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Axonal regeneration is compromised in NFH-LacZ transgenic mice but not in NFH-GFP mice
Neuroscience 228 (2013) 101–108
AXONAL REGENERATION IS COMPROMISED IN NFH-LACZ TRANSGENIC MICE BUT NOT IN NFH-GFP MICE INTRODUCTION
J. CASSEREAU, a G. NICOLAS, a,b,1 P. LONCHAMPT, b M. PINIER, b A. BARTHELAIX, b,c J. EYER b AND F. LETOURNEL b,c*
Injuries to peripheral nerves involve several aetiologies, that are often numerous and complex, ranging from genetic to inflammatory ones, and thus regeneration as well as recovery may be poor (Jivan et al., 2006). Axons have to travel a long distance to reach their target and environment can be unfavourable for regeneration (Eggers et al., 2010). In that perspective, neurotrophic factors (such as nerve growth factor (NGF), glial derived nerve factor (GDNF), brain derived nerve factor (BDNF), neurotrophi 3 (NT3)), cytokines or biomaterials may help axons’ re-growth (Geremia et al., 2010; Girolami et al., 2010; Hu et al., 2010; Siemionow et al., 2010). Monitoring functional restoration is crucial to analyse the different steps of the regeneration process. Besides, improving our knowledge in this field would allow development of innovative treatment strategies. For these purposes animal models, mostly rats or mice, represent valuable tools (Griffin et al., 2010). In particular, transgenic animals expressing fluorescent proteins exhibit several advantages for evaluating the rate of regeneration or the expression of axonally transported proteins (Feng et al., 2000; Letournel et al., 2006). In the peripheral nervous system, axonal injuries can result from transections or crushes. Following transection, regeneration involves first the sprouting of the proximal stump, then the growth through the environment (myelin debris phagocytosis by Schwann cells, expression of cytokine neurotrophic factors, etc.), and finally innervating of the target. On the other hand, following nerve crushes, axons undergo a faster and better regeneration due to the intact basal lamina and the presence of Schwann cells secreting growth factors (Hoke et al., 2006). However, these regenerative processes are often incomplete, the functional recovery is limited, and inappropriate reinnervation often occurs. Regeneration of axons involves molecular and morphological changes allowing the appearance of a growth cone. First, the local cytoskeleton is restructured, and the cytoskeleton polymers are degraded. Second, modification of their synthesis occurs (down or up regulation), that can be monitored using in vivo imaging, as well as synthesis of neurotrophic factors increases (Sahly et al., 2006; Gumy et al., 2010; Sheikh, 2010; Yan et al., 2011). Neurofilaments (NFs) are the major intermediate filaments present in mature neurons. They are obligate heteropolymers and composed of four subunits: neurofilament light (NFL), neurofilament medium (NFM), neurofilament high (NFH) and a-internexin (Yuan et al., 2006; Perrot et al., 2008).
a
LUNAM, Department of Neurology, CHU Angers, 4 rue Larrey, 49033 Angers Cedex 09, France
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LUNAM, UPRES-EA3143, CHU Angers, 4 rue Larrey, 49033 Angers Cedex 09, France
c LUNAM, Neurobiology and Neuropathology Laboratory, 4 rue Larrey, 49033 Angers Cedex 09, France
Abstract—To investigate neurofilament (NF) dynamics during the cytoskeleton reorganization in regenerating axons, and their electrophysiological and histological consequences, we used two transgenic lines of mice: neurofilament high (NFH)-LacZ and NFH-green fluorescent protein (GFP). In NFH-LacZ mice, NFs are retained in cell bodies and deficient in axons (Eyer and Peterson, 1994), while in NFH-GFP mice the fluorescent fusion protein is normally transported along axons (Letournel et al., 2006). Following a crush of the sciatic nerve, conduction recovery in NFHGFP mice is similar to wild-type (wt) mice, but it is reduced in NFH-LacZ mice. Moreover, changes of axonal calibres following regeneration are similar between NFH-GFP and wt mice, but they are systematically reduced in NFH-LacZ mice. Finally, the axonal transport of NFH-GFP fusion protein and NFs is re-initiated after the crush as evidenced by the fluorescent and immunolabelling of axons distal from the crushed point, but NFs and the fusion protein are not transported along axons during regeneration in NFH-LacZ mice. Together, these results argue that the absence of axonal NFs in NFH-LacZ mice compromises the axonal regeneration, and that the NFH-GFP reporter fusion protein represents an efficient model to evaluate the NF dynamics during axonal regeneration. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: neurofilaments, axonal regeneration, electroneuro-myogram, NFH-GFP mice, NFH-LacZ mice, sciatic nerve.
*Correspondence to: F. Letournel, Neurobiology and Neuropathology Laboratory, 4 rue Larrey, 49033 Angers Cedex 09, France. Tel: +33 (0)-241354735; fax: +33 (0)-241354138. E-mail addresses: [email protected] (J. Cassereau), [email protected] (G. Nicolas), [email protected] (P. Lonchampt), [email protected] (M. Pinier), [email protected] (A. Barthelaix), [email protected] (J. Eyer), [email protected] (F. Letournel). 1 Present address: Department of Neurology, Hoˆpital Raymond Poincare´; 104, bd Raymond Poincare´, 92380 GARCHES, France. Abbreviations: ENMG, electroneuromyogram; GFP, green fluorescent protein; NF, neurofilament; NFH, neurofilament high; NFL, neurofilament light; NFM, neurofilament medium; NCV, nerve conduction velocity; SN, sciatic nerve; wt, wild type.
0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.10.011 101
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Radial growth of axons depends mainly on the presence of NFs. Following injuries NFs are locally degraded by a calcium-mediating protease (calpaı¨ n) and their synthesis is down regulated (Spira et al., 2003). In parallel, expression of new tubulin isoforms and an increased axonal transport of microtubules and microfilaments allow formation of the growth cone at the tip of the axon (Sahly et al., 2006). The NF axonal content returns to normal level by day 28 in the L4–L5 dorsal root ganglia (DRG) after mouse sciatic nerve (SN) transections, when assessed by immunohistochemistry (Goldstein et al., 1987). However if an injury persists, as exemplified in human chronic axonal neuropathies, the NF content never returns to normal (Fressinaud et al., 2002). To study the involvement of NFs during regeneration, two transgenic lines of mice were used expressing a fusion protein between the NFH protein and the b-galactosidase reporter for NFH-LacZ mice, or the green fluorescent protein (GFP) for NFH-GFP mice (Eyer and Peterson, 1994; Letournel et al., 2006). Here, we use the NFH-LacZ mice to investigate the possible involvement of NFs in the regeneration process since their axons are deficient for axonal NFs. On the other hand, the NFH-GFP mice were used to analyse in vivo the presence of the GFP during the axonal regeneration. To evaluate the functionality of the injured nerves, we measured electrophysiological, immunohistochemistry and morphological properties of SNs following crushes.
EXPERIMENTAL PROCEDURES
Crush experiments Mice were anesthetized as described above. The area above the right thigh was shaved and asepsis was ensured using Iodin Povidone. One incision (1 cm long) was made in the skin between the gluteus maximus muscle and the biceps femoris muscle. The SN was exposed after tearing apart muscles, without any trauma to muscles or SN. For the crush fine tipped forceps were used. The SN was crushed for 20 s then the forceps were rotated to a final angle of 90° respective to the SN axis and a second crush was done. The nerve was replaced and the incision was sutured. Afterwards, mice were housed in conventional facilities and pain controlled by acetaminophen introduced in the drinking water (50 mg/kg).
