past as a means of identifying the cells of origin
of fibers in nerves and the sources of some
central tracts. As a research method, it has
been supplanted by more informative procedures, which are discussed later.
llerian Degeneration
in ripheral Nerves
that contain phagocytes and Schwann cells.
in Peripheral Nerves
If a neuron is envisaged as a spherical cell body
15 pm in diameter, with an axon I0 cm long
and 2 pm in diameter, a calculation shows that
99.4% of the protoplasm is in the axon, If the
axon of this hypothetical neuron were severed
halfway along its length, the cell would lose
49.7% of its volume. This lost part of the neuron can be regrown when the injury occurs
within the territory of the peripheral nervous
The nucleus is essential for the synthesis of
cytoplasmic proteins, which are transported
distally in the axoplasm to replace proteins
that have been degraded as part of the metabolic activity of the cell. The axon, therefore,
does not survive for long when separated from
the cell body. Simultaneously throughout its
system, a reparative process known as axonal
Iength, the part of the axon distal to the lesion
becomes slightly swollen and irregular within
the Ist day and breaks up into fragments by
the 3rd to 5th day. Muscle contraction on elec-
tion of its axons requires apposition of the cut
ends by placement of sutures through the epineurium. The individual fascicles of the nerve
regeneration. It is important to distinguish
between this use of the word "regeneration"
and its more usual connotation, which is the
replacement of lost cells by mitosis and reorga-
nization of tissue.
If a nerve has been severed, the regenera-
trical stirnulation of a degenerating motor
nerve ceases 2 to 3 days after the nerve is
interrupted. The degeneration includes the
should be realigr.red as accurately as possible.
A crushing injury (or freezing a short length of
nerve in a laboratory animal) transects the
axons but leaves intact the connective tissues
neural components of the sensory and motor
of the nerve, including the perineurial
endings,
sheaths of the fascicles. No surgical interven-
The myelin sheath is converted into short
ellipsoidal segments during the first few days
after interruption of the fiber and gradually
undergoes complete disintegration, Meanwhile cells accumulate in the cylindrical space
tion is needed for this type of injury because
the cells and connective tissue of the endoneurium are there to guide growing axons to
their appropriate destinations.
within the basal lamina of the column of
Schwann cells associated with each nerve fiber. Most of these cells are derived from mono-
nuclear leukocytes that emigrate through the
' Such cells include those of the nucleus thoracicus of the
spinal cord (see Ch. 5) and the accessory cuneate nucleus
of the medulla (see Chs. 7 and I0),
The following description applies to nerves
that have been cleanly cut through and repaired. During the first few days, phagocytes
and fibroblasts fill the interval between the
apposed nerve ends. Regenerating fibers, accompanied by migrating Schwann cells, invade the region by about the 4th day, with
chapter 4:
:ls. The rereath (orthe
rated libers)
stump of a
rularformal Btingner,
wann cells.
:al cell body
l0
L
cm long
shows that
axon. If the
'ere severed
would lose
of the neuJury occurs
ral nervous
1
as
axonal
distinguish
leneration"
vhich is the
and reorgae regenera,n of the cut
rgh the epirf the nerve
as possible,
lrt length of
'ansects the
ctive tissues
perineurial
al interven-
ury because
f the endong axons to
s to nerves
rgh and rephagocytes
retween the
g fibers, acm cells, inh day, with
Response of Nerve
celk to Injury; Nerve Fiber Regeneration; Neuroanatomical Methods
each axon dividing into numerous branches
having enlarged tips. Each tip, known as a
growth cone, is about the same size as a
neuronal cell body and has a convoluted and
constantly moving surface membrane at its
leading edge. The rate of axonal growth is slow
at first; 2 to 3 weeks may elapse before the
growth cones traverse the region of the lesion.
The fine branches may then find their way into
the bands of von Bringner in the distal segments. Several filaments enter each endoneurial tube, and the invasion of a particular
tube leading to a specific type of end organ
appears to be determined only by chance.
