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Long-term protection of axotomized neurons in the dorsal lateral geniculate nucleus in the rat following a single administration of basic fibroblast growth factor or ciliary neurotrophic factor

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THE JOURNAL OF COMPARATIVE NEUROLOGY 392:264–272 (1998)
Long-Term Protection of Axotomized
Neurons in the Dorsal Lateral Geniculate
Nucleus in the Rat Following a Single
Administration of Basic Fibroblast
Growth Factor or Ciliary
Neurotrophic Factor
SEEMA AGARWALA1,2 AND RONALD E. KALIL1,2*
for Neuroscience,University of Wisconsin, Madison, Wisconsin 53706
2Department of Ophthalmology and Visual Sciences, University of Wisconsin,
Madison, Wisconsin 53706
1Center
ABSTRACT
We have studied the long-term effects of basic fibroblast growth factor (bFGF) and ciliary
neurotrophic factor (CNTF) on axotomy-induced cell death in the dorsal lateral geniculate
nucleus (LGN) of adult rats. LGN neurons were axotomized by a visual cortex lesion in 31
adult rats. A gelatin sponge soaked in a solution of bFGF, CNTF, or saline (control) was placed
on the surface of the lesion, and the animals were allowed to survive for 1–12 weeks.
Compared with controls, no major improvement was noted in the mean cross-sectional
area of surviving LGN neurons in rats treated with bFGF or CNTF at any survival time.
However, treatment with either factor significantly increased the number of surviving
neurons at each survival time. At 1 week, the survival of LGN neurons in rats treated with
bFGF or CNTF was 136% and 131% greater, respectively, than in controls. At 12 weeks, the
number of surviving LGN neurons in bFGF- and CNTF-treated rats exceeded that seen in
controls by 114% and 58%, respectively.
Thus, a single administration of bFGF or CNTF following axotomy reduced neuronal
death for long periods of time, but could not prevent atrophy. A single treatment with bFGF or
CNTF, therefore, may block the full execution of a cell death program, but cannot prevent its
initiation. Alternatively, the transduction pathways for maintaining cell size and preventing
cell death may not be identical, and bFGF and CNTF applied as described above may be
effective in activating one pathway but not the other. J. Comp. Neurol. 392:264–272,
1998. r 1998 Wiley-Liss, Inc.
Indexing terms: brain trauma; neural cell death; apoptosis; neurotrophic factors
In the mammalian central nervous system (CNS), neurons usually die if they are disconnected from their targets. For example, in mammals, a lesion of the neocortex
cuts the axons of neurons in the thalamus that project to
the damaged cortical area. When the visual cortex is
damaged in adult animals, the projection neurons in the
dorsal lateral geniculate nucleus (LGN) are axotomized,
which results in their subsequent atrophy and death (see,
e.g., Lashley, 1941; Combs, 1949; Chow and Dewson, 1966;
Wong-Riley, 1972). In contrast, in infant animals, some of
the neurons in the LGN appear to survive a lesion of the
visual cortex (Kalil, 1978; Spear et al., 1980; Kalil, 1984,
1990; Payne and Cornwell, 1994). The reasons for this
r 1998 WILEY-LISS, INC.
age-related difference in the response of neurons to injury
are not well understood. However, considerable evidence
has accumulated in favor of the proposition that the
survival of neurons depends on the availability of specific
trophic factors produced by their target cells (see, e.g.,
Levi-Montalcini, 1987; Hamburger, 1993). Trophic factors
Grant sponsor: National Eye Institute; Grant number: EYO 1331.
*Correspondence to: Ronald Kalil, Center for Neuroscience, University of
Wisconsin, 1300 University Avenue, Madison, WI 53706.
E-mail: rekalil@facstaff.wisc.edu
Received 2 July 1996; Revised 9 October 1997; Accepted 17 October 1997
bFGF AND CNTF PROTECT LGN NEURONS AFTER AXOTOMY
265
Fig. 1. Typical neurons in the dorsal lateral geniculate nucleus (LGN) of a normal adult rat. Scale
bar 5 25 µm.
may be necessary for a neuron’s survival, because they
activate pathways that are essential to normal cell function or, conversely, they hold an endogenous cell death
program in check (Raff, 1992; Raff et al., 1993). Following
axotomy and the consequent disconnection of a cell, death
may follow for either of these reasons, because a critical
trophic factor is no longer available to the cell body (for
reviews, see Levi-Montalcini, 1987; Hamburger, 1993).
