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Effects of advancing age on the central response of rat facial neurons to axotomyLight microscope morphometry.

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THE ANATOMICAL RECORD 228211-219 (1990)
Effects of Advancing Age on the Central
Response of Rat Facial Neurons to Axotomy:
Light Microscope Morphometry
DEBORAH W. VAUGHAN
Department of Anatomy, Boston University School of Medicine, 80 East Concord Street,
Boston, M A 02118
ABSTRACT
Following axotomy, the regrowth of peripheral axons takes
longer in older individuals than in young ones. The present study compares central
responses of facial motor neurons to a crush injury of the facial nerve in 3-monthold and 15-month-old male rats sampled through 28 days post-crush (dpc). Neuronal somata, nuclei, and nucleoli were measured in 30 pm brain stem sections
within subdivisions of the facial nucleus that contain the cell bodies responsible for
the movement of the vibrissae. The temporal patterns of change in the size of the
three structures were interpreted with reference to the re-establishment of functional connections, i.e., the return of voluntary vibrissae activity, which is delayed
by 4 days in the older animals relative to the younger ones. There was no agerelated difference in the pattern of soma1 swelling and recovery, nor was there a n
age-related difference in the response of nuclei and nucleoli to axotomy through 4
dpc. Both nuclei and nucleoli increased in size in animals of both age groups, but
after 4 dpc in the older animals nuclear enlargement was prolonged and the nucleolar increases were less robust compared to the younger animals. The greatest
age difference appeared with the re-establishment of functional connections. In the
3-month-old animals, the resumption of whisker activity coincided with vigorous
transient increases in the sizes of nuclei and nucleoli; in the 15-month-old animals,
there was little nuclear response to functional recovery and a comparatively small
increase in nuclear sizes. Given that signals from the periphery initiate central
metabolic changes when the target is reached, this study suggests that the deteriorative effects of advancing may not simply reside in the neuron’s decreased
resiliency, but may reside in deficiencies in the signals derived from peripheral
supporting cells, the target muscle, or in the reception of those signals.
The regeneration of peripheral nerves after injury
takes longer in animals of advancing age than in young
adults (Drahota and Gutmann, 1961; Black and Lasek,
1979; Pestronk et al., 1980; Komiya, 1981). However,
in the central nervous system of the middle-aged adult
animal, there is little morphological evidence of advancing age, and the neurons perform their normal
functions-those of basic cell maintenance and neurotransmission-quite adequately (e.g., Ishihara and Araki, 1988). The metabolic challenge of axotomy may
expose a n age-related deterioration of motor neurons.
This report is part of a n analysis of the response of the
facial motor system to axon injury in young adult and
middle-aged rats and is concerned with the central
morphological response of the motor neurons.
Axotomy initiates a well-characterized morphological, metabolic, and synthetic reorganization in the cell
bodies of extrinsic neurons, referred to a s the axon reaction (e.g., Lieberman, 1971; Grafstein, 1975; Alberghina and Giuffrida Stella, 1987). Reversible axotomy, that in which the axon successfully regrows to the
target, involves three successive stages (Brattgard et
al., 1957; Engh et al., 1971): Initially, there is a brief 1ci 1990 WILEY-LISS,
INC
to 2-day period of latency as the cell body responds to
the injury and loss of effective contact with its target.
During the second stage of the axon reaction, multiple
fine axons grow from the site of injury and elongate to
the target where they re-establish functional connections. This stage is typically marked by prominent
morphological changes, which reflect the neurons’
switch from a mode of homeostatic cell maintenance
and neurotransmission to a mode of intense biosynthetic activity necessary to replace the axon (Lieberman, 1971; Grafstein, 1983; Austin, 1985; Alberghina
and Giuffrida Stella, 1988). The third stage of the axon
reaction begins with the re-establishment of functional
contact between the motor neuron and target muscle.
At this time, the cell bodies’ synthetic activity produces
cytoskeletal proteins for axon maturation and recovery
to pre-axotomy diameter (Hoffman et al., 1987; Tetzlaff
e t al., 1988) and proteins related to neurotransmission
(Kreutzberg et al., 1984) and the repair and reforma-
Received November 27, 1989; accepted March 8, 1990.