Many filaments miss altogether and grow into
epineurial connective tissue, This is the fate of
all regenerating axons if the severed ends are
too widely separatCd or if dense collagen or
extraneous material intervenes. Such fibers often form complicated whorls (spirals of perroncito), producing a swelling or neuroma
that may be a source of spontaneous pain. At
the other exfteme is the almost perfect regen-
eration of the nerve through the growth of
each fiber along its original endoneurial tube.
This type of regeneration may occur if the
nerve is crushed just enough to interrupt
axons without disruption of the endoneurial
connective tissue, Experimentally, this type of
"ideal" lesion can be achieved by local freezing.
After crossing the region of the lesion and
entering the bands of von Bringner, the axonal
filaments grow along the clefts between columns of Schwann cells and the surrounding
basal laminae, Usually only one branch of
each axon enters a single tube; other sprouts
are drawn back into the shaft of the growing
axon. The rate of growth within the nerve
distal to the lesion is 2 to 4 mm per daysomewhat faster than the slow component of
normal axonal transport. Regenerating fibers
eventually reach motor and sensory endings;
the proportion of endings that are correctly
reinnervated depends on conditions at the site
of the original iqjury. The amount of time that
elapses between nerve suture and the beginning of functional return may be estimated on
the basis of a regeneration rate of I , 5 mm daily,
This value takes into account the time recuired
for the fibers to traverse the lesion and for the
peripheral endings to be reinnervated,
In a human limb, the course of axonal
regeneration can be followed by testing fOr
Tinel's sign, When part of a nerve trunk con_
taining regenerating axons is tapped with a
small hammer, the patient repoits a tingling or
electric sensation in the area of skin no.rnullv
supplied by the nerve,
Meanwhile changes occur along the course of
the regenerating fibers. Each axon becomes
surrounded by the cytoplasm of the Schwann
cells. For axons that are to be myelinated, myelin sheaths are laid down by Schwann ceils
(see Ch. 3 for mechanism), Myelination be_
gins near the lesion and proceeds in a prox_
imodistal direction. Although the myelin
sheath is formed by rhe Schwann cells, iti development is determined by the type of axon.
Experimental studies indicate that all the neu_
roglial cells in a regenerating nerve have the
potential to produce or not to produce niyelin,
irrespective of the nature of the axons with
which they have previously been associated.
Even years after injury and repair, the diameter, internodal length, and conduction ve_
locity of a regenerated nerve fiber are rarelv
more than 80% of the corresponding values for
the original fiber. The motor unit for a regenerated fiber is larger than that of the preexisting
motor unit; that is, the axon supplies more
muscle flbers than it formerly did. These factors contribute to less precise control of reinnervated muscles and to the fact that sensorv
function also is inferior to that mediated by the
uninjured nerve,
onal Degeneration and
Nervous System
The simplest lesion
to
visualize, although
rather rare in clinical practice, is a clean incised
wound of the brain or spinal cord, The space
made by the knife blade fills with blood and
later with collagenous connective
tissue,
which is continuous with the pia mater. The
,
jj
56
Introduction and Neurohistology
astrocytes in the nervous tissue on each side of
the collagenous scar acquire longer and more
numerous cytoplasmic processes, which form
a tangled mass. The number of astrocytes in
the region does not increase appreciably, but
there is a large increase in the total cell popula-
tion, caused mainly by the emigration of
monocytes from blood vessels to form phagocytic cells known as reactive microglia. The
resting microgiia that had been present in the
central nervous tissue also may become phagocytes, but the great majority of such cells
come from the blood. Reactive microglia also
appear in parts of the central nervous system
remote from the lesion but occupied by axons
that are degenerating in consequence ofhaving been severed from their cell bodies. WalIerian degeneration is different from the process described for peripheral nerves because the
degradation and phagocytosis of debris are
carried out much more slowly in the central
nervous system. Degenerating fragments of
myelinated axons are frequently recognizable
several months after the original injury, and
the phagocytic cells that contain the debris
.'persist in situ for many years.
A
,
fundamental difference between the
consequences of injuries to the peripheral and
central nervous systems concerns the regeneration of axons. The proximal stumps of axons
transected within the brain or spinal cord
begin to regenerate, sending sprouts into the
region of the lesion, but this growth ceases
after about 2 weeks. Abortive regeneration of
this type occurs in the central nervous systems
of mammals, birds, and reptiles. Axonal regeneration in fishes and amphibia occurs efficiently in the central nervous system, with
remarkably accurate restoration of synaptic
connections.