The administration of a neurotrophic factor may substitute for an endogenous factor that is no longer available to
a neuron that has had its axon sectioned. Indeed, recent
studies suggest that the application of many neurotrophic
factors, e.g., basic fibroblast growth factor (bFGF), brainderived neurotrophic factor (BDNF), ciliary neurotrophic
factor (CNTF), and glial cell line-derived neurotrophic
factor (GDNF), may mitigate the severity of neuronal loss
after injury or disease (Anderson et al., 1988; Hofer and
Barde, 1988; Otto et al., 1989; Sendtner et al., 1990;
Yamada et al., 1991; Hagg et al., 1992; Agarwala and Kalil,
1993; Hagg and Varon, 1993; Koliatos et al., 1993; Agarwala et al., 1994; Kalil et al., 1995).
Although numerous studies have demonstrated the efficacy of neurotrophic factors as neuroprotectants, relatively few have been conducted in vivo. However, a comparison of the efficacy of neurotrophic factors in vivo and in
vitro suggests that, in some instances, neurotrophic factors that enhance the survival of neurons in vitro may not
be effective in vivo (Oppenheim et al., 1991). Moreover,
with but few exceptions (see, e.g., Mey and Thanos, 1993;
Mansour-Robaey et al., 1994), most studies have not
examined the long-term effects of neurotrophic factors on
neuronal survival but, instead, have focused on relatively
short-term outcomes of 2 weeks or less.
In the present study, we have quantified the neuroprotective effects of bFGF and CNTF by administering these
neurotrophic factors to axotomized neurons in the adult
rat LGN after postoperative survival times that ranged
from 1 week to 12 weeks. The results of these experiments
indicate that a single administration of these trophic
factors at the time of axotomy is capable of reducing neural
cell death in the LGN for as long as 12 weeks, suggesting
that an enduring change in the choice made by a neuron to
survive or die after injury is possible if intervention after
injury is rapid.
MATERIALS AND METHODS
Thirty-one adult, male Holzmann rats (250–300 g) were
used in this study. All animal procedures were performed
in accordance with protocols approved by the Institutional
Animal Use and Care Committee at the University of
Wisconsin-Madison. Rats were anesthetized intramuscularly with a mixture of 90 mg/kg ketamine HCl and 9
mg/kg xylazine. Using aseptic precautions, a unilateral
lesion of visual cortical areas 17, 18, and 18a was made by
266
Fig. 2. A–F: Photomicrographs of lateral geniculate nucleus (LGN)
neurons in rats with a lesion of the visual cortex that were treated
with saline (A,D), basic fibroblast growth factor (B,E), or ciliary
neurotrophic factor (C,F). Postoperative survival times were 1 week
S. AGARWALA AND R.E. KALIL
(A–C) and 12 weeks (D–F). At both postoperative intervals, long
arrows indicate examples of surviving neurons. Dying cells with
condensation of the nuclear chromatin (short arrows) are seen in each
group of rats at 1 week survival. Scale bar 5 25 µm.
bFGF AND CNTF PROTECT LGN NEURONS AFTER AXOTOMY
TABLE 1. Mean Cross-Sectional Area of Surviving LGN Neurons in
bFGF- and CNTF-Treated Animals1
Rat
1-Week survival
bFGF-13
bFGF-14
bFGF-15
Mean 6 s.e.
CNTF-1
CNTF-2
CNTF-9
Mean 6 s.e.
2-Week survival
bFGF-7
bFGF-8
bFGF-9
Mean 6 s.e.
CNTF-15
CNTF-16
CNTF-25
Mean 6 s.e.
4-Week survival
bFGF-4
bFGF-5
bFGF-6
Mean 6 s.e.
CNTF-7
CNTF-10
CNTF-11
CNTF-17
Mean 6 s.e.
8-Week survival
bFGF-16
bFGF-17
bFGF-18
Mean 6 s.e.