212
D.W. VAUGHAN
tion of receptors to re-establish the motor neurons’ afferent synaptic connections (Sumner, 1975; Rotter et
al., 1979; Hoover and Hancock, 1985). The time course
and the intensity of the neuronal responses during the
axon reaction are influenced by animal species (Cammermeyer, 19691, severity of injury (Soreide, 19811,
specific neurons under study (Aldskogius et al., 19801,
the animals’ developmental age (Jones and LaVelle,
19861, and the distance between the cell body and the
injury (Geist, 1933). In this study, the key to understanding the effects of advancing age on the neuron’s
ability to respond to axotomy is to describe and compare the neurons’ response at comparable stages in the
axon reaction, not a t specific days post-injury. In the
rat facial motor system in both the young and the older
animals, all three stages of the axon reaction can be
identified within 28 days following crush-induced axotomy.
The two age groups of animals used in this study are
considered to be “young adult” and “middle aged.” The
age of the younger animals in this study, 3 months,
represents a n age at which early developmental
changes have stabilized (Jones and LaVelle, 1986) and
the central nervous system has reached adult dimensions (Peters et al., 1983). In this species, which has a
mean lifespan of 25 months (Curcio et al., 19841, the
older animals, at 15 months of age, are considered to be
middle-aged. These older animals show no outward
signs of advancing age other than their increased size.
But, despite the weight differences, the skeletal sizes
are similar in the 3- and 15-month-old animals, and the
distance spanned by the regenerating axons to the denervated muscles is the same (40 mm) in both age
groups.
ture concentrated fixatives consisting of paraformaldehyde and glutaraldehyde in 0.1 M cacodylate buffer a t
pH 7.4 (Reese and Karnovsky, 1967). Following perfusion the animal carcass was refrigerated overnight in a
plastic bag to permit fixation to continue in situ, and
the brain stem was removed on the following day. The
block of brain stem containing the caudal two-thirds of
the two facial nuclei was prepared for the serial frozen
sections by cryoprotection with 20% buffered glycerol,
before sectioning in the transverse plane a t 30 pm. A
groove cut into the dorsal surface of the brain stem
marked the control side (Fig. 1). Serial sections
through the caudalmost extent of the nuclei were
mounted in order on glass slides and stained with thionin.
Functional Recovery
The principal target of the motor neurons examined
in this study is the nasolabialis muscle of the whisker
pad (Hinrichsen and Watson, 1984; Klein and Rhoades,
1985; Semba and Eggar, 1986). Functional recovery for
the purpose of this study was determined by the return
of voluntary whisker movement.
Morphornetry
Neurons in the uninjured facial nucleus served as
controls within each animal. Using the caudalmost extent of the right and left facial nuclei in the serial
series, individual brain stem sections were selected for
the samples of control and injured profiles. Specifically,
a level of the nucleus about 15 sections (450 pm) from
the caudal end of the nucleus was identified separately
on both right and left sides and neuronal profiles located in the lateral and intermediate subdivisions of
the facial nucleus were selected for tracing. At least
MATERIALS AND METHODS
90% of the neurons in these subdivisions innervate the
A total of 28 3-month-old and 30 15-month-old muscles responsible for whisker activity (McCall and
Sprague-Dawley-derived male rats (Taconic Farms, Aghajanian, 1979; Semba and Eggar, 1986). Two to
Germantown, NY) was used in this study. The survival three sequentially rostra1 sections were examined to
period of days post-crush (dpc), with the numbers of attain the designated sample number.
For each animal, a t least 45 somata from the control
young and old animals, respectively, were as follows: 1
dpc (two 3-month and two 15-month), 2 dpc (three 3- and injured sides were traced with a camera lucida
month and five 15-month, respectively), 4 dpc (three attachment to a light microscope and a x 40 objective.
3-month and seven 15-month), 10 dpc (four 3-month Only somata with the nucleus fully within the section
and three 15-month), 16 dpc (four 3-month and six 15- were selected for tracing. Since the nucleus does not
month), 21 dpc (six 3-month and four 15-month), and shift to a n eccentric position during the facial motor
28 dpc (three 3-month and three 15-month). The ani- neuron axon reaction (Soreide, 19811, it could be exmals of most sample groups were prepared a s part of a t pected that the maximum profile of the soma was inleast two different series so as to avoid unintentional cluded in the 30 pm thick section. Nuclei, and their
between-sample variation due to factors other than age nucleoli, were traced using a x 100 oil immersion oband survival times. Within each of the series, surgery jective. Since the diameters of these two structures are
was performed on animals of both age groups a t the approximately 18 and 5 pm, respectively, it is possible
same “day zero” so that a t the selected survival times, to select only those which are fully within the tissue
animals of both age groups were perfused and prepared section. The areas of the traced profiles were subsequently quantified using a graphics tablet and a n Apfor microscopy.