The reasons for the failure of axonal regen-
eration in the central nervous systems of
higher animals are unknown. Current hypotheses are concerned with axonal growthpromoting and growth-inhibiting substances,
perhaps derived from the blood or from the
neuroglia, which are absent from or unable to
act in the adult mammalian central nervous
system. Earlier hypotheses, such as the lack of
Schwann cells in the brain and spinal cord and
the obstruction of growing axons by scar tissue
or cyst formation, although perhaps some-
what valid, fail to explain all the available experimental data.
There are a few circumstances in which
axons do regenerate successfully within the
mammalian brain. For example, the neurosecretory axons of the pituitary stalk (see Ch.
I I ) and some monoamine-containing fibers in
the brain stem regenerate effectively, The failure of most axons to regenerate means that
permanent disability follows destruction of
any tract that cannot be bypassed by redistribution of function to alternative pathways.
Transplantation of Neroous Tissue
With some types of injury, notably gunshot
wounds, substantial lengths of peripheral nerve
are lost. The deficit can be repaired by inserting
a graft taken from a thin cutaneous nerve that is
functionally less important than the one to be
repaired. Several strands
cies
differ
]t
Pens
menl
The
1
ters
r
vival
manl
Peop
butu
the rr
brain
peuti
neur(
relati
ient t
be th
unlik
centi
host
synal
regio
recei
NOTIT
of thin nerye are
placed side by side, in the manner of a cable, for
grafting into a large nerve. The process of axonal regeneration in a nerve graft is identical to
that in a transected and sutured nerve, but the
growing axons have to negotiate two sites of
anastomosis. The functional recovery is, therefore, far from perfect. A nerve graft must Lre an
autograft (derived from the same individual) or
an isograft (from an identical twin or an animal
that belongs to the same inbred strain), or itwill
be rejected by the immune system.
The neurons in pieces of adult mammalian
brain or spinal cord do not survive removal and
transplantation. Axons will grow, however, into
and out of tiny fragments of embryonic or fetal
central nervous tissue transplanted into certain
parts of the adult brain, Central axons can also
grow from the brain, spinal cord, or optic nerve
into transplanted pieces of peripheral nerve and
even into some nonneural tissues. Such experiments are contributing importantly to knowledge of groMh-promoting factors that are lost
with the maturation of the central nervous system. Tissues placed in the brain are partly protected from the host's immune system, so for
short-term experiments, it is possible to use
grafts from other individuals of the same spe-
Plas
Con
Alrh(
nerv(
erabl
traur
gions
For
e
cereb
OI SE]
loss
r
week
tion
not tr
<
by or
hemi
clinic
even
spina
F
OVCI,
the n
maln
Chapter 4: Response of Nerve Cells to Injury; Nerve Fiber Regeneration;
Neuroanat\micat
cord and
lar trssue
rs someIable ex-
cies (allografts or homografts) or even from
rografts).
rtly com-
d
e;<peri-
animals.
n which
thin
the
I neulo(see Ch.
;fibers in
The faii-
ans that
ction of
)y redis-
The grafts probably deliver both neurotransmitters and trophic substances that promote survival of postsynaptic neurons. In the late 19gOs,
such grafts in
(see Ch. 12),
ing benefits to
to the human
brain or spinal cord is unlikely to acquire therapeutic significance because (a) the numbers of
rthways.
gunshot
be the normal locations of their cell bodies are
unlikely to generate axbns that will grow several
the
ost-
ral nerve
din
ssue
inserting
,ne
receive afferent synapses appropriate
normal locations of their cell bodies.
cable, for
;s of axo-
Plasticity of Neural
ve that is
to be
erye are
:ntical to
:, but the
r sites of
is, therelst be an
ridual)or
n animal
l,
oritwill
mmalian
roval and
ever, into
ic or fetal
:o certain
can also
rtic nerve
rerve and
:h experio knowlrt are lost
r'ous sys-
artly prom, so for
e to use
rme spe-
not
to
the
Connections
Although axonal regeneration in the central
nervous system occurs only negligibly, considerable functional recovery commonly follows
traumatic or pathological damage in many regions, especially when the lesion is not large.
