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Deletion of p75NTR impairs regeneration of peripheral nerves in mice
Life Sciences 84 (2009) 61–68
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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Deletion of p75NTR impairs regeneration of peripheral nerves in mice Xing-Yun Song, Feng-He Zhang, Fiona H. Zhou, Jinhua Zhong, Xin-Fu Zhou ⁎ Department of Physiology and Centre for Neuroscience, Flinders University, GPO Box 2100, Adelaide 5001, Australia
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Article history: Received 19 March 2008 Accepted 29 October 2008 Keywords: Nerve regeneration Sciatic nerve p75NTR Neurotrophins
a b s t r a c t Aims: After peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves. Main methods: In p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry. Key findings: The results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice. Significance: Our data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves. © 2008 Elsevier Inc. All rights reserved.
Background After injury, peripheral nerves often regenerate and make partial functional connections with their innervated targets. In contrast, central nerves in mammals fail to regenerate and lose their functions. The mechanisms underlying the difference in nerve regeneration between peripheral (PNS) and central nervous system (CNS) are not fully known. Severed axons of neurons from CNS can grow into grafted peripheral nerves but cannot re-enter the central nervous tissues. Studies in recent years have shown that multiple permissive (e.g. growth factors or extracellular matrix) and inhibitory (e.g. myelin associated proteins) factors in their immediate environment may determine the fate of injured neurons. p75NTR binds all neurotrophins with a similar affinity and enhances neurotrophin binding to the trk high affinity receptors augmenting the trk signaling (Lee et al., 1994). p75NTR in neurons also acts as the co-receptor for Nogo receptor mediating inhibitory signals and neurite collapse (Wang et al., 2002; Mi et al., 2004). Therefore, p75NTR may become a therapeutic target for the promotion of nerve regeneration. However, its physiological role in the nerve regeneration is not fully understood. We showed that the simple suppression of p75NTR does not promote the regeneration of the spinal cord (Song et al., 2004). P75NTR is required for the Schwann cell myelination and ⁎ Corresponding author. Tel.: +61 882045814; fax: +61 882045768. E-mail address: xin-fu.zhou@flinders.edu.au (X.-F. Zhou). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.10.013
remyelination during development and after peripheral nerve injury (Cosgaya et al., 2002; Song et al., 2006). Nerve injury is a powerful stimulus for the expression of p75NTR in Schwann cells (SC) in PNS (Taniuchi et al., 1986; Johnson et al., 1988) but not in oligodendrocytes in CNS (Gai et al., 1996). This differential regulation of p75NTR is best demonstrated following dorsal root section at the dorsal root entry zone (DREZ) where a clear demarcation of p75NTR expression is seen between SC (dramatic upregulation) and central glia. Up-regulation of p75NTR is maintained until nerve regeneration re-establishes axon-SC contact and remyelination (Johnson et al., 1988). Sensory and motor neurons also express p75NTR during development and in adult. Nerve injury causes down regulation of p75NTR in sensory neurons but up regulation in motor neurons (Zhou et al., 1996). The differential regulation of p75NTR in peripheral nerves suggests that p75NTR is important in regulation of nerve regeneration in the periphery. However, the question of whether p75NTR plays a positive or negative role in the regeneration of motor neurons remains controversial. Using p75NTR knockout mice as a model one report showed an increase in regeneration of spinal motor nerve (Boyd and Gordon, 2001), while the others showed no effect on the regeneration of facial motor nerve in mutant mice (Gschwendtner et al., 2003) or a decreased regeneration and myelination of motor nerves (Tomita et al., 2007). The role of p75NTR in the regeneration of sensory neurons has not been studied. In the present study, we have examined regeneration of both sensory neurons and facial motor neurons in p75NTR mutant mice.
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Materials and methods
to 10 weeks old, and age matched wild type 129sv mice (n = 24) were anaesthetized with a mixture of 2% isofluorane in O2.
Animals Surgeries Animal experiments were performed under the guidelines of the National Health and Medical Research Council of Australia and approved by the Animal Welfare Committee of Flinders University. p75NTR mutant mice in 129sv background (Lee et al., 1992) (n = 24), 8
Sciatic nerve crush and tracing Under the anaesthesia, the sciatic nerves were crushed at the middle thigh on the left side with a pair of forceps for 2 × 30 s at two
Fig. 1. p75NTR−/− retarded the axon elongation in sciatic nerve in mice after injury. One day after sciatic nerve crush, CTB was injected into the sciatic nerve at the point 5 mm distal to the crush site and animals perfused 3 days after crush. Sciatic nerves were dissected and sectioned for immunohistochemistry of CTB. Photos were taken at segments with different distances (mm) distal to the crush site as shown. White arrow heads indicate crush site.