The facial nerve on the left side was crushed a t its ple IIe computer in conjunction with morphometry and
exit from the stylomastoid foramen between the tips of analytical software designed by Dr. Christine Curcio.
Dumont No. 5 forceps for two 10 second periods. With
Statistical Methods
the first crush the vibrissae were observed to protract,
The mean areas for each animal’s injured and control
after which they remained paralyzed until axons regrew several days later. For the control operation the somata, nuclei, and nucleoli were subjected to a refacial nerve on the right was exposed but not disturbed. peated measures ANOVA using a n SAS (Statistical
At the designated survival time, the animal was Analysis Systems) program. The ANOVA was used to
anesthetized and perfused through the aorta with a determine whether a statistically significant difference
two-stage perfusion of 38°C dilute and room-tempera- existed in the control values of the three structures,
EFFECTS OF AGE ON N E U R O N r m w w E TO AXOTOMY
within and between the two age groups, and then to
determine the statistical significance of the individual
structure's response to the lesion and whether there
was significant interaction between the lesion, the animal ages, and the days post-crush. Where the effects of
the lesion were found to be significant, paired t-tests
were used to determine which individual differences
were statistically significant.
Since there was no statistically significant difference
between the areas of the control structures and no evidence for a contralateral effect, the data have been
presented as percent difference from control.
RESULTS
Functional Recovery
The rodents' characteristic rhythmic whisking motion is normally accomplished by the voluntary contraction of intrinsic muscles in the whisker pad which
draws the vibrissae forward and, with relaxation of the
muscle, the elastic return of the vibrissae back to resting position (Dorfl, 1982). When recovery of function
began, such voluntary movement first appeared as the
ability to protract the vibrissae partially; that is, the
vibrissae swept from their relaxed position against the
side of the animal's head through about 45"rather than
the normally full arc of about 140". During the 3 to 4
day period following the first signs of vibrissae movement, the animals were able to draw the vibrissae progressively further forward until bilaterally symmetrical whisking behavior was attained. Among the
animals of both age groups, the period of recovery was
quite uniform. In the 3-month-old animals, this return
of function occurred during 12 to 14 dpc. The 15-monthold animals uniformly began recovery at 16 dpc, but
full recovery of bilaterally symmetrical whisking was
attained by animals on either 19 or 20 dpc. These periods over which recovery progressively occurred are
indicated by shaded vertical bars on the graphs in Figures 4 through 6.
Light Microscopy
During the 28 day period following axotomy, there
was no striking qualitative difference in the appearance of the soma, nuclei, or nucleoli other than increases in volume. Nuclear eccentricity did not develop; there was no increase in the number of nucleoli;
and there was no apparent diminution of cytoplasmic
basophilia. The volume increases were most evident a t
16 dpc and were apparent even a t low-power magnification, as in Figure 1. The changes appeared to be uniform throughout the population of motor neurons in
the lateral and intermediate subdivisions of the facial
nuclei. Figures 2 and 3 illustrate neuronal cell bodies
of a 3-month-old animal (Fig. 2) and a 15-month-old
animal (Fig. 3) a t 16 dpc.
213
three levels. Table 2 indicates the sample times (dpc) at
which the lesion effects were statistically significant.
The response of neuronal cell body areas to axotomy
is illustrated in Figure 4.In both the 3-month-old and
the 15-month-old animals, following axotomy, the injured neuronal cell body increased in size and reached
a peak a t 16 dpc. As evident in Figure 4,there was no
age-related difference in the magnitude or in the pattern of this response. As seen in Table 1, a statistically
significant interaction existed between the lesion effect
and the days post-crush, but the age groups did not
influence the significance of the response nor was the
interaction of the lesion effects and age and days postcrush statistically significant.
There were age-related differences in the patterns of
response of both the nucleus and the nucleolus. After 4
dpc both the temporal pattern of the response and the
magnitude of the alterations in these two structures
were different between the two age groups. As seen in
Figure 5, in the 3-month-old animals, the injured nuclei rapidly increased in area during the first 4 days,
but afterward they became smaller. Coincident with
the first signs of functional connections, the injured
nuclei once again increased dramatically in size, and
then between 6 and 21 dpc returned toward normal.