CNTF-22
CNTF-23
CNTF-24
Mean 6 s.e.
12-Week survival
bFGF-21
bFGF-22
bFGF-23
Mean 6 s.e.
CNTF-19
CNTF-20
CNTF-21
Mean 6 s.e.
Mean
cell area
(µm2)
Percent of
normal
100.7
85.5
98.1
94.8 6 4.7
113.7
94.8
76.1
94.9 6 10.9
74.6
63.4
72.7
70.2 6 3.5
84.3
70.2
56.4
70.3 6 8.1
75.5
94.6
85.6
85.2 6 5.5
81.4
84.5
82.8
82.9 6 0.9
55.9
70.1
63.5
63.2 6 4.1
60.3
62.6
61.4
61.5 6 0.7
77.0
78.5
77.1
77.5 6 0.5
71.4
88.8
78.3
68.7
76.8 6 4.5
57.1
58.2
57.2
57.5 6 0.3
52.9
65.9
58.0
50.9
56.9 6 3.3
75.3
97.5
85.6
86.1 6 6.4
79.6
61.9
70.1
70.5 6 5.1
55.8
72.3
63.4
63.8 6 4.8
59.0
45.9
52.0
52.3 6 3.8
84.9
68.3
76.8
76.6 6 4.8
78.0
82.1
71.4
77.2 6 3.1
62.9
50.6
56.9
56.8 6 3.6
57.8
60.9
52.9
57.2 6 2.3
1Mean cross-sectional areas of lateral geniculate nucleus (LGN) neurons in basic
fibroblast growth factor (bFGF)- or ciliary neurotrophic factor (CNTF)-treated rats that
received a lesion of the visual cortex. The means for individual animals as well as the
group means at each survival time are shown in the second column. The third column
shows the mean cross-sectional cell area for each animal and the group mean as a
percent of normal. Based on eight normal animals, the mean 6 s.e. cross-sectional area
of LGN neurons is 134.9 6 4.4 µm2.
subpial suction (Paxinos and Watson, 1986). In rats that
received bFGF or CNTF, the lesion site was covered with a
gelatin sponge (Gelfoam; Upjohn, Kalamazoo, MI) soaked
in either 0.26 mg/ml of human bFGF (Promega, Madison,
WI) or in 0.78 mg/ml of rat CNTF (Regeneron Pharmaceuticals, Tarrytown, NY).
Following survival periods of 1–12 weeks, the rats were
deeply anesthetized with 60 mg/kg pentobarbital sodium
and perfused through the heart with a mixture of 4.0%
paraformaldehyde and 0.5% glutaraldehyde in 0.1 M
sodium phosphate buffer, pH 7.2. The brain then was
removed from the skull and stored overnight in fixative.
Serial sections, 50 µm or 75 µm thick, were cut on a
Vibratome in the frontal plane, and a one-in-two or a
one-in-four series was stained with cresyl violet for cell
somata. Corresponding sets of sections in the frontal plane
also were available from the brains of eight unoperated,
normal rats and from 32 control rats that had received a
comparable lesion of the visual cortex but with 0.9% saline
(Agarwala and Kalil, 1997) instead of bFGF or CNTF.
267
Cell measurements
The rate and the magnitude of the neural cell death in
the LGN of rats treated with bFGF or CNTF and in control
rats were determined by counting the number of surviving
neurons and by measuring their cross-sectional areas in
cresyl violet-stained sections. The procedures that were
used to count neurons and measure their sizes in the rat
LGN have been described in detail previously (Kalil, 1978;
Agarwala and Kalil, 1998).
Cross-sectional area
The perikaryal outlines of LGN neurons were drawn
from a single section at the rostrocaudal midpoint of the
LGN by sweeping the microscope field from the dorsolateral to the ventromedial border of the LGN and then
reversing the direction of the sweep until 100 neurons
were obtained (Agarwala and Kalil, 1997). All neurons
with visible nucleoli in a given field were drawn before
advancing the microscope to the next field.