Immunohistochemistry All experiments were performed as previously described (Letournel et al., 2006). Briefly, the SNs were examined under a Leica MacroFluo microscope (with IM500 software) immediately following their dissection from NFH-GFP mice, to assess the presence or absence of NFH-GFP fluorescence. Then, SNs were frozen and stored at 80 °C for further analysis. Seven-micrometre cross sections were performed on SNs proximal (5 mm above) and distal (5 mm under) to the crushed point. Sections were incubated overnight with the first antibody (anti-NFH, 1/500, Sigma, France; anti-NFM, 1/500, Sigma, France; anti-NFL, 1/500, Sigma, France; and antibIII-tubulin, 1/500, Sigma, France) and then with the secondary antibody during 1 h 30 (Alexa 488 or Alexa 568; 1/200; Molecular Probes, France). Slides were mounted and examined with a Leica microscope and the IM500 software.
Morphometric analysis
For the purpose of the study, NFH-GFP and NFH-LacZ mice (Eyer and Peterson, 1994; Letournel et al., 2006) in a FVB background were used with their control littermates. All protocols and experiments were approved by the local Ethics Committee on Animal Experimentation.
The proximal and distal stumps were also examined on semi-thin sections. The axonal calibre and myelin thickness were measured on Toluidine Blue stained semi-thin sections of SN as previously described (Letournel et al., 2006; Perrot et al., 2007).
Electrophysiological examination
Data analysis
Nerve conduction studies were performed on 3–4-month-old mice: 15 NFH-GFP mice (eight males and seven females), 10 NFH-LacZ mice (seven males and three females) and 13 wildtype (wt) (eight males and five females) were investigated. Mice were anesthetized with Ketamine (10%) and Xylazine (2%) (10 ll/g). The room was maintained at a constant temperature (22 °C). Body temperature was monitored with a thermistor using an electrode placed at the left hip, and maintained between 36 and 38 °C using a heating lamp and a heating pad. Electrophysiological studies on SN were based on a standard protocol as previously described (Perrot et al., 2007). Briefly, steel needle electrodes were placed subcutaneously. Proximal stimulation was performed under the right iliac spine and distal stimulation was performed at the ankle. Recording electrodes were placed in the right foot pad and on the second toe. Recordings were made with supramaximal stimulation. The distance between stimulation and recording sites was measured using a compass. Electrophysiological measurements on SN were performed in triplicate and results are expressed as the average of the three measurements. Electrophysiological examinations of the injured mice were performed on the crushed SN at day 2, day 7, day 21 and day 28. At each endpoint, three mice were sacrificed and their tissues were collected for further histological analysis.
All data are expressed as mean ± SEM. Statistical significance was evaluated by Mann–Whitney test (p < 0.05) using GraphPad Prism software (v4).
RESULTS Electrophysiological parameters in NFH-GFP mice are similar to wt mice while they are dramatically reduced in NFH-LacZ transgenic mice (Fig. 1A) NFH-GFP transgenic mice. We previously showed that the expression of the NFH-GFP had no major developmental, morphological and histological consequences on transgenic mice (Letournel et al., 2006). To further examine these animals, we undertook an in vivo analysis of peripheral nerve function by evaluating their electrophysiological parameters. For this purpose we measured nerve conduction velocities (NCVs) and compound motor action potential of SNs from wt and NFH-GFP mice between 3 and 4 months, and compared them to NFH-LacZ. This first step was mandatory to study nerve regeneration thereafter. Motor
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NCV values along the SNs were found to be 42.7 m/s (±1.07) in the wt mice, and 40 m/s (±0.78) in the NFHGFP transgenic mice. Statistical comparisons of the two groups did not reveal significant difference (p = 0.08). Moreover, gender comparison between males and females did not reveal a significant difference. NCV in males NFH-GFP was 45.6 m/s (±0.1) and 41.2 m/s (±0.1) for males wt (p = 0.06). NCV in females NFHGFP was 38.5 m/s (±0.1) and 39.7 m/s (±0.1) for females wt (p = 0.26). Further analysis of the average amplitude of the compound motor action potential revealed no statistical difference between the wt and the NFH-GFP groups. During proximal stimulation, the amplitude of the compound motor action potential was 5.9 mV (±2.8) for NFH-GFP mice and 6.5 mV (±3.1) for wt mice (p = 0.38). Distal compound motor action potential was respectively 7.2 mV (±3.4) and 7.4 mV (±4) (p = 0.76) for NFH-GFP and wt mice (Fig. 1A). NFH-LacZ transgenic mice. As previously described (Perrot et al., 2007), NCV (17.8 ± 1.6 m/s) and compound motor action potential amplitudes (2.1 ± 0.6 mV) in NFH-LacZ mice were constantly and dramatically reduced (Fig. 1A). This represents a reduction of 54% compared to wt mice and of 58% compared to NFH-GFP transgenic mice. Our current results are in line with those previous findings, since amplitude in NFH-LacZ is greatly decreased. Taken together, our results show that the expression of NFH-GFP fusion protein in mice does not alter their
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electrophysiological properties. On the other hand, retention of NF in the cell body and their absence in the axonal compartment dramatically alter nerve conduction. Axonal regeneration in NFH-GFP is similar to wt mice while it is affected in NFH-LacZ mice
Electrophysiological study (Fig. 1B). To investigate nerve regeneration, we performed crushes of SN and followed up compound motor action potential amplitudes over 4 weeks. At day 0 and day 2, the compound motor action potential was not detectable when the crushed SN was proximally stimulated, confirming a complete conduction block. Moreover, distal stimulation allows a normal compound motor action potential to be recorded just after the crush site (8 ± 1.7 mV in wt; 5.9 ± 1.2 mV in NFH-GFP mice; 2.1 ± 0.6 mV in NFH-LacZ mice). However, such a stimulation was not possible at day 7 (0.8 ± 0.4 mV in wt; 0.4 ± 0.2 mV in NFH-GFP mice; 0.03 ± 0.03 mV in NFH-LacZ mice), as a consequence of axonal degeneration (Fig. 1B). Distal amplitudes start to increase 7 days after the crush in NFH-GFP and wt mice (1 ± 0.4 mV in wt; 1.1 ± 0.6 mV in NFH-GFP mice) while in NFH-LacZ mice the increase is delayed up to the 15th day (0.04 ± 0.02 mV at day 15 and 0.42 ± 0.13 mV at day 28) and amplitudes stay much lower than NFH-GFP and controls (Fig. 1B). Interestingly electrophysiological parameters never returned to normal values at day 28. At day 28 we
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Fig. 1. Electroneuromyogram findings in wild-type and transgenic mice. (A) Recording of compound motor action potential in uncrushed sciatic nerve from NFH-GFP, NFH-LacZ and wild-type mice. The compound motor action potential is similar between NFH-GFP mice and their wild-type littermates either after proximal or distal recordings. Compound motor action potential is diminished in NFH-LacZ mice but no temporal dispersion is observed, as expected (Perrot et al., 2007). (B) Recording of compound motor action potential distal to the crush point. The occurrence of Wallerian degeneration could explain the decrease of compound motor action potential in the first week. Increase of compound motor action potential reflects progressive axonal regeneration, that is however incomplete. This period is delayed in NFH-LacZ mice. Note the increase found around day 15 in NFH-GFP and wild-type, while no improvement is observed in NFH-LacZ mice.