For example, destruction of a small area of
cerebral cortex that had a well-defined motor
or sensory function is followed by paralysis or
loss of sensation, with recovery after several
weeks. Similar recovery occurs after transection oftracts offibers, provided the lesions are
not too large. Recovery from paralysis caused
by occlusion of blood vessels in the cerebral
hemispheres (stroke) is common_ly seen in
clinical practice, and functional recovery may
even follow partial transverse lesions of the
spinal cord.
Functional recovery involves the taking
over of the functions of the damaged region of
the nervous system by other regions that re -
main intact. The reorganization of connections
Methods
within the brain is known as plasticity. This
may be an extension of a normally present
adaptability used in the learning of-oiten re-
peated tasks.
Structural changes accompany the func_
tional plasticity that follows injury to rhe ner_
vous system. Thus, when a group of neurons is
deprived of part of its afferent input, the surviving preterminal axons, which mav come
from quite different places, commonly grow
new branches that then form synapses at the
sites denervated by the original lesion. This
event, known as axonal sprouting, may oc_
cur over short distances within a small group
of neurons or over greater distances, as when
the axons of intact dorsal root ganglion cells
extend their axons for three or four segments
up and down the spinal cord after transection
of neighboring dorsal roots. Axonal sprouting
. also occurs in the periphery; the anestheuc
area of skin resulting from a peripheral nerve
injury becomes smaller over several weefs,
even ifthe severed nerve does not regenerate.
The change is considered to be caused by
sprouting of the axons of other cutaneous
nerves within the skin. Comparable sprouting
in partially denervated skeletal mus_
with consequent enlargement of the motor units. Axonal sprouting involves intact
axons and should not be confused with the
occurs
cles,
regeneration oftransected axons. It is probable
that axonal sprouting accounts for functional
plasticity and recovery after lesions in the central nervous system, but a causal relation has
not yet been conclusively proved.
Met\tds for Inuestigating Neural
Pathwags and Funciions
In histological material from normal animals. it
is seldom possible to follow a tract of axons from
its cell bodies of origin to the distant site in
which it terminates. The small diameters and
curved trajectories of axons, together with the
fact that different pathways commonly occupy
the same territory make the direct tiacinq'of
connections impossible. lt is, therefor", n"i""sary to use e><perimental methods to determine
the connections of the many groups of neurons
j7
58
lntroduction and Neurohistology
in the brain and spinal cord. The results of inves-
the time of survival and the conditions of fixation
tigations of neural connectivity in laboratory animals, especially the cat and monkey, may be
applicable to the human brain. This transfer of
data from animals to humans is usually justifiable when there are no major differences between the connections found in taxonomicallv
diverse groups of animals; a pathway present in
rats, dogs, and monkeys is likely to occur also in
of the tissue are criticar.
humans. When variation among species is
found, it is hoped that neuroanatomical information gained from primates, such as monkeys, will be helpful with respect to the human
brain. Sometimes injury and disease in the human nervous system can cause degeneration of
particular tracts of axons, Postmortem e><amination of the degenerated fibers provides information, albeit of a limited accuracy, about normal
human neural connections.
Neuroanatomical Methods Based
on Degeneration
Until the introduction of methods based on axoplasmic transport, fiber tracts were traced by
staining fibers undergoing wallerian degeneration after the placement of a destructive lesion at
a selected site in the central nervous system of
an animal. Although now largely of historical
interest, such methods have contributed importantly to neuroanatomical knowledge.
The Marchitechnique, which is still used on
human postmortem material, depends on the
staining of particles of degenerating myelin with
osmium tetroxide in the presence of an oxidizing agent. The latter suppresses the staining of
normal myelin, so that degenerating fibers appear as lines of black dots on a lighter background. The course of a tract can be followed in
sections taken at appropriate intervals (Fig.
4-2). Silver methods for showing degenerating
unmyelinated axons and synaptic terminals
were rarely applicable to the human nervous
system but were much used for laboratorv
animals.