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opposite angles. A successful crush was confirmed by a transparent band observed at the crush site under the operating microscope. To mark the crush site, a piece of 8-0 silk was sutured on the epineural membrane. The wound was sutured and the animal was allowed to survive for different periods of time. Retrograde labelling of regenerating sensory neurons in the DRG Four days after the crush, the sciatic nerve was exposed again, transected 5 mm distal to the crush site and the proximal stump was inserted in the polyethylene tube sealed at the other end containing 1 μl 4% Fast Blue. The tube was left in situ and the wound sutureclosed. The same procedure was also applied to the contralateral sciatic nerve. Seven days after crush injury, the mice were euthanized by transcardial perfusion with 4% paraformaldehyde and bilateral L5 DRG were dissected and sectioned. The total number of Fast Blue neurons in every third sections was counted. The labeling index was calculated using the total number of labeled neurons in the contralateral side as 100%. As the total number of neurons in the DRG of p75NTR−/− mice is reduced, the count of absolute regenerating neurons will give a biased result. Thus, we used the labelling index in relation to the contralateral side to present the data. Anterograde labelling of regenerating axons The next day after sciatic crush, the mice were re-anaesthetized and the sciatic nerves were exposed. With a glass pipette, a bolus of 0.5 μl of 1% choleratoxin B subunit (CTB) was injected to the crushed sciatic nerves at the point 5 mm proximal to the crush site. After injection, the pipette was held in place for 1 min to prevent any lead of the tracer. Then, the wound was closed and animals were allowed to survive for an additional two days. All mice were perfusion-fixed with 4% paraformaldehyde and the sciatic nerves were dissected for immunohistochemistry. For CTB immunohistochemistry, the nerves were sectioned on a cryostat at 10 μm and mounted on a glass slide. Facial nerve crush and retrograde labelling of facial motor neurons For facial nerve crush, a curved incision (1 cm) was made beneath the right external ear canal and the mastoid process. After separation of subcutaneous tissues and mastoid gland, the facial nerve was exposed. The nerve was crushed for 2 × 30 s with opposite angles at the point 2 mm away from the mastoid process. After the crush, the wound was closed with a suture. Four days after the crush, the crushed nerve was exposed again, transected at the bifurcation distal to the crush site and the proximal stump was inserted in a sealed polyethylene tube containing 1 μl of 4% Fast Blue. At the same time, the contralateral facial nerve was transected at the bifurcation and the proximal stump treated with the same procedure. Seven days after the facial nerve crush, the mice were perfused with 4% paraformaldehyde and brain stems dissected. The total number of neurons was counted from every third sections from ipsilateral and contralateral facial nuclei. The ratio of the ipsilateral number to the contralateral number was calculated.
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GAP-43 and CGRP axons, the photos were taken at 2 mm proximal to and 5 mm distal to the crush site, respectively, from nerve sections of each animal (n = 5). All photos were converted to binary images by NIH ImageJ software. The percentage of positive pixels from proximal images of each animal was used as 100% to calculate the relative immunostaining index. This method of estimation will correct possible basal variations in the staining of these neuronal markers in the mutant animal. Statistical analysis All data were presented as s.e.m. and compared by unpaired Student's t test. Results Effects of p75NTR mutant on axonal regrowth in vivo To examine the roles of p75NTR in the regeneration of peripheral nerve, we compared the axonal growth between the mutant mice and wt mice. Axons were labelled by the anterograde tracer CTB injected into the proximal nerve after the nerve crush. The data showed that 3 days after nerve crush, axons of wild type mice extended their neurites within the degenerated nerve at the speed of 2–3 mm a day. The fastest growing axons in wt mice reached 7.5 mm 3 days after crush (Fig. 1). In contrast in the mutant mice, the axons extended at much slower speed at less than 1 mm/day. The fastest growing axons reached only 2.5 mm 3 days after crush (Fig. 1). The number of regenerating axons in the mutant mice was also reduced compared with the wild type mice control (Figs. 1 and 2). Effects of p75NTR mutant on the number of regenerated neurons in DRG Sensory neurons normally express p75NTR. To examine the role of p75NTR on the regeneration of sensory neurons, we examined the sensory nerve regeneration by retrograde Fast Blue labelling in DRG. As the total number of sensory neurons in the p75NTR mutant mice is reduced (Lee et al., 1992), simply comparison of the total number of Fast Blue labelled neurons between mutant and wt mice would not reveal the regeneration capacity. To overcome the problem, we used the contralateral side as a reference control where total number of labelled neurons was
Immunohistochemistry Seven days after sciatic nerve crush, the sciatic nerve was dissected out and sectioned at 10 μm by a cryostat. The sections were washed, blocked and incubated respectively in goat anti-CTB (List Biologicals) at the dilution of 1:3000, mouse antibody to growth associated protein 43 (Sigma, 1:500), rabbit anti-PGP 9.5 (1:5000), and rabbit anti-calcitonin gene related peptide (CGRP) and appropriate FITC-labelled secondary antibodies. After extensive washes, the sections were observed under fluorescence microscope. For quantification of CTB labeled axon, the total number of labelled fibres at different points distal to the crush site was counted on the best labelled section from each animal. For quantification of PGP 9.5,
Fig. 2. Group data showed p75NTR knockout retarded nerve regeneration after sciatic nerve injury. CTB positive axons in the sciatic nerves at the different points distal to the crush site were counted and analysed. Both the number of regenerating axons and length of regenerating axons distal to the crush sites were reduced in the p75NTR mutant mice (n = 6) compared with wild type mice (n = 6).
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used as 100% and the regeneration index on the ipsilateral side was calculated against the contralateral side. As shown in Fig. 3, the number of Fast Blue labeled neurons was reduced in the p75NTR mutant mice compared with the wt mice. Seven days after sciatic nerve crush, the labelling index in the ipsilateral DRG in wt mice was 77.2 ± 5.4%. In contrast, the labelling index in the mutant mice was 57 ± 5.8% and significantly reduced in comparison with that in wt mice (p b 0.01). The data suggest that the regeneration of sensory neurons was reduced 7 days after sciatic nerve crush.