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Fig. 2. Ultrastructural studies of sciatic nerves. (A) Before crushes, axonal diameter distribution is comparable between NFH-GFP mice and their wild-type littermates. In NFH-LacZ, a reduction of axonal calibre is observed as well as the disappearance of large myelinated axons >8 lm is observed. (B) Typical semi-thin section of sciatic nerves from wild-type mice (B-1) and in NFH-LacZ mice (B-2) showing smaller axons (arrow) and enlarged myelin sheath (arrow head) (final magnification is 630). (C) Twenty-eight days after crushes, a left shift in axonal diameter distribution is observed in NFH-GFP mice and wild-type mice, in contrast to what can be observed in NFH-LacZ mice. (D) Illustration of the previous data is exemplified on these semi-thin sections, 5 mm from the crush point, for wild-type (D-1) and NFH-LacZ mice (D-2) (final magnification is 630).
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calculated that the percentage of recuperation of the compound motor action potential was 53% (wt mice), 46% (NFH-GFP mice) and only 20% for NFH-LacZ mice. These results show that following axonal regeneration the electrophysiological properties are similarly recovered between NFH-GFP and wt mice, but the recovery is much lower in NFH-LacZ mice. This suggests a higher sensibility to injuries of the NF-deficient axons. Morphometric studies (Fig. 2). Before the crush, the size of myelinated calibres in NFH-GFP mice exhibited similar size distribution when compared to control mice, with respectively mean axonal diameters of 4.9 lm (±0.05 lm) and 4.5 lm (±0.05 lm) (Fig. 2A). In contrast and in accordance with previous reports, the size of myelinated axons was significantly smaller in NFH-LacZ mice, with a mean axonal diameter of 2.6 lm (±0.02 lm), p < 0.001 (Perrot et al., 2007). Fig. 2B shows typical semi-thin sections from SNs of control (Fig. 2B-1) and NFH-LacZ (Fig. 2B-2). Axons are smaller (arrow) and myelin sheath is thicker than expected (arrow head). Moreover, there is a loss of large myelinated axons (diameter >8 lm). All these results are in agreement with the lower conduction velocity measured in NFH-LacZ mice. Twenty-eight days after the crush (D28), a decrease of the mean axon diameter occurs both in wt and NFHGFP transgenic mice. A closer look at the size distribution revealed a left shift for NFH-GFP and control mice, indicating an increased number of small axons (respectively 3.6 ± 0.02 lm and 4.2 ± 0.03 lm), typical of axonal regeneration (Fig. 2C). In contrast, NFH-LacZ mice kept the same mean axonal diameter (2.6 ± 0.02 lm). As shown in Fig. 2D, the number of axons at D28 is lower in NFH-LacZ mice (Fig. 2D-1) compared to controls and NFH-GFP mice (Fig. 2D-2). The proportion of large axons (>8 lm) was reduced in NFH-GFP mice and wt mice, representing respectively 4.5% and 1.3% at D28 after the crush, compared to 12% and 11% before the crush. The axonal remyelination was evaluated by measuring myelin thickness and axonal calibre, and thus calculating the g ratio. The average g ratios were similar between NFH-GFP (0.66 ± 0.08) and control mice (0.62 ± 0.09) before the crush and 28 days after the crush (respectively 0.63 ± 0.09 for NFH-GFP and 0.64 ± 0.09 for control samples). The stabilization of the g ratio expresses a proportionally reduced thickness of myelin with a reduced axonal diameter. In NFH-LacZ, myelin thickness in the SN was higher than control mice, before (g = 0.55 ± 0.1) and after crushes at day 28 (g = 0.55 ± 0.1) as already described (Eyer and Peterson, 1994; Perrot et al., 2007). Axonal regeneration can be followed up by detection of NFH-GFP fusion protein (Fig. 3). To further characterize the regeneration, the axonal cytoskeleton was investigated by immunohistochemistry. In parallel, we tested the presence of GFP (Fig. 3). SNs from transgenic mice and wt mice were dissected and analysed at days 2, 7, 15 and 28 following crushes. On
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day 2, the presence of NFH detected by immunohistochemistry is clearly observable in sections proximal to the crush point (Fig. 3A; 1 and 2; asterisk on the right panel), while almost all NFH was undetectable distal from the crush point (Fig. 3A; 3 and 4). Similarly, in the NFH-GFP samples, the GFP signal was high proximal to the crush point whereas the GFP signal was almost undetectable distal to the crush point (Fig. 3A; 1 and 3 lower left panel). This is in sharp contrast with the Thy1-YFP model, another ‘‘crush model’’, where fluorescence is still detectable all along the nerve 7 days after crush (Vyas et al., 2010). Other cytoskeletal proteins were also analysed in parallel to assess the axonal damage and regeneration. NFM and bIII-tubulin were not detected at day 2 in NFH-GFP (Fig. 3A; 5 and 6). The same results were observed for wt mice (Fig. 3A; 7–9). In NFH-LacZ mice NFH and NFM were absent from the axon (Fig. 3A; 10 and 11) as was bIIItubulin (Fig. 3A; 12). The regenerating SN was then tested at day 7, and showed the reappearance of NFH (but also the other cytoskeletal proteins described above) as assessed by the presence of both GFP and the anti-NFH immunohistochemistry signal (data not shown). The same observations were achieved at day 28 (Fig. 3B). On whole-mount SN the fluorescent signal is similar to the one observed in non-crush nerves. However, just proximal to the crush point, a more intense spot is typically observed that could correspond to the accumulation of NFH-GFP (white arrow head on Fig. 3B, middle panel) suggesting an alteration of the axonal transport at this point. NFH content distal to the crush point is comparable between NFH-GFP mice (Fig. 3B; 1 and 2) and wt samples (Fig. 3B-5) but appears to be less intense than before crush (Fig. 3A; 1 and 2). Same results are observed from the evaluation of NFM and bIII-tubulin (Fig. 3B; 3–4–6–7). These data are in accordance with electroneuromyogram (ENMG) findings. Partial recovery of the axonal cytoskeleton proteins content could explain the partial recovery of conduction velocity. In NFH-LacZ mice, the presence of axonal NFH, NFM and bIII-tubulin was hardly detectable, on day 2 (Fig. 3A; 10–12) or day 28 after the crush (Fig. 3B; 8–10). This indicates that NFs stay aggregated in cell bodies even during the regeneration process.