Degenerating axonal terminals also can be
recognized in electron micrographs. When the
general area of projection of a group of neurons
or of a tract is known from light microscopy, the
exact mode of termination of the fibers on the
dendrites, somata, or axons of the postsynaptic
cells may be determined. As with silver degeneration methods, electron microscopy usually
cannot be applied to human material because
Neuroanatomical Methods Based
on,Axoplasmic Transport
Research methods based on degenerating
zxons were replaced in the 1970s by much
more sensitive techniques that reveal both the
cells of origin and the sites of termination of
€xons. The results of the extensive use of
methods based on axoplasmic transport have
necessitated substantial revisions of earlier accounts of neuronal connections in the central
nervous system.
In the autoradiographic method, a smallvolume of a radioactivelv labeled amino acid solution, commonly [3Hlieucine, is injected into the
region that contains the cell bodies of the neurons being investigated. The amino acid is taken
up by the neurons and is incorporated into pro-
teins, which are transported distally along the
axons to the presynaptic boutons. The animal is
killed 24 to 48 hours later and the appropriate
parts of the neryous system are chemically fixed
to immobilize the labeled proteins. Sections are
cut, and autoradiographs are prepared in the
usual way. High concentrations of silver grains,
indicating the presence of tritium in the tissue,
are seen over the site of injection, over the terminal field of projection of the neurons, and often
over the axons between these two regions.
With this technique, it has been possible tci
trace connections previously undetectable by
the use of degeneration methods. It also has the
important advantage that the labeled amino
acid enters only the cell bodies and dendrites of
to be passing
through the site of injection do not take up the
tracer, thus avoiding the confusion that often
complicated the interpretation of areas of terminal degeneration.
Research methods using the axon reaction
and staining degenerating fibers have been
largely replaced by techniques that take advantage of the uptake and axonal transport of proteins and other substances. A histochemically
detectable protein or a suitable fluorescent dye
is injected into the region concerned. The foreign molecules are imbibed by presynaptic terminals in the region and transported retrogradelyto their neuronal perikarya. The process
takes 6 to 72 hours, according to the lengths of
the axons and the substance used as a tracer.
neurons. Axons that happen
Tl-
m(
PI(
MC
the
A'.
j-,
flu,
ua,
ext
ln
ev(
ar(
gir
clr-
tal
Th
ab
US
de
chapter 4:
Response of Nerve
celk to Injury; Nerve Fiber Regeneration; Neuroanalomicat Methods
rf fixation
;ed
enerating
by much
both the
nation of
: use of
port have
larlier acre central
smallvolacid solu-
d into the
the neuid is taken
I
Iinto proalong the
,animal is
rpropriate
cally fixed
ctions are
'ed in the
'er grains,
he tissue,
the termi-
and often
rgions.
ossible to
:ctable by
so has the
:d
amino
:ndrites of
passing
rke up the
that often
:
s of
termi-
n reaction
lave been
Lke
advan-
ort of pro:hemically
:scent dye
L The fornaptic terted retro're process
lengths of
rs a tracer.
The animal is then killed and the tissue is removed and appropriately fixed and sectioned. A
prolein tracer is localized by histochemical
means, thus revealing the neuronal cell bodies
that innervated the site of the injection (Fig.
4-3). A fluorescent tracer is observed directlv bv
fluorescence microscopy.
The first protein to be used srtensively as a
tracer in this way was the en4/me peroxida"e,
e><tracted from the root of the horseradish plant.
In recent years, the method has been made
even more sensitive by the use of lectins, which
are carbohydrate-binding proteins of plant origin. Lectins bind strongly to cell surfaces, including those of axonal terminals, and are then
taken up into the cytoplasm and transported,
The lectin is rendered histochemicallv detectable by its covalent conjugation with u,'r
usually horseradish peroxidase. Other"nrym",
tracers,
detectable by similar methods, include simple
polysaccharides (Fig. 4-4) and some bacterial
toxins.
