Brain Advance Access originally published online on March 11, 2008
Brain 2008 131(6):1492-1505; doi:10.1093/brain/awn039
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Intrinsic neuronal properties control selective targeting of regenerating motoneurons
1Department of Anatomy and Neurobiology, Sir Charles Tupper Building, Dalhousie University, 5850 College St, Halifax, Nova Scotia, Canada B3H 1X5 and 2Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, Program in Cell Biology, Box 290, 1275 York Ave, New York, NY 10021, USA
Correspondence to: Victor F. Rafuse, PhD, Department of Anatomy and Neurobiology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 E-mail: vrafuse{at}dal.ca
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Despite advances in microsurgical techniques, recovery of motor function after peripheral nerve injury is often poor because many regenerating axons reinnervate inappropriate targets. Consequently, surgical repair must include treatment strategies that improve motor axon targeting. Development of such treatments will require a better understanding of the molecular mechanisms governing selective motor axon targeting. This study used a well-established model of nerve transection and repair to examine (1) whether intrinsic differences exist between different pools of motoneurons after peripheral nerve injury, (2) if such differences regulate selective axon targeting, (3) if regenerating motor axons must express polysialic acid (PSA) in order to preferentially reinnervate muscle and (4) whether brief electrical stimulation improves regeneration accuracy because it increases PSA expression on regenerating axons. We found that different motor pools differentially express PSA after injury and that the capacity to re-express PSA appears to be an intrinsic neuronal property established during development. Second, motoneuron pools not up-regulating PSA did not preferentially reinnervate muscle after injury. Third, brief electrical stimulation of the proximal nerve stump immediately after injury only improved selective motor axon targeting if the motoneurons were capable of up-regulating PSA. Finally, the benefits of stimulation were completely abolished if PSA was removed from the regenerating axons. These results indicate that (1) intrinsic neuronal differences between motor pools must be considered in the development of treatments designed to improve axon targeting and (2) therapeutics aimed at increasing PSA levels on regenerating motor axons may lead to superior functional outcomes.
Key Words: axon guidance; sprouting; NCAM; polysialic acid; electrical stimulation
Abbreviations: PSA, polysialic acid; PMR, preferential motor reinnervation; HSD, Honestly significant difference; ENDO-N, Endoneuraminidase-N
Received October 18, 2007. Revised February 15, 2008. Accepted February 20, 2008.
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Introduction |
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Peripheral nerves are capable of robust regeneration following injury. Unfortunately, functional recovery is often poor because many regenerating axons are misdirected to inappropriate end-organs. In the case of motor axons, mistargeting contributes to varying degrees of muscle weakness, movement impairments and physical deformities (Sunderland, 1978
). Normal function is almost never restored even when peripheral nerves are repaired in a timely fashion using advanced microsurgery techniques (Mackinnon and Dellon, 1988). Consequently, surgical repair will have to eventually include strategies that promote better motor axon targeting. In order to develop new treatments the molecular mechanisms governing selective motor axon targeting, in systems where it occurs, must be determined.
The rodent femoral nerve is an invaluable model to explore the mechanisms of axon targeting during regeneration. Originally described in the rat (Brushart, 1988), transected femoral motoneurons preferentially reinnervate the muscle branch of the femoral nerve even though they have equal access to the cutaneous branch. This example of selective motoneuron regeneration is termed preferential motor reinnervation (PMR). Subsequent studies examining the mechanisms underlying PMR have primarily focused on guidance factors expressed by the nerve branches and/or end organs (Brushart, 1993; Martini et al., 1994; Robinson and Madison, 2004, 2005; Hoke et al., 2006; Madison et al., 2007). However, intrinsic properties unique to distinct groups of motoneurons cannot be overlooked as they will likely determine if the regenerating axons respond to the appropriate guidance cues.
One intrinsic property unique to distinct subtypes of developing motoneurons is the expression of polysialic acid (PSA; Landmesser et al., 1988; Allan and Greer, 1998; Soundararajan et al., 2006). Loss of function studies have shown that PSA expression is essential for proper limb muscle innervation in the chick (Tang et al., 1992, 1994). PSA is down-regulated on motoneurons after muscle innervation, but is dramatically up-regulated on many motor axons after nerve injury including those in the femoral nerve (Franz et al., 2005). The functional importance of this re-expression was demonstrated when it was shown that regenerating mouse femoral motor axons must up-regulate PSA in order to exhibit PMR (Franz et al., 2005). Motoneurons not expressing PSA during embryogenesis may not re-express it after nerve injury because many intrinsic neuronal properties expressed in the adult are established during development (Goldberg, 2004). If true, these results would influence our understanding of selective motor axon regeneration and consequently the development of treatment strategies designed to promote better functional recovery after peripheral nerve injury.