Effects of p75NTR mutant on the regeneration of motor neurons As p75NTR is not normally expressed by adult motor neurons, but up regulated after axotomy, their regeneration should be also affected by p75NTR gene knockout. We test the idea using facial nerve as a model. The experimental design was the same as described for the sciatic nerve. Seven days after facial nerve crush, the number of labelled neurons in the facial nuclei in the mutant mice was significantly reduced compared with wt mice (Fig. 4). The labelling index of the ipsilateral motor neurons in wt mice was 77.8 ± 4.5%. In
Fig. 3. p75NTR knockout reduced the regeneration of sensory neurons in L5 DRG as demonstrated by retrograde labelling. Regenerating sensory neurons were labeled by Fast Blue applied to the transected stumps. A: a section from the ipsilateral L5 DRG from wild type mouse. B: a section from the contralateral L5 DRG of the same mouse as in A. C: a section from the ipsilateral L5 DRG from a p75NTR mutant mouse. D: a section from the contralateral L5 DRG of the same mouse as in C. E: the labeling index was calculated from the total number of labeled neurons from each L5 DRG using the number of contralateral L5 DRG as 100% in each animal and the data were averaged from 5 animals. The labeling index was compared between wild type mice and p75NTR mutant mice. ⁎ P b 0.01, Student's t test.
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Fig. 4. p75NTR knockout retarded the regeneration of facial motor neurons in the facial nuclei in mice as demonstrated by retrograde labelling. Regenerating facial motor neurons were labeled by Fast Blue applied to the transected stumps. A: a section from the ipsilateral facial nucleus from wild type mouse. B: a section from the contralateral facial nucleus of the same mouse as in A. C: a section from the ipsilateral facial nucleus from a p75NTR mutant mouse. D: a section from the contralateral facial nucleus of the same mouse as in C. E: the labeling index was calculated from the total number of labeled neurons from each facial nucleus using the number of the contralateral facial nucleus as 100% in each animal and the data were averaged from 5 animals. The labeling index was compared between wild type mice and p75NTR mutant mice. ⁎ P b 0.01, Student's t test.
contrast, the labelling index of ipsilateral motor neurons in the mutant mice was 53.1 ± 5.2% and significantly reduced (p b 0.05) in comparison with that in wt mice. Immunolabeling of regenerating axons To further assess the nerve regeneration, the nerve sections distal to the crush site were stained for CGRP, GAP-43 and PGP 9.5. The regenerating fibres in the nerve 5 mm distal to the crush site were immunoreactive for these markers in both wt and mutant mice. As shown in Fig. 5, the numbers of fibres immunoreactive for GAP-43, PGP 9.5 and CGRP in the segments distal to the crush site in mutant
mice were reduced compared to the wild type mice. Quantitative data showed that the staining areas for GAP43, PGP 9.5, and CGRP in the distal segment in wild type mice were 65.3 ± 5.2, 71.1 ± 4.9 and 68.5 ± 6.1 of their corresponding proximal segment, respectively. As shown in Table 1, the staining areas in distal segment of the mutant animals for all three markers were significantly reduced (p b 0.01 for all three markers). Discussion In the present study, we have demonstrated with different techniques that p75NTR deletion impairs the regeneration of both
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Fig. 5. p75NTR knockout reduced number of nerve fibres immunoreactive for GAP-43, PGP 9.5 and CGRP. One week after sciatic nerve crush, the immunoreactive nerve fibres were examined in the segment of sciatic nerve 5 mm distal to the crush site. A, C and E are from p75NTR mutant mice and stained for GAP-43, PGP 9.5 and CGRP, respectively. B. D and F are from wild type mice and stained for GAP-43, PGP 9.5 and CGRP, respectively.
sensory and motor neurons in the peripheral nerves. With an anterograde tracing technique using CTB we showed that the growing speed of axons in the p75NTR mutant mice was much slower than that of the wild type mice with the same genetic background. With a retrograde tracing technique, we showed that the number of regenerating sensory neurons in the DRG and motor neurons in the facial nuclei was also significantly reduced in p75NTR mutant mice. The data from labeling of CGRP, PGP 9.5 and GAP-43 positive axons
Table 1 Immunostaining index of GAP-43, PGP 9.5 and CGRP in the distal segment of injured sciatic nerve Markers GAP-43 PGP 9.5 CGRP
Labelling index (% of proximal pixels) Wild type mice
p75NTR−/− mice
65.3 ± 5.2 71.1 ± 4.9 68.5 ± 6.1
32.0 ± 3.4* 38.7 ± 4.5* 35.8 ± 35.8*
The labelling index of the distal segment of injured sciatic nerve was calculated with acquired pixel values from respective proximal segments as 100% from individual animals (n = 5). * P b 0.05 compared with wild type mice.