DISCUSSION In this study, we investigated the possible contribution of NFs to an axonal regeneration following an axonal crushes. Crush was selected as an injury model since it does not interrupt axons thus leading to a faster and better regeneration compared to trans-section of SN (Hoke et al., 2006). Herein two transgenic lines of mice exhibiting modification of their NF network were used, the NFHGFP and NFH-LacZ mice, previously described at the histological and biochemical levels (Eyer and Peterson, 1994; Letournel et al., 2006). In the present work we confirmed that the expression of the NFH-GFP protein
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Fig. 3. Immunohistochemical analysis of cytoskeletal proteins present in axons after a sciatic crush. (A) On day 2 after sciatic crush, fluorescent signal has disappeared distal to the crush point (asterisk) in NFH-GFP mice (1–6), wild-type mice (7–9) and NFH-LacZ mice (10–12). (A-1) GFP expression proximal to the crush point. (A-2) Immunodetection of NFH proximal to the crush point, using anti-NFH antibodies. (A-3) GFP disappearance distal to the crush point. (A-4) Detection of NFH distal to the crush point. (A-5) Detection of bIII-tubulin distal to the crush point. (A-6) Detection of NFM distal to the crush point. (A-7) Detection of NFH distal to the crush point in wild-type mice. (A-8) Detection of NFM distal to the crush point. (A-9) Detection of bIII-tubulin distal to the crush point. (A-10) Detection of NFH distal to the crush point, in NFH-LacZ mice. (A-11) Detection of NFM distal to the crush point. (A-12) Detection of bIII-tubulin distal to the crush point. Scale bar = 15 lm. (B) On day 28 after the sciatic crush, fluorescent signal and NFH content distal to the crush point (asterisk) are mostly recovered. However, accumulation of NFH-GFP at the crush point is still present. NFH-GFP mice (1–4), wild-type mice (5–7) and NFH-LacZ mice (8–10). (B-1) GFP expression distal to the crush point. (B-2) Visualization of NFH distal to the crush point. (B-3) Visualization of NFM distal to the crush point. (B-4) Visualization of bIII-tubulin distal to the crush point. (B-5) Visualization of NFH distal to the crush point in wild-type mice. (B-6) Visualization of NFM distal to the crush point. (B-7) Visualization of bIII-tubulin distal to the crush point. (B-8) Visualization of NFH distal to the crush point in NFH-LacZ mice. (B-9) Visualization of NFM distal to the crush point. (B-10) Visualization of bIII-tubulin distal to the crush point. Scale bar = 15 lm.
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does not modify the electrophysiological properties of axons in conducing action potential when compared to wt mice, in a normal situation as well as during axonal regeneration. Thus, a major advantage of the expression of this GFP fusion protein is the accuracy and easiness to observe the axonal regeneration process. This has also been described for other models (Feng et al., 2000). However in these NFH-GFP mice, the fluorescent GFP reporter was fused to a component of the axon. This allows to follow up more precisely the regeneration processes and the time lapse of NF modifications, from their local degradation to their new synthesis and transport. It is particularly interesting to have such an insight into regeneration molecular events, than just studying the modification of expression of a free reporter, driven by a neuronal promoter (Yan et al., 2011). As NFs are expressed late during axon development (Cochard and Paulin, 1984; Chang and Goldman, 2004), the NFH-GFP fusion protein can also be used to investigate late phases of regeneration and might reflect fully recovery axons, at least at the histological level. On day 2 following crush, most of the axonal content for NFs disappeared distal from the crushed point as assessed on whole-mount SN examined at both macroscopic and microscopic levels. This shows a particularly rapid turnover of NFs, as the earlier reappearance of GFP occurs on day 7 after the crush. Interestingly, this parallels the evolution of compound motor action potential amplitudes. However, while fluorescent intensity is close to the level of uninjured nerve, functional and structural analyses are still affected. These data, together with previous reports, suggest that full recovery at the electrophysiological level is delayed or compromised (Xin et al., 1990). Even at day 50, electrophysiological findings showed reduced compound motor amplitude potentials (unpublished data). Therefore, our results confirm that regeneration capacity of axons decreases with time after crush and that it induces permanent lesions even if basal lamina is intact (Eggers et al., 2010). The second model used, NFH-Lac-Z mice, gives insights into the role of NFs when they are deficient from the axon because of the presence of b-galactosidase (Eyer and Peterson, 1994; Eyer et al., 1998; Letournel et al. 2006). Our results are in line with previous work that the absence of NFs in the axonal compartment reduces axon calibres and decreases NCV (Perrot et al., 2007). Interestingly, potential compensatory mechanisms should exist since the myelin sheath is thicker than expected regarding smaller axons observed in NFH-LacZ mice (g ratio <0.6) and the normal distribution of Nav channels along axons (Perrot et al., 2007). ENMG parameters are fundamental to the physiology of the axon: NCVs are proportional to the axon calibre and compound motor action potential correlates with the density of axons in a nerve. Our results show that the absence of NFs in an axon leads to a higher sensitivity of a nerve to a trauma and alters its capacities to regenerate. This might be achieved by a loss of the
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axonal architecture as NFs are involved in the maintenance of the axonal diameter. Indeed the compound motor action potential amplitudes are severely reduced and remain low. The axonal regrowth is compromised in NFH-LacZ mice and is never as efficient as in controls (20% of recovery contrary to 53% in wt mice). These data reinforce the nerve susceptibility theory described by Zochodne et al. (2004), when axons devoid of NFs are submitted to induce diabetes. It is also in accordance with previous results in human beings showing that chronic neuropathies have a sustained expression of NFs even though no or poor clinical recovery is observed (Fressinaud et al., 2002).
CONCLUSION We show in this study that ENMG parameters (NCV and compound motor action potential) of NFH-GFP mice are similar to wt littermates, and thus expression of the fusion protein does not alter the conduction parameters of peripheral nerves of NFH-GFP mice. Moreover, following crushes of the SN, axonal regeneration is similar between wt and NFH-GFP mice, but less efficient in NFH-LacZ mice indicating an altered regenerative process. Taken together, these results show that NFH-GFP mice represent a valuable model to study axonal regeneration by the presence of the GFP and that absence of NFs, in NFH-LacZ mice, reduces nerve conduction and compromises axonal regeneration.
CONFLICT OF INTEREST The authors declare no conflict of interest. Acknowledgements—We gratefully acknowledge R. Amode, J. Antier, L. Denechaud, C. Dumez and I. Viau for their technical assistance. We thank P. Chiron for animal handling.
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Geremia NM, Pettersson LM, Hasmatali JC, Hryciw T, Danielsen N, Schreyer DJ, Verge VM (2010) Endogenous BDNF regulates induction of intrinsic neuronal growth programs in injured sensory neurons. Exp Neurol 223:128–142. Girolami EI, Bouhy D, Haber M, Johnson H, David S (2010) Differential expression and potential role of SOCS1 and SOCS3 in Wallerian degeneration in injured peripheral nerve. Exp Neurol 223:173–182. Goldstein ME, Cooper HS, Bruce J, Carden MJ, Lee VM, Schlaepfer WW (1987) Phosphorylation of neurofilament proteins and chromatolysis following transection of rat sciatic nerve. J Neurosci 7:1586–1594. Griffin JW, Pan B, Polley MA, Hoffman PN, Farah MH (2010) Measuring nerve regeneration in the mouse. Exp Neurol 223:60–71. Gumy LF, Tan CL, Fawcett JW (2010) The role of local protein synthesis and degradation in axon regeneration. Exp Neurol 223:28–37. Hoke A, Redett R, Hameed H, Jari R, Zhou C, Li ZB, Griffin JW, Brushart TM (2006) Schwann cells express motor and sensory phenotypes that regulate axon regeneration. J Neurosci 26:9646–9655. Hu X, Cai J, Yang J, Smith GM (2010) Sensory axon targeting is increased by NGF gene therapy within the lesioned adult femoral nerve. Exp Neurol 223:153–165. Jivan S, Novikova LN, Wiberg M, Novikov LN (2006) The effects of delayed nerve repair on neuronal survival and axonal regeneration after seventh cervical spinal nerve axotomy in adult rats. Exp Brain Res 170:245–254. Letournel F, Bocquet A, Perrot R, Dechaume A, Guinut F, Eyer J, Barthelaix A (2006) Neurofilament high molecular weight-green fluorescent protein fusion is normally expressed in neurons and transported in axons: a neuronal marker to investigate the biology of neurofilaments. Neuroscience 137:103–111.