Here we show that distinct pools of motoneurons differentially express PSA during development and after nerve injury. Furthermore, we show that motoneurons expressing high PSA levels exhibit PMR, while PSA negative motoneurons do not. Finally, brief electrical stimulation, a process known to enhance PMR of femoral motoneurons (Al-Majed et al., 2000b), increases regeneration accuracy because it up-regulates PSA on regenerating axons. Together, these results indicate that phenotypes established during development can determine whether regenerating motoneurons selectively reinnervate their appropriate target after injury.
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Materials and Methods |
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Embryonic day 10 (E10) C57BL/6 mice (Charles River, Wilmington, MA) were used to study PSA expression patterns during development. Nerve regeneration experiments were performed on four different strains of mice. Wild-type C57BL/6 mice and NCAM null (–/–) mice (Cremer et al., 1994
) were bred and housed at Dalhousie University. Mice that express eGFP under the control of an Hb9 promoter (mHb9-Gfp1b mice; Wichterle et al., 2002) were used to visualize regenerating motor axons. NCAMlox/lox/Hb9cre/+ mice, which lack NCAM and PSA expression in motoneurons, were generated by breeding NCAM-floxed (NCAMlox/lox) mice (Bukalo et al., 2004) with mice expressing cre-recombinase under the control of the Hb9 promoter (Yang et al., 2001).
NCAM –/–, NCAM-floxed (Franz et al., 2005) and Hb9 cre mice (Hess et al., 2007) were genotyped as previously described. All procedures were conducted in accordance with the guidelines of the Canadian Council on Animal Care and the policies of Dalhousie University.
Surgeries
One of the three surgeries was performed on 8–12-week old male mice. (1) The femoral nerve was cut between the divergence points of the iliacus and pectineus nerve branches (Fig. 1A); a site that reproducibly elicits PMR in mice (Franz et al., 2005). The proximal and distal stumps were then realigned with a single 11-0 nylon suture (FST, North Vancouver, Canada). The obturator nerve was cut and ligated as a control for the cross-reinnervation surgeries (see later). (2) The obturator nerve was cut proximal to its branch points and sutured to the distal stump of the femoral nerve, which was cut as described earlier (Fig. 1A and B). The proximal end of the femoral nerve was ligated to prevent regeneration (Fig. 1B). (3) The genitofemoral nerve (Fig. 1A) was cut proximal to its lumbo-iguinal and external spermatic nerve branch points, and then sutured to the distal stump of the femoral nerve as described for the obturator cross reinnervation. The femoral nerve was ligated to prevent regeneration.
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Electrical stimulation
In some experiments, the above surgeries were modified such that the proximal end of the cut nerve was stimulated at 20 Hz for 1 h (100 µs pulse duration; 20–60 µA stimulus current) with a biphasic pulse from a S88 Stimulator (Grass, West Warwick, RI) delivered via an polyethylene suction electrode (PE-190; Clay Adams, Sparks, MD). The current was set at 2x the threshold required to evoke muscle contractions. The nerve was then repaired as described earlier. Non-stimulated controls in this experimental group were prepared in the same manner except the stimuli were not applied.
Endoneuraminidase-N (Endo-N) treatment
The femoral nerve was cut, stimulated and repaired as earlier. In addition, immediately after suturing, 0.5 µl of Endo-N (8.7 U/µl) or saline was injected into the lesion site. Endo-N specifically cleaves 
2,8-linked sialic acid polymers with a minimum chain length of eight (Vimr et al., 1984). A single injection of Endo-N completely removes PSA from regenerating axons during its period of peak expression (
1 week after injury; Franz et al., 2005).
Labelling
After 6 weeks, a second operation was performed to assess the accuracy of motoneuron regeneration. The muscle and cutaneous branches were cut and back-labelled with neurotracers to quantify the number of motoneurons innervating each branch (Fig. 1D) according to the methods described by Franz et al. (2005). The mice were allowed to recover for 5 days following application of the dyes to ensure optimal motoneuron labelling after which the animals were anesthetized perfused with 4% paraformaldehyde (PFA). The spinal cords were dissected free, post fixed overnight, cryoprotected, frozen and cut longitudinally at 60 µm. Labelled cells were counted as described previously (Franz et al., 2005) and raw cell counts were corrected by the Abercrombie method (Abercrombie, 1946).