also suggest the retardation in axonal regeneration of peripheral nerves in p75NTR mutant mice. It is likely that enhanced neuronal death in p75NTR−/− mice may cause the reduction of retrogradely labelling of neurons in DRG and in the facial nuclei. As p75NTR is a death receptor and mediates the death of injured neurons as reported in previous studies (Cheema et al., 1996; Bhakar et al., 2003), it is unlikely that the axotomy of neurons would cause enhanced death of DRG neurons and motor neurons. In the present study, we did not attempt to assess the role of p75NTR in the survival of axotomized neurons, because in our previous studies we found that in the absence of p75NTR, sensory neurons are less susceptible to the insult of axotomy and exogenous BDNF (Zhou et al., 2005). Furthermore, the data from the anterograde tracing study have clearly demonstrated the retardation of axonal elongation in p75NTR−/− mice, supporting the role of p75NTR in regeneration. The reduction of GAP-43, CGRP and PGP 9.5 positive fibres in the distal segment of sciatic nerve sections from p75NTR−/− mice further supports our conclusion. Several previous studies attempted to investigate functional roles of p75NTR in the nerve regeneration but the conclusions were controversial. The controversy may be due to different models and different methods used in the analysis of regeneration. The differential
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expression of p75NTR in neurons and glia may also contribute to the controversy (Tomita et al., 2007). In a tibial and common peronneal nerve injury model, Boyd and Gordon showed that there are fewer surviving intact motor neurons but the relative number of regenerating motoneurons is significantly increased in the p75NTR knockout mice (Boyd and Gordon, 2001). In the crushed facial nerve, no difference in the speed of regenerating motor neurons was found between p75NTR knockout and wild type controls(Gschwendtner et al., 2003). In a different model where dorsal roots were transected, more sprouting of monoaminergic nerves and primary afferents was detected in the dorsal horn of p75NTR knockout mice (Scott et al., 2005). These studies point to the conclusion that the presence of p75NTR in neurons is more likely inhibitory to the axonal growth and regeneration. In contrast to these data, we found that the regeneration speed and number of regenerating neurons were significantly reduced in the models of sciatic nerve injury and facial nerve injury in p75NTR mutant mice. Our data indicate that the presence of p75NTR in the periphery appears critical for the rapid growth of sensory and motor axons in the peripheral nerves. Our data are consistent with a recent study by Tomita et al. who found that the chimeric transplantation of p75NTR−/− Schwann cells in the sciatic nerves to nude mice expressing p75NTR in neurons impaired the motor functions and myelination, indicating p75NTR in Schwann cells is critical for both regeneration and myelination (Tomita et al., 2007). Our data are also consistent with that of the development study where p75NTR mutation results in an impairment of axonal elongation and arborization in the peripheral nerves during development (Bentley and Lee, 2000). How p75NTR in the periphery may promote axonal growth? Neurotrophins and p75NTR are upregulated in Schwann cells following injury (Heumann et al., 1987; Bakhit et al., 1991; Funakoshi et al., 1993). The simplest explanation is that p75NTR in Schwann cells plays a role as a neurotrophin-concentrating substrate critical for building up the neurotrophin gradients as described by Johnson et al. (1988) and revisited recently (Zhou and Li, in press). In a previous study we found that p75NTR up-regulation in satellite cells correlates with abnormal noradrenergic nerve sprouting, which invades DRG, forming basket-like structures as a result of peripheral nerve lesion (Zhou et al., 1999). As p75NTR expression is required for the basket formation (Walsh et al., 1999), it is suggested that p75NTR in glia is involved in axonal sprouting and growth. Indeed, the deletion of p75NTR in Schwann cells in the sciatic nerve retarded both motor functions and myelination and reduced regeneration of motor neurons in the spinal cord after sciatic nerve injury (Tomita et al., 2007). It is likely that the absence of p75NTR in Schwann cell surface would lead to the loss of neurotrophin gradients and retard the regenerative axonal growth or collateral sprouting after nerve injury. The alternative interpretation for our data is that p75NTR in Schwann cells and in the peripheral neurons may undergo the receptor-mediated intramembrane proteolysis (RIP) as described previously (Ahmed et al., 2006a,b). It is reported that RIP in cerebella neurons is required for myelin associated protein-induced neurite collapse (Domeniconi et al., 2005). However, RIP in the peripheral tissues such as DRG cultures and in the transplanted sciatic nerves in the eyes may play a positive role in the axonal growth and disinhibit myelin inhibitors on neurite growth (Ahmed et al., 2006a,b). The RIP product of p75NTR in the extracellular space may antagonize the signaling of myelin inhibitors on regenerating neurons and promote nerve regeneration (Zhou and Li, in press). However, the interpretation on our current data is difficult as p75NTR is not only expressed in Schwann cells and satellite glial cells after injury, which may play positive roles in nerve regeneration, but also it is highly expressed by motor neurons and sensory neurons. Neuronal expression of p75NTR would signal both positive and negative signals. As peripheral nerves also express myelin inhibitory factors (Pot et al., 2002), p75NTR in these neurons would respond to
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these inhibitors via NgR/LINGO-1/p75NTR receptor pathways resulting in neurite collapse. We found axonal regeneration in p75NTR mutant mice is retarded, suggesting that the NgR/LINGO-1/p75NTR signaling in the periphery may not dominate. However, further studies are required to differentiate roles of neuronal p75NTR from those of glial p75NTR in the nerve regeneration and neurite growth using conditional mutant models or using in vitro culture models. Conclusions With anterograde and retrograde tracing techniques and immunohistochemical methods we have demonstrated that the deletion of exon III in the p75NTR gene in mice impaired axonal growth of sensory and motor neurons after a crush injury. It is concluded that p75NTR is required for the axonal regeneration in the peripheral nervous system. Acknowledgements This work was supported by NHMRC project grants (375109, 375110) and visiting scholarship from China to FHZ. 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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e
Deletion of p75NTR impairs regeneration of peripheral nerves in mice Xing-Yun Song, Feng-He Zhang, Fiona H. Zhou, Jinhua Zhong, Xin-Fu Zhou ⁎ Department of Physiology and Centre for Neuroscience, Flinders University, GPO Box 2100, Adelaide 5001, Australia
a r t i c l e
i n f o
Article history: Received 19 March 2008 Accepted 29 October 2008 Keywords: Nerve regeneration Sciatic nerve p75NTR Neurotrophins
a b s t r a c t Aims: After peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves. Main methods: In p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry. Key findings: The results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice. Significance: Our data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves. © 2008 Elsevier Inc. All rights reserved.