In each treated animal, the mean cross-sectional area of
neurons in the LGN ipsilateral to the cortical lesion was
compared with the mean cross-sectional area of LGN
neurons obtained from normal adult rats and with the
mean size of ipsilateral LGN neurons in control rats that
had survived for the same postoperative period. The
statistical significance of the observed differences in cell
size were evaluated with a two-tailed t test, using the
mean cross-sectional area for LGN cells in each animal as
a single value.
Cell number
Relative cell loss at different survival times in treated
rats was estimated by comparing the number of neurons in
the LGN ipsilateral to the cortical lesion with the cell
number in normal animals and in control rats. For each
brain, the number of neurons in the LGN was estimated as
described previously (Agarwala and Kalil, 1998). In brief,
cell counts were made with a 1003 oil-immersion objective
from two sections near the rostrocaudal midpoint of the
LGN. This was done by first positioning a Whipple eyepiece grid near the dorsolateral border of the LGN and
then sweeping it in steps ventromedially, until three
sweeps were made across the LGN in each section. The cell
counts were corrected for split-cell error (Abercrombie,
1946), multiplied by the section thickness (50 µm or 75
µm), and adjusted for the measured volume of the LGN to
obtain an estimate of the total number of neurons in the
nucleus. The percentage of spared neurons at each survival time was determined by comparing the estimated
number of LGN neurons in each treated rat with the
estimated number of neurons in the LGN of normal rats.
Differences in cell number between the treated and control
rats and between the treated and normal rats at each
survival period were evaluated with a two-tailed t-test.
RESULTS
Neurons in the LGN of the normal adult rat are shown
in Figure 1 and provide a basis for comparison with those
in operated rats (Fig. 2). After a lesion of the visual cortex,
the numbers and sizes of LGN neurons remain essentially
unchanged for up to 3 days. This period of seeming
quiescence is followed by the sudden and rapid death and
atrophy of LGN neurons, and, by the end of the first
268
S. AGARWALA AND R.E. KALIL
Fig. 3. Histograms showing mean cross-sectional areas at different survival times of lateral geniculate
nucleus (LGN) neurons in rats that received a lesion of the visual cortex and were treated with basic
fibroblast growth factor (bFGF) or ciliary neurotrophic factor (CNTF) as a percent of the size of LGN cells
in control rats.
postoperative, week nearly 70% of the neurons in the LGN
have died (Agarwala and Kalil, 1998). Figure 2A,D shows
LGN neurons in control rats that survived for 1 week and
12 weeks, respectively. The number of surviving neurons
(Fig. 2, long arrows) at each of these postoperative intervals is reduced dramatically from normal (cf. Fig. 1).
However, the results of the present experiments indicate
that, when bFGF or CNTF is administered at the site of
the cortical lesion immediately after it is made, a significant reduction in the death of axotomized LGN neurons
results. The neuroprotective effects of bFGF at postoperative survivals of 1 week and 12 weeks are shown in Figure
2B,E, and the effects of CNTF at comparable survival
times are illustrated in Figure 2C,F. Although dying
neurons, which are marked by condensation of the nuclear
chromatin (Fig. 2, short arrows), are evident at 1 week of
survival in rats treated with bFGF or CNTF, as they also
are in controls, many surviving neurons (Fig. 2, long
arrows) are apparent. Twelve weeks after removal of the
visual cortex, it is very clear that the numbers of surviving
LGN neurons in bFGF- (Fig. 2E) or CNTF-treated rats
(Fig. 2F) are much greater than in control operated rats
(Fig. 2D).
Cross-sectional area
The mean cross-sectional area of neurons for each
animal treated with bFGF or CNTF is presented in Table
1. The mean cross-sectional area of LGN neurons after the
administration of bFGF or CNTF is shown as a percent of
normal in Figure. 3. At 1 week, when cell area is reduced to
66% of normal in control rats, the mean neuronal areas are
70% and 71% of normal in bFGF- or CNTF-treated rats,
respectively (P . 0.05). By 2 weeks, when mean LGN cell
areas in control rats are approximately 57% of normal, the
neurons in the bFGF- or CNTF-treated rats are 63% and
61% of normal, respectively. These differences are not
statistically significant, nor are any of the cell size differences observed between treated and control rats at 4, 8, or
12 weeks of survival significant. It appears, therefore, that
neither bFGF nor CNTF is capable of protecting axotomized LGN neurons in the rat from atrophying when
administered as in these experiments.