Immunohistochemistry and optical analysis
PFA-fixed E10 mice embryos were embedded, cryoprotected and sectioned coronally at 40 µm. Sections were incubated overnight at 4°C with the monoclonal anti-PSA IgM antibody 5A5 (1 : 10 000 ascites; Developmental Hybridoma Bank, Iowa) and a monoclonal anti-βIII-tubulin IgG antibody (1 : 1000; MMS-435P; Covance, Berkeley, CA). After rinsing, sections were incubated for 1 h with rhodamine-conjugated goat anti-mouse IgM (1 : 500; M7019; Sigma, Oakville, Canada) and Alexa Fluor 488 goat anti-mouse IgG (1 : 500; A11001; Invitrogen, Burlington, Canada), rinsed, and mounted in glycerol/PBS containing 0.03 mg/ml 
-phenylenediamine.
To examine PSA on regenerating axons we perfused the mice 5 days after nerve transection and removed a 4 mm nerve segment immediately proximal to the lesion site. Nerves were sectioned coronally at 14 µm, incubated for 12 h at RT with 5A5 (1 : 50 000) and anti-βIII-tubulin (1 : 1000), rinsed, incubated for 1 h with rhodamine-conjugated goat anti-mouse IgM antibody (1 : 500; M7019; Sigma) and Alexa Fluor 488 goat anti-mouse IgG (1 : 500), rinsed, and finally mounted as described earlier. Images were acquired with a digital camera (C4742; Hamamatsu, Hamamatsu, Japan) using IPLab acquisition software (BD Biosciences, Rockville, MD). Exposure times and adjustments for the captured images were identical for each nerve analysed.
Fluorescence quantification was used to compare changes in the expression of PSA between experimental conditions as described by Hanson and Landmesser (2004). Optical quantification was chosen because PSA is only expressed on a very discrete region of the small distal nerve (Fig. 3). Second, fluorescent quantification can estimate relative differences in PSA expression at the cellular level (Fu et al., 2006; Gascon et al., 2007), such as at the membrane where PSA is known to reduce cell–cell adhesive activity (Rutishauser, 2008). To compare changes in the expression of PSA, two channels of fluorescence were captured and aligned. Anti-ßIII-tubulin was acquired in the first channel and acted as an internal control to compensate for variability in section thickness, orientation of the nerve, and density of axons within each section. The second channel recorded levels of PSA. Pixel intensity was measured along a linear region of interest that extended across the entire length of each representative nerve section using IPLab (Fig. 4A; white line). The pixel intensity profile on the PSA channel appeared as many sharp peaks and valleys along the linear region of interest (e.g. Fig.4B). This distribution of pixel intensities reflects the fact that PSA-NCAM is abundantly expressed at the cell-surface (Rutishauser, 2008). To compare changes in the expression of PSA at the cell surface the mean intensity of the peaks was divided by the mean intensity of the internal control (i.e. ßIII-tubulin). The average ratio of mean pixel intensity of the cut and cut/stimulated nerve groups was then normalize to the mean pixel intensity of the uncut control nerve within each experimental group. Three nerve sections from 3 different animals were measured for each condition.
Anatomical analysis of axonal sprouts
Regenerating motor axons were quantitatively compared in mHb9-Gfp1b mice that were injected with Endo-N or saline after being either electrically stimulated, or sham stimulated (Modified Surgery #1; see above). Three weeks after injection the femoral nerves were excised and processed as described earlier. In order to enhance the eGFP signal, the nerve sections were incubated with a rabbit GFP antibody (1 : 1000; AB3080P; Chemicon, Temecula, CA) and visualized with a FITC-conjugated goat anti-rabbit IgG secondary antibody (1 : 500; F-0382; Sigma). Nerve sections were digitally photographed and the number of motor axon collateral sprouts distal to the lesion site was analysed with Image J software (Version 1.36b, National Institute of Health, USA). The lengths of lateral eGFP+ axonal deflections crossing the injury site were measured using Axiovision software.