Many neurons in the brain have axons thar
colors are commonly used for this purpose. lf
both tracers are present in a single ceil body,
that neuron has axonal branches that go to both
sites of injection.
hoteins and dyes also are taken up and
transported retrogradely by injured axons of
passage, so care must be taken not to cause
undue physical damage when injecting into an
area that contains nerve endings whose cells of
origin are to be identified. Uptake by injured
axons may be deliberately studied by applying
protein tracers at the sites of transection ol
tracts or to cut peripheral neryes.
jg
60
Introduction and Neurohistology
,[
G
tf
Y
Figure 4-3. Transverse section through the ventral part
of the medulla of a rat
in which horseradish peroxidase was injected into the cortex of one cerebellar
hemisphere 24 hours before the animal was killed, The section was treated for
histochemical detection of peroxidase actMty, revealed as a dark blue deposit
that appears black in these photomicrographs. (A) Labeled cell bodies in the
inferio
(x 30
Other
red, a
Wth the development of increased sensitivity in methods for the histochemical detection
of peroxidase, it has become possible to study
the anterograde as well as the retrograde transport of tracer proteins, The amount of protein
taken up by cell bodies and dendrites is less
than that absorbed by presynaptic terminals.
However, an appreciable amount does enter the
cell bodies and is transported orthogradely in
the rapid component of the axoplasmic trans-
port system, The protein is detected histochemically in the terminal and preterminal parts
of axons, which have an appearance quite different from that of labeled perikarya. The method
provides, for a smaller investment of time and
effort, results comparable to those obtained by
the autoradiographic method. Some lectins are
especially suitable for anterograde tracing and
provide remarkably clear delineation of the ter-
minal branches of exons.
chapter 4:
Response of Nerve cells to
pathwags
.Trarasgnaptic Tracing of
ln soine viral diseases, such as rabies, the infec-
tive agent spreads through the central nervous
system by being passed from one neuron to
d
histonal parts
te differ-
method
ime and
ained by
ctins are
:ing and
f the ter-
lnjury; Nerve Fiber Regeneration; Neuroanatomical Methods
with2
in the
given
labele
chemically detectable enryme, or the viral pro-
systep is made highly active; for example, its
visual system may be stimulated by lighl or its
auditory system by sound. The radioactive
sugar accumulates in all the neurons in the
active system and may be detected there autoradiographically. In the visual system, for example, actMty is detected in the retina, in certain
Metabolic M arking M ethods
The sugar 2-deory-o-glucose is an analogue of
ordinary o-glucose. It enters cells in the same
way as glucose but cannot be metabolized.
When cells are active, their glucose uptake increases. Therefore, if an active cell is supplied
structures in the brain that are active when a
particular system of pathways is in use. It may
thus be possible to determine which of a multitude of connections demonstrated bv neuro-
synapses. The virus can be modified to make
the cells that harbor it synthesize a histotein may be stained immunohistochemically.
6l
62
Intrcduction and Neurohistology
anatomical tracing methods are the most important in relation to function,
Certain en4/mes used in the metabolic activities of all cells can be demonstrated histochemically. Cytochrome oxidase is a notable
example, and in regions that contain functionally active neurons, the activity of this enzyme is higher than in adjacent quiescent areas,
Cytochrome oxidase histochemistry has been
used with great success in the demonstration of
columns of cells that respond to different visual
stimuli in the cortsr of the occipital lobe of the
brain (see Ch, 14),
'.
that destroy the cytoplasm. The result is a lesion
more selective than one produced by physical
methods. Cells that use monoamines as synaptic transmitters are selectively intoxicated by analogues ofthese substances. Thus neurons that
make use of dopamine or norepinephrine are
intoxicated by 6-hydroxydopamine, and sero-
tonin cells are similarly sensitive to 5,6-dihydroxytryptamine,
Some poisonous lectins (notably ricin-60
from the castor bean) and other compounds
(notably the antibiotic doxorubicin, which is
Regional Cerebral Blood FIow
and Metaboltsm
In humans, it is possible to monitor blood flow in
the cerebral corte>< by computing regionalvariations in the gamma radiation detected around
the head after injection of a suitable radioactive
tracer. Sudden increases in blood flow are associated with activity in the underlying cortex. ln
used to treat some types of cancer) are taken up
by axonal endings and by injured axons of paJsage and transported retrogradely to the neuro-
clinical neurology, the technique is used to identify abnormally high or low blood flow caused by
selective lesions to provide e<perimental models
of diseases in which certain populations of neu-
disease.