Perrot R, Lonchampt P, Peterson AC, Eyer J (2007) Axonal neurofilaments control multiple fiber properties but do not influence structure or spacing of nodes of Ranvier. J Neurosci 27:9573–9584. Perrot R, Berges R, Bocquet A, Eyer J (2008) Review of the multiple aspects of neurofilament functions, and their possible contribution to neurodegeneration. Mol Neurobiol 38:27–65. Sahly I, Khoutorsky A, Erez H, Prager-Khoutorsky M, Spira ME (2006) On-line confocal imaging of the events leading to structural dedifferentiation of an axonal segment into a growth cone after axotomy. J Comp Neurol 494:705–720. Sheikh KA (2010) Non-invasive imaging of nerve regeneration. Exp Neurol 223:72–76. Siemionow M, Bozkurt M, Zor F (2010) Regeneration and repair of peripheral nerves with different biomaterials: review. Microsurgery 30:574–588. Spira ME, Oren R, Dormann A, Gitler D (2003) Critical calpaindependent ultrastructural alterations underlie the transformation of an axonal segment into a growth cone after axotomy of cultured Aplysia neurons. J Comp Neurol 457:293–312. Vyas A, Li Z, Aspalter M, Feiner J, Hoke A, Zhou C, O’Daly A, Abdullah M, Rohde C, Brushart TM (2010) An in vitro model of adult mammalian nerve repair. Exp Neurol 223:112–118. Xin L, Richardson PM, Gervais F, Skamene E (1990) A deficiency of axonal regeneration in C57BL/6J mice. Brain Res 510:144–146. Yan Y, Sun HH, Mackinnon SE, Johnson PJ (2011) Evaluation of peripheral nerve regeneration via in vivo serial transcutaneous imaging using transgenic Thy1-YFP mice. Exp Neurol 232:7–14. Yuan A, Rao MV, Sasaki T, Chen Y, Kumar A, Veeranna Liem RK, Eyer J, Peterson AC, Julien JP, Nixon RA (2006) Alpha-internexin is structurally and functionally associated with the neurofilament triplet proteins in the mature CNS. J Neurosci 26:10006–10019. Zochodne DW, Sun HS, Cheng C, Eyer J (2004) Accelerated diabetic neuropathy in axons without neurofilaments. Brain 127:2193–2200.
(Accepted 4 October 2012) (Available online 16 October 2012)
AXONAL REGENERATION IS COMPROMISED IN NFH-LACZ TRANSGENIC MICE BUT NOT IN NFH-GFP MICE INTRODUCTION
J. CASSEREAU, a G. NICOLAS, a,b,1 P. LONCHAMPT, b M. PINIER, b A. BARTHELAIX, b,c J. EYER b AND F. LETOURNEL b,c*
Injuries to peripheral nerves involve several aetiologies, that are often numerous and complex, ranging from genetic to inflammatory ones, and thus regeneration as well as recovery may be poor (Jivan et al., 2006). Axons have to travel a long distance to reach their target and environment can be unfavourable for regeneration (Eggers et al., 2010). In that perspective, neurotrophic factors (such as nerve growth factor (NGF), glial derived nerve factor (GDNF), brain derived nerve factor (BDNF), neurotrophi 3 (NT3)), cytokines or biomaterials may help axons’ re-growth (Geremia et al., 2010; Girolami et al., 2010; Hu et al., 2010; Siemionow et al., 2010). Monitoring functional restoration is crucial to analyse the different steps of the regeneration process. Besides, improving our knowledge in this field would allow development of innovative treatment strategies. For these purposes animal models, mostly rats or mice, represent valuable tools (Griffin et al., 2010). In particular, transgenic animals expressing fluorescent proteins exhibit several advantages for evaluating the rate of regeneration or the expression of axonally transported proteins (Feng et al., 2000; Letournel et al., 2006). In the peripheral nervous system, axonal injuries can result from transections or crushes. Following transection, regeneration involves first the sprouting of the proximal stump, then the growth through the environment (myelin debris phagocytosis by Schwann cells, expression of cytokine neurotrophic factors, etc.), and finally innervating of the target. On the other hand, following nerve crushes, axons undergo a faster and better regeneration due to the intact basal lamina and the presence of Schwann cells secreting growth factors (Hoke et al., 2006). However, these regenerative processes are often incomplete, the functional recovery is limited, and inappropriate reinnervation often occurs. Regeneration of axons involves molecular and morphological changes allowing the appearance of a growth cone. First, the local cytoskeleton is restructured, and the cytoskeleton polymers are degraded. Second, modification of their synthesis occurs (down or up regulation), that can be monitored using in vivo imaging, as well as synthesis of neurotrophic factors increases (Sahly et al., 2006; Gumy et al., 2010; Sheikh, 2010; Yan et al., 2011). Neurofilaments (NFs) are the major intermediate filaments present in mature neurons. They are obligate heteropolymers and composed of four subunits: neurofilament light (NFL), neurofilament medium (NFM), neurofilament high (NFH) and a-internexin (Yuan et al., 2006; Perrot et al., 2008).
a
LUNAM, Department of Neurology, CHU Angers, 4 rue Larrey, 49033 Angers Cedex 09, France
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LUNAM, UPRES-EA3143, CHU Angers, 4 rue Larrey, 49033 Angers Cedex 09, France
c LUNAM, Neurobiology and Neuropathology Laboratory, 4 rue Larrey, 49033 Angers Cedex 09, France
Abstract—To investigate neurofilament (NF) dynamics during the cytoskeleton reorganization in regenerating axons, and their electrophysiological and histological consequences, we used two transgenic lines of mice: neurofilament high (NFH)-LacZ and NFH-green fluorescent protein (GFP). In NFH-LacZ mice, NFs are retained in cell bodies and deficient in axons (Eyer and Peterson, 1994), while in NFH-GFP mice the fluorescent fusion protein is normally transported along axons (Letournel et al., 2006). Following a crush of the sciatic nerve, conduction recovery in NFHGFP mice is similar to wild-type (wt) mice, but it is reduced in NFH-LacZ mice. Moreover, changes of axonal calibres following regeneration are similar between NFH-GFP and wt mice, but they are systematically reduced in NFH-LacZ mice. Finally, the axonal transport of NFH-GFP fusion protein and NFs is re-initiated after the crush as evidenced by the fluorescent and immunolabelling of axons distal from the crushed point, but NFs and the fusion protein are not transported along axons during regeneration in NFH-LacZ mice. Together, these results argue that the absence of axonal NFs in NFH-LacZ mice compromises the axonal regeneration, and that the NFH-GFP reporter fusion protein represents an efficient model to evaluate the NF dynamics during axonal regeneration. Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: neurofilaments, axonal regeneration, electroneuro-myogram, NFH-GFP mice, NFH-LacZ mice, sciatic nerve.
*Correspondence to: F. Letournel, Neurobiology and Neuropathology Laboratory, 4 rue Larrey, 49033 Angers Cedex 09, France. Tel: +33 (0)-241354735; fax: +33 (0)-241354138. E-mail addresses: [email protected] (J. Cassereau), [email protected] (G. Nicolas), [email protected] (P. Lonchampt), [email protected] (M. Pinier), [email protected] (A. Barthelaix), [email protected] (J. Eyer), [email protected] (F. Letournel). 1 Present address: Department of Neurology, Hoˆpital Raymond Poincare´; 104, bd Raymond Poincare´, 92380 GARCHES, France. Abbreviations: ENMG, electroneuromyogram; GFP, green fluorescent protein; NF, neurofilament; NFH, neurofilament high; NFL, neurofilament light; NFM, neurofilament medium; NCV, nerve conduction velocity; SN, sciatic nerve; wt, wild type.