Cell number
The number of LGN neurons estimated for each animal
that received bFGF or CNTF is presented in Table 2. The
bFGF AND CNTF PROTECT LGN NEURONS AFTER AXOTOMY
TABLE 2. Number of Surviving LGN Neurons in bFGF- and CNTFTreated Animals1
Rat
1-Week survival
bFGF-13
bFGF-14
bFGF-15
Mean 6 s.e.
CNTF-1
CNTF-2
CNTF-9
Mean 6 s.e.
2-Week survival
bFGF-7
bFGF-8
bFGF-9
Mean 6 s.e.
CNTF-15
CNTF-16
CNTF-25
Mean 6 s.e.
4-Week survival
bFGF-4
bFGF-5
bFGF-6
Mean 6 s.e.
CNTF-7
CNTF-10
CNTF-11
CNTF-17
Mean 6 s.e.
8-Week survival
bFGF-16
bFGF-17
bFGF-18
Mean 6 s.e.
CNTF-22
CNTF-23
CNTF-24
Mean 6 s.e.
12-Week survival
bFGF-21
bFGF-22
bFGF-23
Mean 6 s.e.
CNTF-19
CNTF-20
CNTF-21
Mean 6 s.e.
Number
of neurons
Percent of
normal
19,109
30,324
29,319
26,250 6 3,583
30,316
23,848
23,174
25,779 6 2,276
57.3
91.0
88.0
78.8 6 10.7
91.0
71.6
69.5
77.3 6 6.8
16,706
20,297
18,435
18,479 6 1,037
22,968
22,630
12,616
19,404 6 3,396
50.1
60.9
55.3
55.4 6 3.1
68.9
67.9
37.9
58.2 6 10.2
17,668
14,677
14,636
15,661 6 1,004
14,350
21,102
15,763
16,680
16,974 6 1,457
53.0
44.0
43.9
47.0 6 3.0
43.1
63.3
47.3
50.0
50.9 6 4.4
20,002
16,048
16,228
17,426 6 1,289
17,151
14,550
16,806
16,169 6 815
60.0
48.1
48.7
52.3 6 3.9
51.5
43.7
50.4
48.5 6 2.4
17,897
9,479
12,923
13,433 6 2,443
10,315
10,934
8,433
9,894 6 752
53.7
28.4
38.8
40.3 6 7.3
30.9
32.8
25.3
29.7 6 2.3
1LGN cell numbers in bFGF- or CNTF-treated rats that received a lesion of the visual
cortex. The estimated number of LGN cells in each animal and the group mean 6 s.e. at
each survival time are shown in the second column. Cell numbers are expressed as a
percent of normal in the third column 3. Based on eight normal animals, the mean 6 s.e.
number of neurons is 33,330 6 2,168.
mean number of LGN neurons for animals in each treatment group is shown as a percent of normal in Figure 4. At
1 week after axotomy, when neuronal numbers have fallen
rapidly to 33% of normal in control rats, approximately
78% of the neurons in the LGN in rats that received bFGF
or CNTF survive, i.e., at 1 week of survival, rats treated
with bFGF or CNTF show a 136% improvement in neuron
survival, respectively, compared with controls (Table 3). At
2 weeks, about 55% and 58% of the neurons in the LGN
survive in bFGF- or CNTF-treated rats, respectively. This
is equivalent to a 62% and a 70% improvement in cell
survival, respectively, compared with control rats, in which
only 34% of the neurons in the LGN are alive at 2 weeks
after axotomy. Substantial increases in neuronal survival
in the LGN following a single treatment with bFGF or
CNTF are also seen at each of the later survival periods, 4,
8, and 12 weeks, that were studied. At 12 weeks, for
example, bFGF-treated animals retain 114% more neurons in the LGN than control rats (40% vs. 19%), and, in
animals treated with CNTF, neuronal survival is improved
by 58% compared with controls (30% vs. 19%).