Background After injury, peripheral nerves often regenerate and make partial functional connections with their innervated targets. In contrast, central nerves in mammals fail to regenerate and lose their functions. The mechanisms underlying the difference in nerve regeneration between peripheral (PNS) and central nervous system (CNS) are not fully known. Severed axons of neurons from CNS can grow into grafted peripheral nerves but cannot re-enter the central nervous tissues. Studies in recent years have shown that multiple permissive (e.g. growth factors or extracellular matrix) and inhibitory (e.g. myelin associated proteins) factors in their immediate environment may determine the fate of injured neurons. p75NTR binds all neurotrophins with a similar affinity and enhances neurotrophin binding to the trk high affinity receptors augmenting the trk signaling (Lee et al., 1994). p75NTR in neurons also acts as the co-receptor for Nogo receptor mediating inhibitory signals and neurite collapse (Wang et al., 2002; Mi et al., 2004). Therefore, p75NTR may become a therapeutic target for the promotion of nerve regeneration. However, its physiological role in the nerve regeneration is not fully understood. We showed that the simple suppression of p75NTR does not promote the regeneration of the spinal cord (Song et al., 2004). P75NTR is required for the Schwann cell myelination and ⁎ Corresponding author. Tel.: +61 882045814; fax: +61 882045768. E-mail address: xin-fu.zhou@flinders.edu.au (X.-F. Zhou). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.10.013
remyelination during development and after peripheral nerve injury (Cosgaya et al., 2002; Song et al., 2006). Nerve injury is a powerful stimulus for the expression of p75NTR in Schwann cells (SC) in PNS (Taniuchi et al., 1986; Johnson et al., 1988) but not in oligodendrocytes in CNS (Gai et al., 1996). This differential regulation of p75NTR is best demonstrated following dorsal root section at the dorsal root entry zone (DREZ) where a clear demarcation of p75NTR expression is seen between SC (dramatic upregulation) and central glia. Up-regulation of p75NTR is maintained until nerve regeneration re-establishes axon-SC contact and remyelination (Johnson et al., 1988). Sensory and motor neurons also express p75NTR during development and in adult. Nerve injury causes down regulation of p75NTR in sensory neurons but up regulation in motor neurons (Zhou et al., 1996). The differential regulation of p75NTR in peripheral nerves suggests that p75NTR is important in regulation of nerve regeneration in the periphery. However, the question of whether p75NTR plays a positive or negative role in the regeneration of motor neurons remains controversial. Using p75NTR knockout mice as a model one report showed an increase in regeneration of spinal motor nerve (Boyd and Gordon, 2001), while the others showed no effect on the regeneration of facial motor nerve in mutant mice (Gschwendtner et al., 2003) or a decreased regeneration and myelination of motor nerves (Tomita et al., 2007). The role of p75NTR in the regeneration of sensory neurons has not been studied. In the present study, we have examined regeneration of both sensory neurons and facial motor neurons in p75NTR mutant mice.
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Materials and methods
to 10 weeks old, and age matched wild type 129sv mice (n = 24) were anaesthetized with a mixture of 2% isofluorane in O2.
Animals Surgeries Animal experiments were performed under the guidelines of the National Health and Medical Research Council of Australia and approved by the Animal Welfare Committee of Flinders University. p75NTR mutant mice in 129sv background (Lee et al., 1992) (n = 24), 8
Sciatic nerve crush and tracing Under the anaesthesia, the sciatic nerves were crushed at the middle thigh on the left side with a pair of forceps for 2 × 30 s at two
Fig. 1. p75NTR−/− retarded the axon elongation in sciatic nerve in mice after injury. One day after sciatic nerve crush, CTB was injected into the sciatic nerve at the point 5 mm distal to the crush site and animals perfused 3 days after crush. Sciatic nerves were dissected and sectioned for immunohistochemistry of CTB. Photos were taken at segments with different distances (mm) distal to the crush site as shown. White arrow heads indicate crush site.