Statistical analysis
Mean values (±standard error of the mean; SEM) are shown throughout. A one-way ANOVA was used to make comparisons between the average peak pixel intensities, number of eGFP positive fibres distal and length of lateral axonal deflections. Tukey's Honestly Significant Difference (HSD) Post Hoc test was used to determine where the significant differences occurred if the F-value exceeded F-critical. Unpaired student's t-tests were used to make comparisons between motoneuron numbers.
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PSA is differentially expressed by subsets of developing motoneurons
While it is well established that PSA is expressed by motoneurons during embryonic development, its level of expression differs between motoneuron pools in the chick (Landmesser et al., 1988
). Whether different groups of mouse motoneurons differentially express PSA during neuromuscular development is not known. To test this possibility, we sectioned E10 mouse hindlimbs at the level of the lumbosacral plexus and double immunolabelled the sections with antibodies directed against neurofilament (Fig. 2A; NF) and PSA (Fig. 2B). Figure 2A shows a cross-section of the developing hindlimb containing three major nerve branches as they exit the developing spinal cord (Fig. 2A; asterisk). The dorsal (Fig. 2A; D) and ventral nerve trunks (Fig. 2A; V) innervate limb muscles while motor axons in the ventral flank nerve (Fig. 2A; VF) innervate muscles in the groin. Interesting, while PSA was expressed on limb innervating nerve trunks, no PSA was detectable on the ventral flank nerve (Fig. 2B; large arrow). This differential pattern of PSA expression is slightly different in the developing chick embryo where axons in the dorsal nerve trunk, which includes femoral motoneurons (Jones, 1979), express significantly more PSA compared to both the ventral nerve trunk and ventral flank nerve of the lumbosacral plexus (Tang et al., 1992).
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PSA is differentially up-regulated on different motoneuron pools after nerve injury
Peripheral neurons up-regulate many developmentally expressed growth associated molecules after injury (Goldberg, 2004
; Bosse et al., 2006; Rossi et al., 2007). Consequently, our present results raised the possibility that PSA is differentially expressed by different pools of regenerating motoneurons. To determine whether this is true, we transected the muscle branch of the femoral nerve (derived from the dorsal nerve trunk during development), the obturator nerve (derived from the ventral nerve trunk) and the genitofemoral nerve (derived from the ventral flank nerve) (Jones, 1979). The mice were killed 5 days later because previous studies have shown that PSA levels peak near the lesion site at this time point (Franz et al., 2005). A 4 mm segment from each nerve, immediately proximal to the lesion, was isolated, cut in cross-section, and processed for PSA and ßIII-tubulin immunohistochemistry. Immediately upon inspection of the immunolabelled sections we noticed that there was a gradation of PSA expression along the length of the transected nerves. This gradient is shown in Fig. 3 where the cartoon illustrates the site of femoral nerve transection and suture, as well as the location of the two representative nerve sections (i and ii, where i is closer to the spinal cord). As observed during development (e.g. Soundararajan et al., 2006), we found that PSA expression was higher on the nerve near the growth cones compared to more proximal sites. These results indicate that PSA is likely required at the level of the distal axon and/or growth cone, but not along the proximal axonal shaft. Furthermore, it highlights the necessity to compare PSA expression in sections taken from the same region of the regenerating nerve. Consequently, the location of each section along the length of the nerve was recorded and sections from the same level were used for comparison.
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To establish baseline levels of PSA expression, a comparable segment from each nerve was dissected from un-operated, age-matched control mice and immunolabelled for PSA. Nerves from each group were stained simultaneously to ensure that variability in incubation times and antibody concentrations were minimized. Figure 3 shows that PSA was expressed at similarly low levels on uncut control femoral (Fig. 3A), obturator (Fig. 3D) and genitofemoral nerves (Fig. 3G). Interestingly, while PSA was up-regulated on the femoral nerve 5 days after nerve transection (Fig. 3B; see also Franz et al., 2005
), its expression was not different from control levels on transected obturator (Fig. 3E) or genitofemoral nerves (Fig. 3H).
To quantitatively compare PSA levels between nerves we used optical measurements of fluorescence intensity (Hanson and Landmesser, 2004; see Methods for details). Fluorescence was quantified by drawing a region of interest across the diameter of the nerve cross-section (Fig. 4A; white line). Because PSA is predominately expressed on the cell membrane (Cremer et al., 1994) the pixel intensity profiles consist of many sharp peaks and valleys (e.g. Fig. 4B). Figure 4C (black bars) shows that the intensity of the peaks of the uncut femoral, obturator and genitofemoral nerves was not significantly different from each other. In addition, fluorescence quantification confirmed that PSA was significantly up-regulated on the femoral, but not obturator or genitofemoral nerves, 5 days after nerve transaction (Fig. 4C; gray bars). These results demonstrate that up-regulation of PSA after axotomy is not an inherent property of all regenerating motor nerves. Instead, re-expression is intrinsic to a subset of motoneurons including those forming the femoral nerve.