269
DISCUSSION
The experiments reported here demonstrate that a
single administration of bFGF or CNTF directly to axotomized LGN neurons immediately after their axons are
sectioned reduces the death of these neurons significantly,
but does not prevent them from atrophying. The increase
in the survival of axotomized LGN neurons by bFGF and
CNTF is substantial and long lasting. In rats that received
a single dose of these neurotrophic factors after axotomy
and then survived for 12 weeks, 60–110% more neurons
are seen in the LGN than are observed in rats treated only
with saline.
bFGF and CNTF prevent neuron death
but not atrophy of LGN neurons
Despite the marked effect of neurotrophic factors on the
survival of injured neurons, neither of the trophic factors
used in this study was effective in preventing neuronal
atrophy after axotomy. Many laboratories have examined
the protective effects of neurotrophic factors and have
concluded that the prevention of neuronal death and
atrophy do not necessarily go hand in hand (Otto et al.,
1989; Li et al., 1994; Yan et al., 1995). For example,
although nerve growth factor (NGF) and bFGF are capable
of preventing the death of axotomized medial septal neurons, only NGF prevents their atrophy as well (Otto et al.,
1989). Further complexity is implied by the fact that a
specific neurotrophic factor may prevent the atrophy of
neurons in one system but fails to do so in another. Thus,
although GDNF reverses the atrophy of axotomized motor
neurons, it does not prevent the atrophy of injured substantia nigra and other mesencephalic dopaminergic neurons
(Henderson et al., 1994; Beck et al., 1995; Oppenheim et
al., 1995; Yan et al., 1995). The reasons for these differential neuroprotective effects are not clear. They may be due
to simple differences among experiments in methods, such
as doses or regimens, or in modes of delivery, or they may
indicate complex interactions between specific factors and
populations of neurons that are not yet understood because the appropriate signaling mechanisms remain to be
clarified.
It is reasonable to speculate that the proper activation of
one signaling mechanism may be required to protect a
damaged neuron from dying, whereas another pathway
may be involved in preventing cell atrophy. For example,
during normal neural development, a young neuron must
first succeed in surviving before growth and differentiation
are initiated. The successful completion of each step
during development may involve different neurotrophic
factors acting in a carefully orchestrated, temporal sequence to produce a mature neuron. In this light, it is
perhaps not surprising that a single administration of one
neurotrophic factor to injured neurons may fail to preserve
the size of these neurons, although it is sufficient to protect
them from dying.
Enhanced survival of axotomized
LGN neurons
In these experiments, as in other studies that have
described the short-term effects of trophic factor treatment
of injured neurons (see, e.g., Anderson et al., 1988; Knusel
et al., 1992; Morse et al., 1993; Mitsumoto et al., 1994), a
substantial improvement (65–135%) was seen in the numbers of surviving LGN neurons between 1 week and 4
270
S. AGARWALA AND R.E. KALIL
Fig. 4. Histograms showing mean number of surviving lateral geniculate nucleus (LGN) neurons in
rats that received a lesion of the visual cortex and were treated with basic fibroblast growth factor (bFGF)
or ciliary neurotrophic factor (CNTF) as a percent of the number of LGN cells in control rats.
TABLE 3. Percent Improvement in the Number of Surviving LGN
Neurons in Rats Treated With bFGF or CNTF Compared With Controls1
Postoperative
survival time
1 Week
2 Weeks
4 Weeks
8 Weeks
12 Weeks
Neurotrophic factor
bFGF
CNTF
136
62
66
167
114
131
70
79
147
58
1The percent improvement in the number surviving LGN neurons in rats that received a
lesion of visual cortex following treatment with bFGF or CNTF compared with the
number of surviving cells in operated rats that received only saline.
weeks postoperatively. However, many of the studies that
have extended observations beyond 4 weeks report that, at
these longer survival times, the numbers of surviving
neurons in trophic factor-treated animals begin to decline
and approach those in untreated animals (Mey and Thanos,
1993; Mansour-Robaey et al., 1994; Vejsada et al., 1995). In
the current experiments, significantly greater numbers of
LGN neurons survived for as long as 12 weeks with bFGF
or CNTF treatment (30–40% of normal compared with
19% of normal in controls), indicating, respectively, a 110%
and a 60% improvement in neuronal survival. The reason
why bFGF-treated brains show greater cell survival at 12
weeks compared with CNTF-treated brains is not clear,
but it may be related to differences in the effectiveness of
the interaction between LGN cells and specific neurotrophic factors (discussed above) or to the different doses
that were administered for bFGF and CNTF. Regardless of
the reason, these results suggest that cell death may be
blocked permanently in some injured neurons when all of
the conditions of neurotrophic factor treatment, e.g., concentration, timing, and duration, are optimized.