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opposite angles. A successful crush was confirmed by a transparent band observed at the crush site under the operating microscope. To mark the crush site, a piece of 8-0 silk was sutured on the epineural membrane. The wound was sutured and the animal was allowed to survive for different periods of time. Retrograde labelling of regenerating sensory neurons in the DRG Four days after the crush, the sciatic nerve was exposed again, transected 5 mm distal to the crush site and the proximal stump was inserted in the polyethylene tube sealed at the other end containing 1 μl 4% Fast Blue. The tube was left in situ and the wound sutureclosed. The same procedure was also applied to the contralateral sciatic nerve. Seven days after crush injury, the mice were euthanized by transcardial perfusion with 4% paraformaldehyde and bilateral L5 DRG were dissected and sectioned. The total number of Fast Blue neurons in every third sections was counted. The labeling index was calculated using the total number of labeled neurons in the contralateral side as 100%. As the total number of neurons in the DRG of p75NTR−/− mice is reduced, the count of absolute regenerating neurons will give a biased result. Thus, we used the labelling index in relation to the contralateral side to present the data. Anterograde labelling of regenerating axons The next day after sciatic crush, the mice were re-anaesthetized and the sciatic nerves were exposed. With a glass pipette, a bolus of 0.5 μl of 1% choleratoxin B subunit (CTB) was injected to the crushed sciatic nerves at the point 5 mm proximal to the crush site. After injection, the pipette was held in place for 1 min to prevent any lead of the tracer. Then, the wound was closed and animals were allowed to survive for an additional two days. All mice were perfusion-fixed with 4% paraformaldehyde and the sciatic nerves were dissected for immunohistochemistry. For CTB immunohistochemistry, the nerves were sectioned on a cryostat at 10 μm and mounted on a glass slide. Facial nerve crush and retrograde labelling of facial motor neurons For facial nerve crush, a curved incision (1 cm) was made beneath the right external ear canal and the mastoid process. After separation of subcutaneous tissues and mastoid gland, the facial nerve was exposed. The nerve was crushed for 2 × 30 s with opposite angles at the point 2 mm away from the mastoid process. After the crush, the wound was closed with a suture. Four days after the crush, the crushed nerve was exposed again, transected at the bifurcation distal to the crush site and the proximal stump was inserted in a sealed polyethylene tube containing 1 μl of 4% Fast Blue. At the same time, the contralateral facial nerve was transected at the bifurcation and the proximal stump treated with the same procedure. Seven days after the facial nerve crush, the mice were perfused with 4% paraformaldehyde and brain stems dissected. The total number of neurons was counted from every third sections from ipsilateral and contralateral facial nuclei. The ratio of the ipsilateral number to the contralateral number was calculated.
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GAP-43 and CGRP axons, the photos were taken at 2 mm proximal to and 5 mm distal to the crush site, respectively, from nerve sections of each animal (n = 5). All photos were converted to binary images by NIH ImageJ software. The percentage of positive pixels from proximal images of each animal was used as 100% to calculate the relative immunostaining index. This method of estimation will correct possible basal variations in the staining of these neuronal markers in the mutant animal. Statistical analysis All data were presented as s.e.m. and compared by unpaired Student's t test. Results Effects of p75NTR mutant on axonal regrowth in vivo To examine the roles of p75NTR in the regeneration of peripheral nerve, we compared the axonal growth between the mutant mice and wt mice. Axons were labelled by the anterograde tracer CTB injected into the proximal nerve after the nerve crush. The data showed that 3 days after nerve crush, axons of wild type mice extended their neurites within the degenerated nerve at the speed of 2–3 mm a day. The fastest growing axons in wt mice reached 7.5 mm 3 days after crush (Fig. 1). In contrast in the mutant mice, the axons extended at much slower speed at less than 1 mm/day. The fastest growing axons reached only 2.5 mm 3 days after crush (Fig. 1). The number of regenerating axons in the mutant mice was also reduced compared with the wild type mice control (Figs. 1 and 2). Effects of p75NTR mutant on the number of regenerated neurons in DRG Sensory neurons normally express p75NTR. To examine the role of p75NTR on the regeneration of sensory neurons, we examined the sensory nerve regeneration by retrograde Fast Blue labelling in DRG. As the total number of sensory neurons in the p75NTR mutant mice is reduced (Lee et al., 1992), simply comparison of the total number of Fast Blue labelled neurons between mutant and wt mice would not reveal the regeneration capacity. To overcome the problem, we used the contralateral side as a reference control where total number of labelled neurons was
Immunohistochemistry Seven days after sciatic nerve crush, the sciatic nerve was dissected out and sectioned at 10 μm by a cryostat. The sections were washed, blocked and incubated respectively in goat anti-CTB (List Biologicals) at the dilution of 1:3000, mouse antibody to growth associated protein 43 (Sigma, 1:500), rabbit anti-PGP 9.5 (1:5000), and rabbit anti-calcitonin gene related peptide (CGRP) and appropriate FITC-labelled secondary antibodies. After extensive washes, the sections were observed under fluorescence microscope. For quantification of CTB labeled axon, the total number of labelled fibres at different points distal to the crush site was counted on the best labelled section from each animal. For quantification of PGP 9.5,
Fig. 2. Group data showed p75NTR knockout retarded nerve regeneration after sciatic nerve injury. CTB positive axons in the sciatic nerves at the different points distal to the crush site were counted and analysed. Both the number of regenerating axons and length of regenerating axons distal to the crush sites were reduced in the p75NTR mutant mice (n = 6) compared with wild type mice (n = 6).
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used as 100% and the regeneration index on the ipsilateral side was calculated against the contralateral side. As shown in Fig. 3, the number of Fast Blue labeled neurons was reduced in the p75NTR mutant mice compared with the wt mice. Seven days after sciatic nerve crush, the labelling index in the ipsilateral DRG in wt mice was 77.2 ± 5.4%. In contrast, the labelling index in the mutant mice was 57 ± 5.8% and significantly reduced in comparison with that in wt mice (p b 0.01). The data suggest that the regeneration of sensory neurons was reduced 7 days after sciatic nerve crush.