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Brief electrical stimulation increases PSA expression in a subset of regenerating motoneurons
PMR is enhanced in rats when the proximal stump of the transected femoral nerve is electrically stimulated for 1 h prior to surgical repair (Al-Majed et al., 2000b
). The molecular mechanisms underlying this improvement involves several factors (Al-Majed et al., 2000a; English et al., 2007; Maehlen and Nja, 1982; Manivannan and Terakawa, 1994; see Discussion), one of which is likely PSA because its expression is increased with activity in excitable cells (Kiss et al., 1994; Rafuse and Landmesser, 1996). To determine whether brief electrical stimulation modulates PSA expression on regenerating motor axons we stimulated the proximal end of the femoral, obturator and genitofemoral nerves immediately after transection (1 h stimulation at 20 Hz). The mice were killed and nerves processed 5 days later. Immunohistochemical analysis showed that the previously stimulated regenerating femoral nerves expressed relatively more PSA (Fig. 3C) compared to unstimulated cut femoral axons (Fig. 3B). PSA expression was also up-regulated on cut obturator nerves after electrical stimulation (Fig. 3F) even though PSA levels did not increase following transection alone (Fig. 3E). Interestingly, the level of PSA on the stimulated cut obturator nerve was very similar to the level expressed by the regenerating femoral nerves that were not stimulated. In contrast to the stimulation-induced increase in PSA levels on femoral and obturator nerves, brief electrical stimulation did not alter the relative levels of PSA expressed by regenerating genitofemoral nerves (Fig. 3I). When peak pixel intensities were quantified from three mice per condition (3 sections/mice) we found the average intensity of the electrically stimulated femoral nerve was significantly higher than all other conditions studied (Fig. 4C; open bar, ##P < 0.01). Interestingly, the relative amount of PSA expressed on the transected femoral nerves (Fig. 4C; grey bar) was not significantly different from the electrically stimulated obturator nerves (Fig. 4C; open bars), however both were statistically higher than the remaining groups including the electrically stimulated genitofemoral nerve (Fig. 4C; open bars, #P < 0.01). Taken together, these results indicate that up-regulation of PSA after nerve injury, or after nerve injury with brief electrical stimulation, is an intrinsic property that is not shared by all motoneuron subtypes.
PMR does not occur when foreign motoneurons cross-reinnervate the femoral nerve
Mouse femoral motor axons must express PSA is order to preferentially reinnervate muscle fibres (i.e. exhibit PMR) after nerve injury when the nerve is transected 3–5 mm proximal to the bifurcation of the cutaneous and muscle nerve branches (Franz et al., 2005). Consequently, it seemed reasonable to hypothesize that cut motoneurons incapable of up-regulating PSA do not exhibit PMR. To test this hypothesis, the obturator or genitofemoral nerves were cut and sutured onto the distal femoral nerve stump (Fig. 1B) and motor axon regeneration accuracy was compared to when the femoral nerve was cut and simply repaired onto itself. The femoral to femoral nerve surgeries were performed as described in Franz et al. (2005) except that the obturator nerve was cut and ligated as a control. In the case of the obturator and genitofemoral nerve cross-reinnervation surgeries the proximal femoral nerve was ligated in order to prevent self-reinnervation of its distal stump. The mice were allowed to recover for 6 weeks before a second operation was performed to apply fluorescent dyes to the muscle and cutaneous nerve branches (Fig. 1D and F). As shown previously (Robinson and Madison, 2003, 2005; Franz et al., 2005), PMR occurred when PSA+ femoral motoneurons reinnervated their own distal nerve stump (Fig. 5A; Standard Repair; P = 0.009; n = 6). In contrast, regenerating PSA-obturator motoneurons only showed a trend for PMR (P = 0.056; n = 6) and the genitofemoral (P = 0.346; n = 8) motoneurons reinnervated the muscle and cutaneous pathways with equal preference (Fig. 5A; Standard Repair). The lower number of reinnervated genitofemoral motoneurons in Fig. 5A reflects the fact that the genitofemoral, obturator and femoral nerves contain 90.5 ± 9.2 (n = 3 animals), 174 ± 9.7 (n = 3) and 173 ± 10.3 motoneurons (Franz et al., 2005), respectively.