The mechanisms that might be responsible for the
long-term survival of axotomized LGN neurons following a
single administration of bFGF or CNTF are not well
understood. In vitro experiments with sympathetic neurons suggest that a short hiatus usually intervenes between a traumatic insult and a neuron’s commitment to
die. The administration of trophic factors as well as other
neuroprotective substances, such as cycloheximide, during
this period, but not later, can prevent the death of sympathetic neurons, (Deckwerth and Johnson, 1993; Estus et
al., 1994). The in vivo experiments described previously
bFGF AND CNTF PROTECT LGN NEURONS AFTER AXOTOMY
(Agarwala and Kalil, 1998) are similar, in that the soma
sizes and numbers of LGN neurons appear normal for up
to 3 days after axotomy before any cytological signs of
injury become apparent. The experiments reported here
show that, if neurotrophic factors are delivered to axotomized LGN neurons during this critical period, before
these neurons have committed to die, then many of these
neurons will survive.
Neurotrophic factors may be conveyed by axons to their
cell somata from target cells by receptor-mediated retrograde transport (for reviews, see Korsching, 1993; Lindsay
et al., 1994). When they are injected into the brain of adult
rats, specific receptor-mediated uptake of neurotrophic
factors is known to occur for bFGF and CNTF (Ferguson et
al., 1991; Curtis et al., 1993). Radiolabeled bFGF appears
in the cell bodies of neurons that project to the injection
site within 18 hours, suggesting a transport rate of 1.7
mm/hour (Ferguson et al., 1990, 1991). Given the short
geniculocortical path length in the rat, the trophic factors
administered to the damaged cortex in the present experiments may have reached the cell somata of injured LGN
neurons in less than 1 day following axotomy, which
appears to be quick enough to influence the fate of at least
some neurons that otherwise might die were these factors
not present.
It also is possible that bFGF or CNTF may have been
available to axotomized LGN neurons for longer than
might be considered likely for proteins applied extraneously to the brain. For example, it has been suggested that,
following uptake and transport to the cell soma, bFGF
undergoes proteolytic cleavage at a rate that is relatively
slow compared with that of some other neurotrophic
factors, such as NGF (Ferguson et al., 1990), which could
prolong the effective duration of the neurotrophic activity
of bFGF. Furthermore, both bFGF and CNTF and their
receptors sometimes are up-regulated in the vicinity of a
CNS injury (Frautschy et al., 1991; Ip et al., 1993; Lindsay
et al., 1994), and CNTF transport from a damaged area
may be enhanced for several days following an injury (Ip et
al., 1993; Curtis et al., 1993). Recent evidence also indicates that some neurons may protect themselves from
dying by invoking autocrine mechanisms for the production of neurotrophic factors (Acheson et al., 1995). An
interesting possibility that cannot be ruled out is that
injury in other neurons may trigger similar autocrine
loops, but only after being primed by an appropriate
neurotrophic factor.
On balance, these findings suggest the possibility that,
following the administration of neurotrophic factors in
vivo, different mechanisms may act synergistically to
enhance or extend the functional effectiveness of the
trophic factor. It is interesting to consider that such
cooperativity may play an important role in promoting
neuronal survival, but, until the basic signaling pathways
used by neurotrophic factors in neurons in the intact brain
are better understood, it must remain a speculation.
ACKNOWLEDGMENTS
We thank Jennifer Luehring for excellent technical
assistance and support and Regeneron Pharmaceuticals,
Inc., for providing the CNTF used in these experiments.
271
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dorsal, growth, neurotrophic, following, long, administration, axotomized, rat, factors, geniculate, terms, basic, protection, lateral, single, ciliary, neurons, nucleus, fibroblasts
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