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 factorкод для вставкиСкачать
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: email@example.com 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. 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