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Electrical stimulation improves regeneration accuracy in motoneurons capable of up-regulating PSA
Whether electrical stimulation improves PMR because it increases PSA levels on regenerating motor axons is not known. To address this issue we cut, electrically stimulated and later back-labelled the femoral, obturator or genitofemoral nerves (see Methods for details). Figure 5A shows that electrical stimulation dramatically improved femoral motoneuron regeneration accuracy over non-stimulated femoral nerves (P < 0.003; n = 6). There was no difference in the total number of regenerated motoneurons (P = 0.931), so this stimulation-induced improvement was attributed to significantly more motoneurons reinnervating the muscle pathway (Fig. 5A and B; P = 0.036) and significantly fewer motoneurons reinnervating the cutaneous pathway (Fig. 5A and C; P = 0.013). Because stimulated femoral motor axons express more PSA compared to non-stimulated femoral axons (Figs 3 and 4), these results suggest that the degree of PMR is correlated to the level of PSA expressed by the regenerating nerve.
Obturator motoneurons elicited PMR when electrically stimulated (P < 0.001; n = 7) even though no PMR was evident using standard repair conditions (Fig. 5A). This improvement was primarily due to significantly fewer motoneurons reinnervating the cutaneous pathway after stimulation (P = 0.032; Fig. 5C). The overall number of regenerating motoneurons was not significantly different from non-stimulated obturator nerves (P = 0.816) because there was a small increase in the number of motoneurons reinnervating the motor pathway (Fig. 5A and B). These results indicate that foreign axons are capable of responding to guidance cues that preferentially guide them down the motor pathway when cross-reinnervated onto the distal femoral nerve stump provided they express PSA (Figs 3 and 4).
Genitofemoral motoneurons did not exhibit PMR when electrically stimulated (Fig. 5; P = 0.066; n = 7). Once again, stimulation did not effect the number of regenerating motoneurons (P = 0.812; Fig. 5). Overall, these findings indicate that the extent of PMR is directly correlated with the amount of PSA expressed by the regenerating motoneurons, irrespective of their origin.
Electrical stimulation does not improve regeneration accuracy if PSA is removed from the reinnervating motoneurons
Brief electrical stimulation accelerates and enhances the expression of several regeneration-associated genes (Al-Majed et al., 2000a, 2004). To dissociate the functional roles of PSA from other regeneration-associated genes we transected and stimulated the femoral nerve of NCAM –/– mice, that also lack PSA, and wild-type mice whose femoral nerve was injected with Endo-N to remove PSA at the time of nerve transection. For comparison, the femoral nerve was cut and stimulated in wild-type mice injected with saline. PMR was assessed 6 weeks later as described earlier.
Figure 6 shows PSA immunolabelled cross sections, from the femoral nerve muscle pathway, that were harvested from saline injected wild-type (Fig. 6A), NCAM –/– (Fig. 6B), Endo-N injected (Fig. 6C) and NCAMlox/lox/Hb9cre/+ mice (Fig. 6D; see later) 5 days after nerve transection/stimulation. Not surprisingly, PSA was only expressed by the stimulated/saline injected femoral nerves. Also, as expected, stimulated/saline injected femoral nerves in wild-type mice displayed robust PMR (Fig. 6E; P = 0.01; n = 5). In contrast, PMR was absent when the femoral motoneurons were stimulated in NCAM –/– mice (Fig. 6E; P = 0.155; n = 6) and wild-type mice injected with Endo-N (Fig. 6E; P = 0.603; n = 5). The lack of PMR was likely due to the absence of PSA, rather than NCAM, because regeneration accuracy was equally impaired in the NCAM –/– and Endo-N-treated mice.
