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Neurotrophins such as ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) and growth factors such as fibroblast growth factor (FGF-2) play important roles in neuronal survival and in axonal outgrowth during development. However, whether they can modulate regeneration after optic nerve injury in the adult animal is less clear. The present study investigates the effects of application of these neurotrophic factors on the speed, number, and distribution of regenerating axons in the frog Rana pipiens after optic nerve crush. Optic nerves were crushed and the factors, or phosphate-buffered saline, were applied to the stump or intraocularly. The nerves were examined at different times after axotomy, using anterograde labeling with biotin dextran amine and antibody against growth-associated protein 43. We measured the length, number, and distribution of axons projecting beyond the lesion site. Untreated regenerating axons show an increase in elongation rate over 3 weeks. CNTF more than doubles this rate, FGF-2 increases it, and BDNF has little effect. In contrast, the numbers of regenerating axons that have reached 200 μm at 2 weeks were more than doubled by FGF-2, increased by CNTF, and barely affected by BDNF. The regenerating axons were preferentially distributed in the periphery of the nerve; although the numbers of axons were increased by neurotrophic factor application, this overall distribution was substantially unaffected. © 2013 Wiley Periodicals, Inc.
Regeneration of the adult mammalian central nervous system (CNS) is very limited in part because of an unfavorable environment: the presence of inhibitory molecules (Schwab et al., 1985; Fawcett et al., 2012) and physical barriers at the lesion site (debris or astrocytic glial scar; Sandvig et al., 2004; Fawcett et al., 2012). Thus the mammalian optic nerve shows regeneration of its axons only after a peripheral nerve graft onto the cut end (So and Aguayo, 1985; Villegas-Perez et al., 1988). In contrast, lower vertebrates such as amphibians (Sperry, 1944; Scalia et al., 1985) and fish (Wanner et al., 1995; Ankerhold et al., 1998) regenerate successfully, partially because the environment of the CNS, and in particular the optic nerve, does not exhibit the same inhibitory properties. After injury in mammals, optic nerve axons are disconnected from their targets, and the supply of trophic factors is interrupted, resulting in a decline in their levels in the retina and eventual cell death (Barde, 1989; Raff et al., 1993; Mey and Thanos, 1993; Peinado-Ramon et al., 1996; Pettmann and Henderson, 1998; Lebrun-Julien and Di Polo, 2008). Fish retinal ganglion cells (RGCs) do not suffer the same fate, but in frogs there is approximately 50% cell loss (Scalia et al., 1985). We have investigated in previous studies the mechanisms by which the application of growth factors can increase this survival rate in the frog visual system (Blanco et al., 2000, 2008; Ríos-Muñoz et al., 2005). However, because of an the lack of inhibitory environment, this system is also a good model for exploring whether application of growth factors influences optic nerve regeneration.
Neurotrophic factors such as brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF-2), and ciliary neurotrophic factor (CNTF) play important roles in neuron survival and in axonal outgrowth during development and in vitro. However, whether they can modulate regeneration after optic nerve injury in the adult animal is less clear.
In rats, BDNF transiently increases the outgrowth of regenerating axons from early developing RGCs and has a subtle effect on adult RGC explants (Avwenagha et al., 2003). BDNF application to zebrafish RGCs in culture increases axonal outgrowth and growth cone chemoattraction during development (Chen et al., 2013). FGF-2 application stimulates axonal growth during Xenopus visual system development (McFarlane et al., 1995, 1996). We have shown that in adult Rana pipiens FGF-2 increases the levels of the growth-associated protein 43 (GAP-43), a protein upregulated during axonal regeneration (Soto et al., 2003, 2006a). FGF-2 also upregulates the expression of BDNF and its receptor TrkB during axonal regeneration (Soto et al., 2006b; Blanco et al., 2008). For rats with optic nerve injury in vivo, it has been shown that FGF-2 gene delivery via recombinant adeno-associated viruses (AAV) stimulates axonal growth of a small number of axons (Sapieha et al., 2003).