Effects of p75NTR mutant on the regeneration of motor neurons As p75NTR is not normally expressed by adult motor neurons, but up regulated after axotomy, their regeneration should be also affected by p75NTR gene knockout. We test the idea using facial nerve as a model. The experimental design was the same as described for the sciatic nerve. Seven days after facial nerve crush, the number of labelled neurons in the facial nuclei in the mutant mice was significantly reduced compared with wt mice (Fig. 4). The labelling index of the ipsilateral motor neurons in wt mice was 77.8 ± 4.5%. In
Fig. 3. p75NTR knockout reduced the regeneration of sensory neurons in L5 DRG as demonstrated by retrograde labelling. Regenerating sensory neurons were labeled by Fast Blue applied to the transected stumps. A: a section from the ipsilateral L5 DRG from wild type mouse. B: a section from the contralateral L5 DRG of the same mouse as in A. C: a section from the ipsilateral L5 DRG from a p75NTR mutant mouse. D: a section from the contralateral L5 DRG of the same mouse as in C. E: the labeling index was calculated from the total number of labeled neurons from each L5 DRG using the number of contralateral L5 DRG as 100% in each animal and the data were averaged from 5 animals. The labeling index was compared between wild type mice and p75NTR mutant mice. ⁎ P b 0.01, Student's t test.
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Fig. 4. p75NTR knockout retarded the regeneration of facial motor neurons in the facial nuclei in mice as demonstrated by retrograde labelling. Regenerating facial motor neurons were labeled by Fast Blue applied to the transected stumps. A: a section from the ipsilateral facial nucleus from wild type mouse. B: a section from the contralateral facial nucleus of the same mouse as in A. C: a section from the ipsilateral facial nucleus from a p75NTR mutant mouse. D: a section from the contralateral facial nucleus of the same mouse as in C. E: the labeling index was calculated from the total number of labeled neurons from each facial nucleus using the number of the contralateral facial nucleus as 100% in each animal and the data were averaged from 5 animals. The labeling index was compared between wild type mice and p75NTR mutant mice. ⁎ P b 0.01, Student's t test.
contrast, the labelling index of ipsilateral motor neurons in the mutant mice was 53.1 ± 5.2% and significantly reduced (p b 0.05) in comparison with that in wt mice. Immunolabeling of regenerating axons To further assess the nerve regeneration, the nerve sections distal to the crush site were stained for CGRP, GAP-43 and PGP 9.5. The regenerating fibres in the nerve 5 mm distal to the crush site were immunoreactive for these markers in both wt and mutant mice. As shown in Fig. 5, the numbers of fibres immunoreactive for GAP-43, PGP 9.5 and CGRP in the segments distal to the crush site in mutant
mice were reduced compared to the wild type mice. Quantitative data showed that the staining areas for GAP43, PGP 9.5, and CGRP in the distal segment in wild type mice were 65.3 ± 5.2, 71.1 ± 4.9 and 68.5 ± 6.1 of their corresponding proximal segment, respectively. As shown in Table 1, the staining areas in distal segment of the mutant animals for all three markers were significantly reduced (p b 0.01 for all three markers). Discussion In the present study, we have demonstrated with different techniques that p75NTR deletion impairs the regeneration of both
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Fig. 5. p75NTR knockout reduced number of nerve fibres immunoreactive for GAP-43, PGP 9.5 and CGRP. One week after sciatic nerve crush, the immunoreactive nerve fibres were examined in the segment of sciatic nerve 5 mm distal to the crush site. A, C and E are from p75NTR mutant mice and stained for GAP-43, PGP 9.5 and CGRP, respectively. B. D and F are from wild type mice and stained for GAP-43, PGP 9.5 and CGRP, respectively.
sensory and motor neurons in the peripheral nerves. With an anterograde tracing technique using CTB we showed that the growing speed of axons in the p75NTR mutant mice was much slower than that of the wild type mice with the same genetic background. With a retrograde tracing technique, we showed that the number of regenerating sensory neurons in the DRG and motor neurons in the facial nuclei was also significantly reduced in p75NTR mutant mice. The data from labeling of CGRP, PGP 9.5 and GAP-43 positive axons
Table 1 Immunostaining index of GAP-43, PGP 9.5 and CGRP in the distal segment of injured sciatic nerve Markers GAP-43 PGP 9.5 CGRP
Labelling index (% of proximal pixels) Wild type mice
p75NTR−/− mice
65.3 ± 5.2 71.1 ± 4.9 68.5 ± 6.1
32.0 ± 3.4* 38.7 ± 4.5* 35.8 ± 35.8*
The labelling index of the distal segment of injured sciatic nerve was calculated with acquired pixel values from respective proximal segments as 100% from individual animals (n = 5). * P b 0.05 compared with wild type mice.