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Gordon and colleagues (Al-Majed et al., 2000b
) applied TTX proximal to the transection site while electrically stimulating the cut rat femoral nerve to prevent action potentials from reaching the motoneuron cell body. PMR enhancement was dramatically attenuated in these rats indicating that stimulation mediates its effect via the motoneurons and not the surrounding glia. To confirm that PSA is up-regulated on femoral motoneurons and not the surrounding glia or basal lamina after electrical stimulation, we conditionally ablated NCAM and PSA in motoneurons by breeding NCAM-floxed mice (Bukalo et al., 2004) with mice in which cre-recombinase was expressed under the control of the Hb9 promoter (Yang et al., 2001; Hess et al., 2007; see Methods for details). The very low level of PSA expression in the muscle pathway of the femoral nerve 5 days after nerve transection in NCAMlox/lox/Hb9cre/+ mice indicates that the vast majority of PSA expressed in the cut femoral nerve is associated with regenerating motoneurons (Fig. 6D). Interestingly, the low levels of PSA expression also indicates that muscle afferents, which do not express the Hb9 gene, do not up-regulate PSA after injury. To conclusively determine whether PSA expression by motoneurons regulates PMR we cut, electrically stimulated and repaired the femoral nerve in NCAMlox/lox/Hb9cre/+ mice as described earlier and assessed PMR 6 weeks later. As anticipated, PMR was absent in the reinnervated NCAMlox/lox/Hb9cre/+ mice (Fig. 6E; P = 0.2; n = 6) indicating that increased PSA expression by motoneurons, and not glia, is necessary to enhance PMR after brief electrical stimulation.
Motor axon sprouting is enhanced by electrical stimulation and requires the expression of PSA
Cut motor axons form multiple collateral sprouts as they extend across the injury site (Ramon y Cajal, 1928). The formation of these supernumerary collaterals is believed to increase the probability that at least one sprout will innervate an appropriate basal lamina tube in the distal nerve stump (Brushart, 1988, 1993; Madison et al., 1999). Because regeneration accuracy is compromised with reduced sprouts and fields of arborization (Franz et al., 2005) it seemed reasonable to assume that PMR will be improved if they increase. Unfortunately, it is experimentally difficult to simply increase the number of collaterals sprouts and/or their fields of arborization. However, regenerating sympathetic ganglion cells in vivo (Maehlen and Nja, 1982; Manivannan and Terakawa, 1994) and dorsal root ganglion cells in vitro (Itoh et al., 1995) form supernumerary collateral sprouts when electrically stimulated. Consequently, we used brief electrical stimulation to determine whether regeneration accuracy is improved because it increases both the number of sprouts and fields of arborization. Second, if stimulation increases motor axon sprouting and/or arborization size, we wished to determine whether PSA is involved in these two processes.
The number of collateral sprouts and changes in arborization size were quantified in mHb9-Gfp1b mice because their motoneurons express eGFP (Wichterle et al., 2002; Franz et al., 2005; Wilson et al., 2005). The femoral nerve was transected, injected with either saline or Endo-N, and in some cases electrically stimulated at 20 Hz for 1 h immediately before it was sutured to its distal stump. The nerves were allowed to regenerate for 3 weeks after which time the femoral nerve was removed and sectioned from the surgical repair site to another site 1 mm distally. For comparison, cross-sections were taken from the same level of the femoral nerve in un-operated mHb9-Gfp1b mice. The number of eGFP+ axonal sprouts and their length of lateral deflections were quantified from representative cross-sections from each mouse. Figure 7A shows that the femoral nerve in uncut control mHb9-Gfp1b mice contains two groups of motor axons that are distributed in a very stereotypic manner at the level of the nerve injury (also see; Franz et al., 2005). As reported previously (Franz et al., 2005), there was a significant increase in the number of eGFP+ motor axons distal to the lesion site after nerve transection and standard repair (Fig. 7B, I; Saline). Furthermore, the reinnervated eGFP+ axons were evenly distributed throughout the nerve cross-section (Fig. 7B). As hypothesized, brief electrical stimulation further increased the number of eGFP+ collateral sprouts in the distal nerve stump (Fig. 7C, I; Saline + Stim). This increase in sprouting was dramatically attenuated when Endo-N was injected into the transected nerve when stimulated (Fig. 7D, I; Endo-N + Stim).