Exogenous application of CNTF in rats induces a temporal enhancement in RGC survival in vivo (Mey and Thanos, 1993) and promotes regeneration of a few axons through peripheral nerve grafts (Cui et al., 1999; Cui and Harvey, 2000). CNTF can induce moderate axonal regeneration in vitro and in vivo but only when the effects of the inhibitory environment are suppressed (Lingor et al., 2008). The objectives of the present study were to take advantage of the permissive environment of the frog optic nerve to determine the effects that CNTF, BDNF, and FGF-2 have on the speed and number of regenerating axons after optic nerve crush.
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- MATERIALS AND METHODS
This study establishes a model for optic nerve regeneration in the frog Rana pipiens, first to measure the intrinsic capabilities of regeneration and second to test how axonal extension and the number of regenerating fibers are affected when exogenous neurotrophic factors are applied. This model using experimental nerve crush simulates some aspects of real clinical conditions that damage optic nerve axons, such as traumatic optic neuropathy (Wu et al., 2008), ischemic optic neuropathy (Hayreh, 2009), optic neuritis (Guy, 2008), and glaucoma (Lebrun-Julien and Di Polo, 2008). In this as in previous studies (Ríos-Muñoz et al., 2005; Soto et al., 2006b), we have used a single application of growth factor to the nerve or eyeball. It is not known how long BDNF, CNTF, and FGF-2 persist at these sites in the frog, but the half-life of CNTF in mammalian eyeball has been measured in minutes (Dittrich et al., 1994), although BDNF in rodent brain can have a half-life of 3 hr in embryos (Fukumitsu et al., 2006) or last for up to 4 days in adults (Nawa et al., 1995). However, it is certain that the prolonged effects of growth factor application that we observe here, and have observed in the past, must far outlast the persistence of the actual factors themselves. Whether methods for sustained delivery will greatly potentiate these effects remains to be investigated.
Elongation of Rana pipiens Axons After Optic Nerve Crush
Lower vertebrates such as frogs and fish possess the ability to regenerate the optic nerve. In the case of fish, all RGCs survive and regenerate their axons after axotomy (Wanner et al., 1995; Ankerhold et al., 1998), but, in the case of Rana pipiens, only 50% of the RGCs survive after axotomy, and these are capable of regenerating and restoring visual function (Sperry, 1944; Blanco et al., 2000). The present study measured the length of the longest 100 axons over different time periods after injury. These measurements suggest that Rana pipiens axons show a small acceleration in their regrowth, increasing in speed from 130 μm/week to 189 μm/week at 3 weeks. These regeneration speeds are consistent with our previous studies, which have shown optic axons innervating the tectum at 6 weeks (Soto et al., 2006a). This seems somewhat slower than the results reported from other studies (Stelzner et al., 1986; Humphrey, 1988); however, in those cases the animals were kept at higher, more variable temperatures, and the axon lengths were not measured directly, as in this study.
BDNF Increases Elongation of Regenerating Axons
Among the three growth factors tested, BDNF has the least effect. It has no significant effect on the numbers of regrowing axons when applied to the nerve, but it elicits a small (20%) increase when applied to the cell bodies. In frogs, few RGCs have died by 2 weeks after axotomy (Blanco et al., 2000), so this axon count is a reasonably accurate measure of regenerating RGCs, with the caveat that regenerating axons can produce collateral branches (Stelzner et al., 1986). In addition, the growth factor increases the rate of elongation of the fastest regrowing axons irrespective of the site of application. There are some reports of BDNF having stimulatory effects on regenerating axon outgrowth in rats, pigs, and even humans (Sawai et al., 1996; Takano et al., 2002; Bonnet et al., 2004); however, in other cases BDNF may promote RGC survival but prevent outgrowth (Pernet and Di Polo, 2006).
FGF-2 Doubles the Number of Regenerating Axons and Increases Their Rate of Elongation
FGF-2 is the most effective of the three neurotrophic factors in increasing the number of regenerating axons, more than doubling them when applied intraocularly and increasing them by 72% when applied to the nerve. It is also effective at increasing the elongation rate of the fastest regrowing axons, although in this case the site of application makes no difference.