0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuroscience.2012.10.011 101
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Radial growth of axons depends mainly on the presence of NFs. Following injuries NFs are locally degraded by a calcium-mediating protease (calpaı¨ n) and their synthesis is down regulated (Spira et al., 2003). In parallel, expression of new tubulin isoforms and an increased axonal transport of microtubules and microfilaments allow formation of the growth cone at the tip of the axon (Sahly et al., 2006). The NF axonal content returns to normal level by day 28 in the L4–L5 dorsal root ganglia (DRG) after mouse sciatic nerve (SN) transections, when assessed by immunohistochemistry (Goldstein et al., 1987). However if an injury persists, as exemplified in human chronic axonal neuropathies, the NF content never returns to normal (Fressinaud et al., 2002). To study the involvement of NFs during regeneration, two transgenic lines of mice were used expressing a fusion protein between the NFH protein and the b-galactosidase reporter for NFH-LacZ mice, or the green fluorescent protein (GFP) for NFH-GFP mice (Eyer and Peterson, 1994; Letournel et al., 2006). Here, we use the NFH-LacZ mice to investigate the possible involvement of NFs in the regeneration process since their axons are deficient for axonal NFs. On the other hand, the NFH-GFP mice were used to analyse in vivo the presence of the GFP during the axonal regeneration. To evaluate the functionality of the injured nerves, we measured electrophysiological, immunohistochemistry and morphological properties of SNs following crushes.
EXPERIMENTAL PROCEDURES
Crush experiments Mice were anesthetized as described above. The area above the right thigh was shaved and asepsis was ensured using Iodin Povidone. One incision (1 cm long) was made in the skin between the gluteus maximus muscle and the biceps femoris muscle. The SN was exposed after tearing apart muscles, without any trauma to muscles or SN. For the crush fine tipped forceps were used. The SN was crushed for 20 s then the forceps were rotated to a final angle of 90° respective to the SN axis and a second crush was done. The nerve was replaced and the incision was sutured. Afterwards, mice were housed in conventional facilities and pain controlled by acetaminophen introduced in the drinking water (50 mg/kg).
Immunohistochemistry All experiments were performed as previously described (Letournel et al., 2006). Briefly, the SNs were examined under a Leica MacroFluo microscope (with IM500 software) immediately following their dissection from NFH-GFP mice, to assess the presence or absence of NFH-GFP fluorescence. Then, SNs were frozen and stored at 80 °C for further analysis. Seven-micrometre cross sections were performed on SNs proximal (5 mm above) and distal (5 mm under) to the crushed point. Sections were incubated overnight with the first antibody (anti-NFH, 1/500, Sigma, France; anti-NFM, 1/500, Sigma, France; anti-NFL, 1/500, Sigma, France; and antibIII-tubulin, 1/500, Sigma, France) and then with the secondary antibody during 1 h 30 (Alexa 488 or Alexa 568; 1/200; Molecular Probes, France). Slides were mounted and examined with a Leica microscope and the IM500 software.
Morphometric analysis
For the purpose of the study, NFH-GFP and NFH-LacZ mice (Eyer and Peterson, 1994; Letournel et al., 2006) in a FVB background were used with their control littermates. All protocols and experiments were approved by the local Ethics Committee on Animal Experimentation.
The proximal and distal stumps were also examined on semi-thin sections. The axonal calibre and myelin thickness were measured on Toluidine Blue stained semi-thin sections of SN as previously described (Letournel et al., 2006; Perrot et al., 2007).
Electrophysiological examination
Data analysis
Nerve conduction studies were performed on 3–4-month-old mice: 15 NFH-GFP mice (eight males and seven females), 10 NFH-LacZ mice (seven males and three females) and 13 wildtype (wt) (eight males and five females) were investigated. Mice were anesthetized with Ketamine (10%) and Xylazine (2%) (10 ll/g). The room was maintained at a constant temperature (22 °C). Body temperature was monitored with a thermistor using an electrode placed at the left hip, and maintained between 36 and 38 °C using a heating lamp and a heating pad. Electrophysiological studies on SN were based on a standard protocol as previously described (Perrot et al., 2007). Briefly, steel needle electrodes were placed subcutaneously. Proximal stimulation was performed under the right iliac spine and distal stimulation was performed at the ankle. Recording electrodes were placed in the right foot pad and on the second toe. Recordings were made with supramaximal stimulation. The distance between stimulation and recording sites was measured using a compass. Electrophysiological measurements on SN were performed in triplicate and results are expressed as the average of the three measurements. Electrophysiological examinations of the injured mice were performed on the crushed SN at day 2, day 7, day 21 and day 28. At each endpoint, three mice were sacrificed and their tissues were collected for further histological analysis.
All data are expressed as mean ± SEM. Statistical significance was evaluated by Mann–Whitney test (p < 0.05) using GraphPad Prism software (v4).
RESULTS Electrophysiological parameters in NFH-GFP mice are similar to wt mice while they are dramatically reduced in NFH-LacZ transgenic mice (Fig. 1A) NFH-GFP transgenic mice. We previously showed that the expression of the NFH-GFP had no major developmental, morphological and histological consequences on transgenic mice (Letournel et al., 2006). To further examine these animals, we undertook an in vivo analysis of peripheral nerve function by evaluating their electrophysiological parameters. For this purpose we measured nerve conduction velocities (NCVs) and compound motor action potential of SNs from wt and NFH-GFP mice between 3 and 4 months, and compared them to NFH-LacZ. This first step was mandatory to study nerve regeneration thereafter. Motor
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NCV values along the SNs were found to be 42.7 m/s (±1.07) in the wt mice, and 40 m/s (±0.78) in the NFHGFP transgenic mice. Statistical comparisons of the two groups did not reveal significant difference (p = 0.08). Moreover, gender comparison between males and females did not reveal a significant difference. NCV in males NFH-GFP was 45.6 m/s (±0.1) and 41.2 m/s (±0.1) for males wt (p = 0.06). NCV in females NFHGFP was 38.5 m/s (±0.1) and 39.7 m/s (±0.1) for females wt (p = 0.26). Further analysis of the average amplitude of the compound motor action potential revealed no statistical difference between the wt and the NFH-GFP groups. During proximal stimulation, the amplitude of the compound motor action potential was 5.9 mV (±2.8) for NFH-GFP mice and 6.5 mV (±3.1) for wt mice (p = 0.38). Distal compound motor action potential was respectively 7.2 mV (±3.4) and 7.4 mV (±4) (p = 0.76) for NFH-GFP and wt mice (Fig. 1A). NFH-LacZ transgenic mice. As previously described (Perrot et al., 2007), NCV (17.8 ± 1.6 m/s) and compound motor action potential amplitudes (2.1 ± 0.6 mV) in NFH-LacZ mice were constantly and dramatically reduced (Fig. 1A). This represents a reduction of 54% compared to wt mice and of 58% compared to NFH-GFP transgenic mice. Our current results are in line with those previous findings, since amplitude in NFH-LacZ is greatly decreased. Taken together, our results show that the expression of NFH-GFP fusion protein in mice does not alter their
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electrophysiological properties. On the other hand, retention of NF in the cell body and their absence in the axonal compartment dramatically alter nerve conduction. Axonal regeneration in NFH-GFP is similar to wt mice while it is affected in NFH-LacZ mice
Electrophysiological study (Fig. 1B). To investigate nerve regeneration, we performed crushes of SN and followed up compound motor action potential amplitudes over 4 weeks. At day 0 and day 2, the compound motor action potential was not detectable when the crushed SN was proximally stimulated, confirming a complete conduction block. Moreover, distal stimulation allows a normal compound motor action potential to be recorded just after the crush site (8 ± 1.7 mV in wt; 5.9 ± 1.2 mV in NFH-GFP mice; 2.1 ± 0.6 mV in NFH-LacZ mice). However, such a stimulation was not possible at day 7 (0.8 ± 0.4 mV in wt; 0.4 ± 0.2 mV in NFH-GFP mice; 0.03 ± 0.03 mV in NFH-LacZ mice), as a consequence of axonal degeneration (Fig. 1B). Distal amplitudes start to increase 7 days after the crush in NFH-GFP and wt mice (1 ± 0.4 mV in wt; 1.1 ± 0.6 mV in NFH-GFP mice) while in NFH-LacZ mice the increase is delayed up to the 15th day (0.04 ± 0.02 mV at day 15 and 0.42 ± 0.13 mV at day 28) and amplitudes stay much lower than NFH-GFP and controls (Fig. 1B). Interestingly electrophysiological parameters never returned to normal values at day 28. At day 28 we
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Fig. 1. Electroneuromyogram findings in wild-type and transgenic mice. (A) Recording of compound motor action potential in uncrushed sciatic nerve from NFH-GFP, NFH-LacZ and wild-type mice. The compound motor action potential is similar between NFH-GFP mice and their wild-type littermates either after proximal or distal recordings. Compound motor action potential is diminished in NFH-LacZ mice but no temporal dispersion is observed, as expected (Perrot et al., 2007). (B) Recording of compound motor action potential distal to the crush point. The occurrence of Wallerian degeneration could explain the decrease of compound motor action potential in the first week. Increase of compound motor action potential reflects progressive axonal regeneration, that is however incomplete. This period is delayed in NFH-LacZ mice. Note the increase found around day 15 in NFH-GFP and wild-type, while no improvement is observed in NFH-LacZ mice.