Such a bimodal response was not present in the 15month-old animals: their injured nuclei showed a gradual increase in area through 10 dpc, followed by a gradual decline. Even a t their maximum size, the older
nuclei enlarged no more than the initial increase of the
younger ones. Unlike the younger animals' nuclear response, there was little change in the nuclear response
coincident with the return of function in the older animals. As seen in Table 1, age itself was not a significant factor in the lesion effect (age may cause little
more than the delay in response). The interaction between the lesion, the animals' age, and days post-crush
was highly significant.
Figure 6 compares the patterns of the nucleolar responses. In the 3-month-old animals the injured nucleoli increased in size gradually through 10 dpc, then
dramatically increased through 16 dpc, after which
they returned toward control dimensions. The pattern
of injured nucleolar change in the 15-month-old animals was bimodal, showing one peak in area a t 4 dpc
and a second peak a t 21 dpc, followed by a return toward normal. The two peaks in the size of the 15month-old nucleoli were similar in magnitude and corresponded in magnitude to the peak reached by the
3-month-old nucleolar profiles a t 4 dpc. Table 1 shows
that for the nucleoli there was a significant interaction
between animal age and the lesion effect. As with the
other two structures analyzed, there was highly significant interaction between the lesion and days postcrush, and the lesion, days, and age.
Morphometry
DISCUSSION
The ANOVA of the mean areas of the control somata,
nuclei, and nucleoli during the post-crush period
showed no statistically significant differences either
within or between the two age groups. Table 1 presents, for each of the three structures, the P values
derived from the ANOVA for the interactions between
the lesion effect and 1)the days post-crush survival, 2)
the two animal age groups, and 3) the interaction of all
This is the first detailed morphological examination
of the effects of advancing age on central changes
brought about by peripheral axon injury. Older animals require more time to regenerate injured peripheral axons than young adults, but the reason is uncertain. It has been suggested that advancing age causes
a delay in the initial period preceding regrowth (Drahota and Gutmann, 1961) or advancing age causes a
214
D.W. VAUGHAN
Figs. 1-3
E F F E C T S OF AGE ON NEURON RESPONSE TO AXOTOMY
TABLE 1. P values of interactions
Interactions
Lesion . day post-crush
Lesion . age
Lesion day . age
‘
Soma
Nucleus
Nucleolus
<.0001
= .3
= .9
<.0001
= .9
<.0001
<.0001
= ,007
<.0001
reduced rate of regeneration (Black and Lasek, 19791,
or age is responsible for both initial delay and reduced
rate (Komiya, 1981).With the exception of a study of
axotomized facial motor neurons in rabbits by Cammermeyer (1963), in which he reports the early
changes were generally more conspicuous and the recovery process less intense in very old animals, we
know of no other morphological study of the effects of
advancing age on the central changes that accompany
the axon reaction.
No age difference was detected between the 3-monthold and 15-month-old animals in the initial response of
the soma, nuclei, and nucleoli to axotomy. Each of
these three structures began increasing in size very
soon after axotomy so that by 24 hours each was enlarged. Thus there was no evidence that advancing age,
at least through middle age, caused a delay in the neurons’ initial response to injury. However, if there were
a n increased latency on the order of that which Drahota and Gutmann (1961) detected in regenerating dorsal root ganglion-1 day versus 1.5 days-the sample
points of this study may not have been adequate to
detect such a difference.
The second stage of the axon reaction, t h a t of axon
outgrowth, lasts until the axon re-establishes contact
with its target and therefore is of different duration in
the two groups of animals. For this study, we roughly
equated muscle fiber reinnervation with recovery of
function, but recognized that some neurons may reestablish contact with muscle fibers before or after “full
recovery” of function. Regardless of that, for the
present study we considered that a t the time bilaterally symmetrical whisking occurred the muscle was
equivalently reinnervated in both the old and the
young animals.
This second stage of the axon reaction is the period
during which the injured neurons become dedicated to
regrowing axons. Acceleration of rRNA synthesis and
protein synthesis have been correlated with somal, nuclear, and nucleolar enlargement (Whitnall and Grafstein, 1982; Goessens, 1984; Jones and LaVelle, 1986;
Hall and Borke, 1988; Wells and Vaidya, 1989). There
Fig. 1. Thirty micrometer section of brain stem from 3-month-old
animal, 16 dpc. The notch indicates t h e control side of t h e brain stem.
Neuronal cell bodies of the facial nuclei a r e evident in t h e ventrolatera1 regions of the brain stem. The boxed areas indicate the lateral
and intermediate regions of the nuclei from which t h e neurons were
selected for analysis. x 20.