also suggest the retardation in axonal regeneration of peripheral nerves in p75NTR mutant mice. It is likely that enhanced neuronal death in p75NTR−/− mice may cause the reduction of retrogradely labelling of neurons in DRG and in the facial nuclei. As p75NTR is a death receptor and mediates the death of injured neurons as reported in previous studies (Cheema et al., 1996; Bhakar et al., 2003), it is unlikely that the axotomy of neurons would cause enhanced death of DRG neurons and motor neurons. In the present study, we did not attempt to assess the role of p75NTR in the survival of axotomized neurons, because in our previous studies we found that in the absence of p75NTR, sensory neurons are less susceptible to the insult of axotomy and exogenous BDNF (Zhou et al., 2005). Furthermore, the data from the anterograde tracing study have clearly demonstrated the retardation of axonal elongation in p75NTR−/− mice, supporting the role of p75NTR in regeneration. The reduction of GAP-43, CGRP and PGP 9.5 positive fibres in the distal segment of sciatic nerve sections from p75NTR−/− mice further supports our conclusion. Several previous studies attempted to investigate functional roles of p75NTR in the nerve regeneration but the conclusions were controversial. The controversy may be due to different models and different methods used in the analysis of regeneration. The differential
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expression of p75NTR in neurons and glia may also contribute to the controversy (Tomita et al., 2007). In a tibial and common peronneal nerve injury model, Boyd and Gordon showed that there are fewer surviving intact motor neurons but the relative number of regenerating motoneurons is significantly increased in the p75NTR knockout mice (Boyd and Gordon, 2001). In the crushed facial nerve, no difference in the speed of regenerating motor neurons was found between p75NTR knockout and wild type controls(Gschwendtner et al., 2003). In a different model where dorsal roots were transected, more sprouting of monoaminergic nerves and primary afferents was detected in the dorsal horn of p75NTR knockout mice (Scott et al., 2005). These studies point to the conclusion that the presence of p75NTR in neurons is more likely inhibitory to the axonal growth and regeneration. In contrast to these data, we found that the regeneration speed and number of regenerating neurons were significantly reduced in the models of sciatic nerve injury and facial nerve injury in p75NTR mutant mice. Our data indicate that the presence of p75NTR in the periphery appears critical for the rapid growth of sensory and motor axons in the peripheral nerves. Our data are consistent with a recent study by Tomita et al. who found that the chimeric transplantation of p75NTR−/− Schwann cells in the sciatic nerves to nude mice expressing p75NTR in neurons impaired the motor functions and myelination, indicating p75NTR in Schwann cells is critical for both regeneration and myelination (Tomita et al., 2007). Our data are also consistent with that of the development study where p75NTR mutation results in an impairment of axonal elongation and arborization in the peripheral nerves during development (Bentley and Lee, 2000). How p75NTR in the periphery may promote axonal growth? Neurotrophins and p75NTR are upregulated in Schwann cells following injury (Heumann et al., 1987; Bakhit et al., 1991; Funakoshi et al., 1993). The simplest explanation is that p75NTR in Schwann cells plays a role as a neurotrophin-concentrating substrate critical for building up the neurotrophin gradients as described by Johnson et al. (1988) and revisited recently (Zhou and Li, in press). In a previous study we found that p75NTR up-regulation in satellite cells correlates with abnormal noradrenergic nerve sprouting, which invades DRG, forming basket-like structures as a result of peripheral nerve lesion (Zhou et al., 1999). As p75NTR expression is required for the basket formation (Walsh et al., 1999), it is suggested that p75NTR in glia is involved in axonal sprouting and growth. Indeed, the deletion of p75NTR in Schwann cells in the sciatic nerve retarded both motor functions and myelination and reduced regeneration of motor neurons in the spinal cord after sciatic nerve injury (Tomita et al., 2007). It is likely that the absence of p75NTR in Schwann cell surface would lead to the loss of neurotrophin gradients and retard the regenerative axonal growth or collateral sprouting after nerve injury. The alternative interpretation for our data is that p75NTR in Schwann cells and in the peripheral neurons may undergo the receptor-mediated intramembrane proteolysis (RIP) as described previously (Ahmed et al., 2006a,b). It is reported that RIP in cerebella neurons is required for myelin associated protein-induced neurite collapse (Domeniconi et al., 2005). However, RIP in the peripheral tissues such as DRG cultures and in the transplanted sciatic nerves in the eyes may play a positive role in the axonal growth and disinhibit myelin inhibitors on neurite growth (Ahmed et al., 2006a,b). The RIP product of p75NTR in the extracellular space may antagonize the signaling of myelin inhibitors on regenerating neurons and promote nerve regeneration (Zhou and Li, in press). However, the interpretation on our current data is difficult as p75NTR is not only expressed in Schwann cells and satellite glial cells after injury, which may play positive roles in nerve regeneration, but also it is highly expressed by motor neurons and sensory neurons. Neuronal expression of p75NTR would signal both positive and negative signals. As peripheral nerves also express myelin inhibitory factors (Pot et al., 2002), p75NTR in these neurons would respond to
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these inhibitors via NgR/LINGO-1/p75NTR receptor pathways resulting in neurite collapse. We found axonal regeneration in p75NTR mutant mice is retarded, suggesting that the NgR/LINGO-1/p75NTR signaling in the periphery may not dominate. However, further studies are required to differentiate roles of neuronal p75NTR from those of glial p75NTR in the nerve regeneration and neurite growth using conditional mutant models or using in vitro culture models. Conclusions With anterograde and retrograde tracing techniques and immunohistochemical methods we have demonstrated that the deletion of exon III in the p75NTR gene in mice impaired axonal growth of sensory and motor neurons after a crush injury. It is concluded that p75NTR is required for the axonal regeneration in the peripheral nervous system. Acknowledgements This work was supported by NHMRC project grants (375109, 375110) and visiting scholarship from China to FHZ. 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