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Fig. 2. Ultrastructural studies of sciatic nerves. (A) Before crushes, axonal diameter distribution is comparable between NFH-GFP mice and their wild-type littermates. In NFH-LacZ, a reduction of axonal calibre is observed as well as the disappearance of large myelinated axons >8 lm is observed. (B) Typical semi-thin section of sciatic nerves from wild-type mice (B-1) and in NFH-LacZ mice (B-2) showing smaller axons (arrow) and enlarged myelin sheath (arrow head) (final magnification is 630). (C) Twenty-eight days after crushes, a left shift in axonal diameter distribution is observed in NFH-GFP mice and wild-type mice, in contrast to what can be observed in NFH-LacZ mice. (D) Illustration of the previous data is exemplified on these semi-thin sections, 5 mm from the crush point, for wild-type (D-1) and NFH-LacZ mice (D-2) (final magnification is 630).
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calculated that the percentage of recuperation of the compound motor action potential was 53% (wt mice), 46% (NFH-GFP mice) and only 20% for NFH-LacZ mice. These results show that following axonal regeneration the electrophysiological properties are similarly recovered between NFH-GFP and wt mice, but the recovery is much lower in NFH-LacZ mice. This suggests a higher sensibility to injuries of the NF-deficient axons. Morphometric studies (Fig. 2). Before the crush, the size of myelinated calibres in NFH-GFP mice exhibited similar size distribution when compared to control mice, with respectively mean axonal diameters of 4.9 lm (±0.05 lm) and 4.5 lm (±0.05 lm) (Fig. 2A). In contrast and in accordance with previous reports, the size of myelinated axons was significantly smaller in NFH-LacZ mice, with a mean axonal diameter of 2.6 lm (±0.02 lm), p < 0.001 (Perrot et al., 2007). Fig. 2B shows typical semi-thin sections from SNs of control (Fig. 2B-1) and NFH-LacZ (Fig. 2B-2). Axons are smaller (arrow) and myelin sheath is thicker than expected (arrow head). Moreover, there is a loss of large myelinated axons (diameter >8 lm). All these results are in agreement with the lower conduction velocity measured in NFH-LacZ mice. Twenty-eight days after the crush (D28), a decrease of the mean axon diameter occurs both in wt and NFHGFP transgenic mice. A closer look at the size distribution revealed a left shift for NFH-GFP and control mice, indicating an increased number of small axons (respectively 3.6 ± 0.02 lm and 4.2 ± 0.03 lm), typical of axonal regeneration (Fig. 2C). In contrast, NFH-LacZ mice kept the same mean axonal diameter (2.6 ± 0.02 lm). As shown in Fig. 2D, the number of axons at D28 is lower in NFH-LacZ mice (Fig. 2D-1) compared to controls and NFH-GFP mice (Fig. 2D-2). The proportion of large axons (>8 lm) was reduced in NFH-GFP mice and wt mice, representing respectively 4.5% and 1.3% at D28 after the crush, compared to 12% and 11% before the crush. The axonal remyelination was evaluated by measuring myelin thickness and axonal calibre, and thus calculating the g ratio. The average g ratios were similar between NFH-GFP (0.66 ± 0.08) and control mice (0.62 ± 0.09) before the crush and 28 days after the crush (respectively 0.63 ± 0.09 for NFH-GFP and 0.64 ± 0.09 for control samples). The stabilization of the g ratio expresses a proportionally reduced thickness of myelin with a reduced axonal diameter. In NFH-LacZ, myelin thickness in the SN was higher than control mice, before (g = 0.55 ± 0.1) and after crushes at day 28 (g = 0.55 ± 0.1) as already described (Eyer and Peterson, 1994; Perrot et al., 2007). Axonal regeneration can be followed up by detection of NFH-GFP fusion protein (Fig. 3). To further characterize the regeneration, the axonal cytoskeleton was investigated by immunohistochemistry. In parallel, we tested the presence of GFP (Fig. 3). SNs from transgenic mice and wt mice were dissected and analysed at days 2, 7, 15 and 28 following crushes. On
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day 2, the presence of NFH detected by immunohistochemistry is clearly observable in sections proximal to the crush point (Fig. 3A; 1 and 2; asterisk on the right panel), while almost all NFH was undetectable distal from the crush point (Fig. 3A; 3 and 4). Similarly, in the NFH-GFP samples, the GFP signal was high proximal to the crush point whereas the GFP signal was almost undetectable distal to the crush point (Fig. 3A; 1 and 3 lower left panel). This is in sharp contrast with the Thy1-YFP model, another ‘‘crush model’’, where fluorescence is still detectable all along the nerve 7 days after crush (Vyas et al., 2010). Other cytoskeletal proteins were also analysed in parallel to assess the axonal damage and regeneration. NFM and bIII-tubulin were not detected at day 2 in NFH-GFP (Fig. 3A; 5 and 6). The same results were observed for wt mice (Fig. 3A; 7–9). In NFH-LacZ mice NFH and NFM were absent from the axon (Fig. 3A; 10 and 11) as was bIIItubulin (Fig. 3A; 12). The regenerating SN was then tested at day 7, and showed the reappearance of NFH (but also the other cytoskeletal proteins described above) as assessed by the presence of both GFP and the anti-NFH immunohistochemistry signal (data not shown). The same observations were achieved at day 28 (Fig. 3B). On whole-mount SN the fluorescent signal is similar to the one observed in non-crush nerves. However, just proximal to the crush point, a more intense spot is typically observed that could correspond to the accumulation of NFH-GFP (white arrow head on Fig. 3B, middle panel) suggesting an alteration of the axonal transport at this point. NFH content distal to the crush point is comparable between NFH-GFP mice (Fig. 3B; 1 and 2) and wt samples (Fig. 3B-5) but appears to be less intense than before crush (Fig. 3A; 1 and 2). Same results are observed from the evaluation of NFM and bIII-tubulin (Fig. 3B; 3–4–6–7). These data are in accordance with electroneuromyogram (ENMG) findings. Partial recovery of the axonal cytoskeleton proteins content could explain the partial recovery of conduction velocity. In NFH-LacZ mice, the presence of axonal NFH, NFM and bIII-tubulin was hardly detectable, on day 2 (Fig. 3A; 10–12) or day 28 after the crush (Fig. 3B; 8–10). This indicates that NFs stay aggregated in cell bodies even during the regeneration process.