Fig. 2. Neuronal cell bodies from a brain stem section of 3-monthold, 16 dpc animal, photographed with Nomarski optics. a: Control; b:
lesion x 4 7 5 .
Fig. 3. Neuronal cell bodies from brain stem section of 15-month-old,
16 dpc animal, photographed with Nomarski optics. a: Control; b:
lesion. x 475.
215
was no significant age difference detected in the response of the neuronal cell bodies during this time. The
unimodal increase and decrease in somal volume have
been reported by others (Aldskogius e t al., 1980; Hall
and Borke, 19881, although Brattgard and coworkers
(1957) find that the rabbit hypoglossal neuronal cell
volume did not return to its original value in a linear
fashion after attaining maximum volume a t day 6. The
volumes of the hypoglossal somata decreased slightly
after day 6, then increased a second time, though by a
smaller amount, a t 48 days, before regaining original
values after 90 days.
The pattern of nuclear change during this stage in
the 15-month-old animals was similar to that of the
3-month-old animals in that the relative percent increase and subsequent condensation were comparable,
but the process was delayed in the older animals. Thus,
while nuclei appeared to undergo the same changes in
both age groups, they required more time to reach their
peak in the older animals. Studies have shown some
proteins generated a t this time in the axon reaction are
not normally found in the perikaryal cytoplasm of
adult neurons, but represent a reappearance of proteins formally present during developmental stages
(Griffith and LaVelle, 1971; Skene and Willard, 1981).
Furthermore, a P-tubulin mRNA that normally disappears when developmental stages are complete has
been found to reappear in regenerating adult dorsal
root ganglion cells (Hoffman and Cleveland, 1988) and
a n embryonic a-tubulin mRNA has been found to be
rapidly induced in regenerating adult facial nuclei,
then down-regulated following functional reinnervation (Miller et al., 1989). The ability of regenerating
neurons to re-express genes which have been repressed
since the end of development is a n intriguing concept
as i t relates to this study, for the nuclear differences
observed in the regenerating older neurons may be related to a difficulty in re-expressing genes that have
been repressed for a long time. The nuclear enlargement observed during this stage could reflect the transient transcription of the mRNA, and the older animals
simply take longer to access those parts of the genome
in which are located these genes.
Age differences in the pattern of nucleolar change
during this stage of axon regrowth may be explained by
the greater length of this period in the older animals.
Enlargement followed by condensation may be a normal nucleolar response that follows the production of
necessary ribosomes. Langford and coworkers (1980)
described a bimodal pattern in the rate of ribosomal
RNA synthesis in the crushed rat nodose ganglion
cells: a rapid early increase peaking a t 3 days, a fall to
control levels, then a second peak during the second
week after axotomy. In this study, the second stage of
the axon reaction was about 14 days duration in the
15-month-old animals, but only 10 days duration in the
3-month-old animals. In the young adult animal there
may not have been enough time to reveal nucleolar
condensation because a new set of demands, signalled
by the axon reaching its target, stimulated a second
nucleolar expansion shortly after the demands for
growing the axon had been met.
The third phase of the axon reaction begins with the
re-establishment of functional contact between the
neuron and target muscle, when the neurons’ synthetic
50
--
40
--
Soma A r e d
A
Percent
D 1f f e re nc e 30
3 month animals
-&
15 m o n t h aniicals
--
70
--
lo
--
:
.
1
I
t
1
,
I
I
I
I
I
I
I
Nucleus Area
60
50
40
Percent 3o
Difference
PO
10
70
60
50
40
rt
i
Nucleolus Area
3
/
,
,
Percent
Difference
30
20
10
Fins 4-6
,
I
,
I
- - - - A -3- m~ o~n t h animals
+ 15 month a n i m a l s
217
EFFECTS OF AGE O N N E U R O N RESPONSE TO AXOTOMY
TABLE 2. Significance of the lesion at specific days post-crush
Soma
3 month
15 month
Nucleus
3 month
15 month
Nucleolus
3 month
15 month
1
2
ns
ns
**
ns
ns
ns
ns
*
***
4:
g
*:g
:c *: :,:
;*:%
~
:*:,:
Day
10
4
:*:,:
+.