FGF-2 has been implicated as a trophic factor that promotes survival and axonal regeneration. FGF-2 application upon nerve crush potentiates the injury-evoked upregulation of growth-associated protein (GAP-43) levels in RGCs (Soto et al., 2003). Also, previous observations have shown that FGF-2 application upregulates mRNA for BDNF and its receptor TrkB after optic nerve injury (Soto et al., 2006b; Blanco et al., 2008). It has long been known that FGF-2 stimulates axonal growth of Xenopus RGCs during development (McFarlane et al., 1995, 1996), but there is less evidence for effects in adult RGCs. However, it has been shown that viral delivery of FGF-2 to RGCs in rats with optic nerve injury stimulates axonal elongation, but only in a few of the surviving neurons (Sapieha et al., 2003).
CNTF Doubles the Speed of Regeneration
CNTF, applied either to the cut axons or to the cell bodies, elicits a very potent effect on axonal elongation, more than doubling the speed of regeneration of the fastest-growing axons. CNTF was also quite effective in increasing the numbers of regrowing axons, but more so when applied intraocularly. In adult mice, two intraocular injections of CNTF greatly increase the numbers of RGC axons that have reached the end of a peripheral nerve graft 3 weeks after nerve crush (Cui and Harvey, 2000), and a continuous supply via adenoviral transfection of the neurons themselves, or of neighboring Müller glia, is even more effective (Leaver et al., 2006; Pernet et al., 2013). However, in these studies, the numbers of axons at different distances were counted, making it difficult to distinguish effects on the rate of axonal elongation from increases in the numbers of surviving, regenerating RGCs.
Differential Effects of the Neurotrophic Factors Possibly Indicate the Involvement of Different Signaling Pathways
Concomitant application of blocking antibodies against the appropriate receptors blocks at least 90% of the effects of the neurotrophic factors on both axon elongation and axon numbers. In the case of BDNF, the TrkB antibody blocks completely, whereas the antibodies against CNTFRα and FGR1 are only 90% effective. For FGF-2, this perhaps indicates that the response could also be partially mediated by FGFR3, which is present in the RGCs (Duprey-Díaz et al., 2012); for CNTF, it is not clear what other receptors may be present in this system.
For axonal elongation, the order is CNTF > > FGF-2 > BDNF, whereas, for axon numbers, it is FGF-2 > CNTF > > BDNF. These differential effects of the growth factors on axon elongation and regenerating axon numbers perhaps suggest that different downstream signaling pathways are involved in these different processes. However, our observation that the neurotrophic cocktail is no more effective than CNTF alone in the first case or FGF-2 in the second indicates that there is also some degree of overlap or cross-talk of the signaling pathways, so that a single growth factor can saturate the response.
CNTF is known to activate three signaling pathways in RGCs: the Janus kinase/signal transducer and activators of transcription (JAK/STAT), mitogen-activated protein kinase (MAPK), and phosphatidylinositide 3-kinase (PI3/Akt) signaling pathways (Park et al., 2004; Lingor et al., 2008; Fischer and Leibinger, 2012; Wen et al., 2012). FGF-2 activates MAPK and PKA pathways (Soto et al., 2006b), and BDNF is known to activate MAPK and Akt signaling (Nakazawa et al., 2002; Bonnet et al., 2004). There is a good deal of potential overlap in these pathways, and it is not possible to point out which is clearly responsible for CNTF's strong effect on axonal elongation or FGF-2's predominant effect on axon numbers. It has been suggested that only the JAK/STAT and PI3/Akt, and not the MAPK, pathways are directly involved in stimulating axon growth (Müller et al., 2009), which would account for the strong effect of CNTF that we observe but does not explain the effects of FGF-2. In contrast, the stimulatory effect of virally delivered FGF-2 on rat RGC axon regrowth was shown to be due mostly to MAPK activation (Sapieha et al., 2006). However, BDNF also activates MAPK in RGCs, yet has rather small effects on axon outgrowth unless potentiated by cyclic AMP activation (Hu et al., 2010). Clearly there is a need to investigate in more detail the signaling pathways involved in promoting axon elongation and numbers of regenerating axons.
We conclude that both CNTF and FGF-2 have strong facilitating effects on axon regeneration in the frog optic nerve. Future studies will concentrate on elucidating the intracellular signaling pathways involved.