.
acid binds to glutamate receptors, the calcium
channels of the postsynaptic cells are opened
for unduly long times. Calcium ions that diffuse
into the neurons activate proteolytic enzymes
Similar information can be obtained by positron emission tomography (see Ch, 16), which
provides pictorial and quantitative information
about o),rygen utilization or glucose uptake at
sites deep within the brain.
nal cell bodies, where they inhibit nucleic acid
and protein synthesis, This strategy, known as
"suicide transport", is also useful for producing
rons degenerate spontaneously.
SUGGESTED READING
and observing the destination of nerve impulses
by recording the potentials evoked elsewhere.
The accurate measurement of the time elapsed
between stimulation and recording provides information that may help to determine the
number of neurons, or synaptic delays, that are
Berry M: Regeneration of axons in the central nervous system. In Navaratnam V, HarriSon Ri
(eds): hogress inAnatomy, vol 3, pp 213-23).
New York, Cambridge University Press, 1983
Cotman CW, Nieto-Sampedro M, Harris EW: Synapse replacement in the nervous system of adult
vertebrates. Physiol Rev 6I1684-784, l98l
Crutcher I(A: Sympathetic sprouting in the central
nervous system: A model for studies of axonal
growth in the mammalian brain. Brain Res Rev
called "physiological neuronography."
Several toxic substances are used in laboratory animals as adjuncts to the study of neuroanatomy. For example, nicotine was first used
a century ago by Langley to block synapses
and thus establish their locations in autonomic
L2:203-23), 1987
Fawcett JW, Keynes RJ: Peripheral nerve regenerati.on. Annu Rev Neurosci 13l.4)-60, I99O
Franklin RJM, Blakemore WF: The peripheral nervous system-central nervous system regeneration dichotomy: A role for glial cell transplantation. J Cell Sci 95:I85-190, 1990
Phgsiolog ical and Pharmacolog ical M ethods
Anatomical studies of neuronal pathways are
often supplemented by stimulating neurons
included in the pathway, This procedure is
ganglia.
Local injection of kainic acid or ibotenic acid
kills many types of neurons without causing
transection of passing fibers. These substances
are analogues of glutamic acid, which is an
ercitatory transmitter. When kainic or ibotenic
Hall SM: Regeneration in the peripheral nervous
system. Neuropathol Exp Neurobiol I5:513529, t989
Kiernan JA: Hypotheses concerned with axonal regeneration in the mammalian nervous system.
Biol Rev 54:155-197, 1979
r .-
!lp
:\ ll
Chapter 4: Respottse of Nerve Celk to Injury; Nerve Fiber Regeneration; Neuroanatomical
T)
Id
;e
)S
In
al
)at
'e
)f
,0
ls
is
p
t)-
d
ts
g
ls
.t-
ri.J
J,
)
l-
It
.l
rl
I-
t-
ltls
l.
Landau WM: Artificial intelligence: The brain transplant cure for parkinsonism. Neurology 40:
733-740, 1990
Lipton SA: Growth factors for neuronal survival and
plocess regeneration. Arch Neurol 46:124It248, 1989
Mclean JH, Shipley MT, Bernstein DI: Golgi-like
transneuronal retrograde labelling with CNS
injections of herpes simplex virus type l. Brain
Res
Bull 22:867=88I, 1989
Methods 63
Oorschot DE, Jones DG: Axonal regeneration in the
mammalian central nervous system. Adv Anat
Embryol Cell Biol
tt9 t-t2t,
t990
Purves D: Assessing some dynamic properties of the
living nervous system. euart J Exp physiol
74:1089-I 105, 1989
Schwab ME: Myelin-associated inhibitors of neurite growth. Exp Neurol lo9
2-j,
I99O
Sunderland S: Nerves and Nerve Injuries, 2nd ed.
Edinburgh, Churchill-Livingstone, I 978