:,:i:
ns
***
16
21
s: 1:
**1:
:*:i:
1:
* 1:
1:*
*1:
:* *
ns
I:
28
:p:c
:j:
*
*
ns
ns
**.#
*:**
ns
ns, not significant; *P < .05; **P < .01; ***P < .001.
activities must switch again. Following functional recovery, many of the parameters found to have changed
during the axon reaction begin to return to original
levels, such as the mass of the nucleus and its nucleic
acid content, and the mass of the cell body and its nucleic acid content (Watson, 1970; Aldskogius et al.,
1980). Several investigators have suggested that interaction with the denervated muscle signals a variety of
central responses in the neuronal cell body (Brattgard
et al., 1957; Engh et al., 1971; Cragg, 1979; Kreutzberg
e t al., 1984; Smith et al., 1984; Johnson et al., 1985;
Hall and Borke, 1988), including induction of a neurofilament gene that is expressed at this time (Hoffman
and Cleveland, 1988) and turning off of the a-tubulin
mRNA expression referred to above (Miller et al.,
1989).
Among the 3-month-old animals of this study, dramatic changes in the nucleus and nucleolus accompanied the re-establishment of functional connections: a
vigorous increase in the size of both of these structures
followed by a n equally vigorous condensation toward
normal levels. Such a n acute morphological response
has not previously been reported, but there are a t least
three explanations for why this striking response is
seen in the present study.
1. Experimental design. Many studies of the axon
reaction are designed to reduce or prevent the chance of
successful axon regeneration, and so a second peak in
the response of central structures simply may not occur
because it is the contact with the target that signals
the second response (e.g., Engh e t al., 1971). LaVelle
Flg. 4. Percent difference in the cross-sectional areas of cell bodies
for 3-month-old animals (dotted lines, and 15-month-old animals
(solid lines). The data are presented as means 2 S.E.M. The stippled
vertical bars represent the periods over which the animals recover
bilaterally symmetrical whisking behavior. This occurs between 12 to
14 dpc in the 3-month-old and 16 to 19.5 dpc in the 15-month-old
animals.
Fig. 5 . Percent difference in the areas of nuclei for 3-month-old
(dotted lines) and 15-month-old (solid lines) animals. The data are
presented as means i S.E.M. The stippled vertical bars represent the
periods of functional recovery as described in Figure 4.
Fig. 6. Percent difference in the areas of nucleoli for 3-month-old
(dotted lines) and 15-month-old (solid lines) animals. The data are
presented as means i S.E M The stippled vertical bars represent the
periods of functional recovery a s described for Figure 4.
and LaVelle (1958) prevented regeneration after axotomy of hamster facial neurons and they report that the
peak central response occurred 4 days after they severed the facial nerve. In the present study, if axons had
been prevented from reaching the denervated muscle
fibers, the vigorous second response of nuclei and nucleoli would probably not occur, and the peak response
would perhaps also occur a t 4 days post-injury.
2. Uniform distance to target. The neuron’s acute
response to reaching its target is detectable in this
study because the population of neurons examined is
relatively uniform with regard to the distance the axons must grow to reach the target. At least 90% of the
neurons in the lateral and intermediate subdivisions of
the facial nucleus project to the muscles of the whisker
pad (Klein and Rhoades, 1985; Semba and Eggar,
19861, and they are furthermore all likely to re-innervate their original targets because the crush injury
does not interrupt the external (basal) lamina tubes
which guide their direction of growth (e.g., Thomas,
1989). All the motor neurons reach their intended target at about the same time. In a more heterogeneous
population of neurons, and with longer distances to
grow, as in the case of spinal cord motor neurons responding to sciatic nerve injury, sampling a t a given
post-operative time would record a blur of responses
from different neurons reaching targets over a broader
period of time.
3. Selection of post-operative survival times. In the
present study, 1 week after recovery of function, the
sizes of nuclei and nucleoli are similar in magnitude to
what they were just preceding the reinnervation. The
increase in size was detected at about the time of functional recovery. As a n example of this importance of
sampling time, Aldskogius and coworkers (1980) may
have missed a similar second response in their examination of the transected rat hypoglossal nerve: They
sampled up through 14 days post-surgery and then
again a t day 28. Reinnervation of the tongue occurs,
apparently, by day 21 (Hall and Borke, 1988), and so it
is not surprising they detected only unimodal changes
in the sizes of the nuclei and nucleoli.
In this study, differences attributable to advancing
age were most substantial a t the beginning of the final
stage of the axon reaction. In the older animals, the
peak nuclear increase recorded 2 days following recovery of function was considerably less than that of the
3-month-old animals recorded a t a comparable time.
218
D.W. VAUGHAN
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ACKNOWLEDGMENTS
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