DISCUSSION In this study, we investigated the possible contribution of NFs to an axonal regeneration following an axonal crushes. Crush was selected as an injury model since it does not interrupt axons thus leading to a faster and better regeneration compared to trans-section of SN (Hoke et al., 2006). Herein two transgenic lines of mice exhibiting modification of their NF network were used, the NFHGFP and NFH-LacZ mice, previously described at the histological and biochemical levels (Eyer and Peterson, 1994; Letournel et al., 2006). In the present work we confirmed that the expression of the NFH-GFP protein
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Control mice
NFH-LacZ mice
Fig. 3. Immunohistochemical analysis of cytoskeletal proteins present in axons after a sciatic crush. (A) On day 2 after sciatic crush, fluorescent signal has disappeared distal to the crush point (asterisk) in NFH-GFP mice (1–6), wild-type mice (7–9) and NFH-LacZ mice (10–12). (A-1) GFP expression proximal to the crush point. (A-2) Immunodetection of NFH proximal to the crush point, using anti-NFH antibodies. (A-3) GFP disappearance distal to the crush point. (A-4) Detection of NFH distal to the crush point. (A-5) Detection of bIII-tubulin distal to the crush point. (A-6) Detection of NFM distal to the crush point. (A-7) Detection of NFH distal to the crush point in wild-type mice. (A-8) Detection of NFM distal to the crush point. (A-9) Detection of bIII-tubulin distal to the crush point. (A-10) Detection of NFH distal to the crush point, in NFH-LacZ mice. (A-11) Detection of NFM distal to the crush point. (A-12) Detection of bIII-tubulin distal to the crush point. Scale bar = 15 lm. (B) On day 28 after the sciatic crush, fluorescent signal and NFH content distal to the crush point (asterisk) are mostly recovered. However, accumulation of NFH-GFP at the crush point is still present. NFH-GFP mice (1–4), wild-type mice (5–7) and NFH-LacZ mice (8–10). (B-1) GFP expression distal to the crush point. (B-2) Visualization of NFH distal to the crush point. (B-3) Visualization of NFM distal to the crush point. (B-4) Visualization of bIII-tubulin distal to the crush point. (B-5) Visualization of NFH distal to the crush point in wild-type mice. (B-6) Visualization of NFM distal to the crush point. (B-7) Visualization of bIII-tubulin distal to the crush point. (B-8) Visualization of NFH distal to the crush point in NFH-LacZ mice. (B-9) Visualization of NFM distal to the crush point. (B-10) Visualization of bIII-tubulin distal to the crush point. Scale bar = 15 lm.
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does not modify the electrophysiological properties of axons in conducing action potential when compared to wt mice, in a normal situation as well as during axonal regeneration. Thus, a major advantage of the expression of this GFP fusion protein is the accuracy and easiness to observe the axonal regeneration process. This has also been described for other models (Feng et al., 2000). However in these NFH-GFP mice, the fluorescent GFP reporter was fused to a component of the axon. This allows to follow up more precisely the regeneration processes and the time lapse of NF modifications, from their local degradation to their new synthesis and transport. It is particularly interesting to have such an insight into regeneration molecular events, than just studying the modification of expression of a free reporter, driven by a neuronal promoter (Yan et al., 2011). As NFs are expressed late during axon development (Cochard and Paulin, 1984; Chang and Goldman, 2004), the NFH-GFP fusion protein can also be used to investigate late phases of regeneration and might reflect fully recovery axons, at least at the histological level. On day 2 following crush, most of the axonal content for NFs disappeared distal from the crushed point as assessed on whole-mount SN examined at both macroscopic and microscopic levels. This shows a particularly rapid turnover of NFs, as the earlier reappearance of GFP occurs on day 7 after the crush. Interestingly, this parallels the evolution of compound motor action potential amplitudes. However, while fluorescent intensity is close to the level of uninjured nerve, functional and structural analyses are still affected. These data, together with previous reports, suggest that full recovery at the electrophysiological level is delayed or compromised (Xin et al., 1990). Even at day 50, electrophysiological findings showed reduced compound motor amplitude potentials (unpublished data). Therefore, our results confirm that regeneration capacity of axons decreases with time after crush and that it induces permanent lesions even if basal lamina is intact (Eggers et al., 2010). The second model used, NFH-Lac-Z mice, gives insights into the role of NFs when they are deficient from the axon because of the presence of b-galactosidase (Eyer and Peterson, 1994; Eyer et al., 1998; Letournel et al. 2006). Our results are in line with previous work that the absence of NFs in the axonal compartment reduces axon calibres and decreases NCV (Perrot et al., 2007). Interestingly, potential compensatory mechanisms should exist since the myelin sheath is thicker than expected regarding smaller axons observed in NFH-LacZ mice (g ratio <0.6) and the normal distribution of Nav channels along axons (Perrot et al., 2007). ENMG parameters are fundamental to the physiology of the axon: NCVs are proportional to the axon calibre and compound motor action potential correlates with the density of axons in a nerve. Our results show that the absence of NFs in an axon leads to a higher sensitivity of a nerve to a trauma and alters its capacities to regenerate. This might be achieved by a loss of the
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axonal architecture as NFs are involved in the maintenance of the axonal diameter. Indeed the compound motor action potential amplitudes are severely reduced and remain low. The axonal regrowth is compromised in NFH-LacZ mice and is never as efficient as in controls (20% of recovery contrary to 53% in wt mice). These data reinforce the nerve susceptibility theory described by Zochodne et al. (2004), when axons devoid of NFs are submitted to induce diabetes. It is also in accordance with previous results in human beings showing that chronic neuropathies have a sustained expression of NFs even though no or poor clinical recovery is observed (Fressinaud et al., 2002).
CONCLUSION We show in this study that ENMG parameters (NCV and compound motor action potential) of NFH-GFP mice are similar to wt littermates, and thus expression of the fusion protein does not alter the conduction parameters of peripheral nerves of NFH-GFP mice. Moreover, following crushes of the SN, axonal regeneration is similar between wt and NFH-GFP mice, but less efficient in NFH-LacZ mice indicating an altered regenerative process. Taken together, these results show that NFH-GFP mice represent a valuable model to study axonal regeneration by the presence of the GFP and that absence of NFs, in NFH-LacZ mice, reduces nerve conduction and compromises axonal regeneration.
CONFLICT OF INTEREST The authors declare no conflict of interest. Acknowledgements—We gratefully acknowledge R. Amode, J. Antier, L. Denechaud, C. Dumez and I. Viau for their technical assistance. We thank P. Chiron for animal handling.
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(Accepted 4 October 2012) (Available online 16 October 2012)