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P < 0.01, as compared to all groups. (K–M) Proposed model showing that a single PSA+ femoral motoneuron forms multiple collateral sprouts that explore the muscle (green fascicles) and cutaneous (red fascicles) pathways in the distal stump after standard repair (K). Enhancing PSA expression increases both the number of sprouts and field of arborization (L) while decreasing PSA expression has the opposite effect (M).The sagittal section in Fig. 7E shows the complex axonal trajectories eGFP+ femoral motor axons take as they traverse across the surgical repair site (delineated by the two opaque lines). These elaborate paths establish the field of arborization and appear as lateral axon excursions in cross-sections taken from the site of nerve injury (Fig. 7F, arrow; see also Witzel et al., 2005
). Nerves containing long excursions reflect the presence of motor axons with large fields of arborization (Witzel et al., 2005). Lateral excursion lengths in transected and electrically stimulated femoral nerves in mHb9-Gfp1b mice were quantified and compared with mHb9-Gfp1b mice that had undergone standard repair without electrical stimulation or received electrical stimulation/repair followed by enzymatic removal of PSA. The cross-section in Fig. 7G shows multiple long lateral excursions traversing the repair site 3 weeks after brief electrical stimulation and nerve repair. The mean length of the excursions was significantly greater in stimulated animals (Fig. 7J; Saline + Stim) compared to animals receiving standard nerve repair (Fig. 7J; Saline). In contrast, motor axon collaterals had noticeably shorter lateral excursions when PSA was removed immediately after electrical stimulation (Fig. 7H; Endo-N + Stim). In fact, when quantified, the mean length of the lateral excursions was significantly shorter in the stimulated/Endo-N-injected mice (Fig. 7J; Endo-N + Stim) than the unstimulated/saline-injected animals (Fig. 7J; Saline). Taken together, these results strongly suggest that PMR is enhanced with brief electrical stimulation because it increases the number of collateral sprouts and arborization size and that these increases require up-regulation of PSA over normal expression levels (Figs. 7K–M). |
Discussion |
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Regenerating femoral motoneurons preferentially reinnervate the quadriceps femoris muscle after nerve injury even though they have equal access to the skin (Brushart, 1988
, 1993). Although the molecular mechanisms underlying this example of PMR are complex, it is generally accepted that selective motor axon regeneration is promoted by guidance factors expressed by the regenerating axons and distal targets (Madison et al., 2007). Interestingly, an underlying assumption in PMR studies is that all regenerating motoneurons, regardless of origin, respond to guidance factors in a similar manner. The results in the present study challenge this presumption by showing that motoneurons are only capable of PMR if they increase PSA expression after injury. In addition, brief electrical stimulation enhances PMR because it increases PSA expression on regenerating axons, which in turn promotes more collateral sprouting and larger fields of arborization.
PMR: a universal phenomenon?
PMR of femoral motoneurons has been observed in rats (Brushart, 1988, 1993; Al-Majed et al., 2000a, b), non-human primates (Madison et al., 1999; Krarup et al., 2002) and mice (Robinson and Madison, 2003; Franz et al., 2005; Eberhardt et al., 2006). However, despite the cross-species prevalence of this phenomenon, its occurrence in mice has not been absolute (Mears et al., 2003; Ahlborn et al., 2007). For instance, while Eberhardt et al. (2006) reported PMR in C57BL/6 mice, some of the co-authors later reported a lack of PMR in the same strain of mice (Ahlborn et al., 2007). The underlying reasons for the conflicting results between (and within) labs is not clear, but likely involves several compounding factors. First, the transection site differs between studies. For example, Mears et al. (2003) cut the femoral nerve between the divergence of the iliacus and obturator nerves (T.M. Brushart, personal communication) while we cut the nerve more distally after the divergence of the iliacus. Higher transections result in the regeneration of multiple motoneuron pools, some of which may not preferentially target the quadriceps muscles. If so, PMR of the femoral motoneurons may be masked by those motor pools not exhibiting PMR. This hypothesis is supported by the fact that PMR in the rat is more robust when the transection and repair occurs closer to the bifurcation (Madison et al., 1996). Second, while the proximal and distal nerves stumps were opposed end to end in the present study, Ahlborn et al. (2007) left a 2 mm gap between the stumps and confined them within a polyethylene tube. This surgical strategy may reduce PMR because previous studies in mice have shown that regeneration accuracy is enhanced if the nerve stumps are opposed and repaired with a fibrin sealant (Robinson and Madison, 2003). Finally, while most PMR studies randomly applied retrograde tracers to the muscle and cutaneous branches (e.g. Al-Majed et al., 2000a, b; Robinson and Madsion, 2003; Franz et al., 2005), others did not (Ahlborn et al., 2007). Alternating dyes is required to control for possible differences in retrograde uptake and transport of the dyes that could bias the cell counts. Taken together, future studies using mice genetics to examine the molecular basis for preferential growth of regenerating motoneurons will have to standardize their experimental techniques in order to make justifiable comparison between laboratories.
Molecular differences exists between motor pools
One of the most striking findings in the present study was the observation that obturator and genitofemoral motoneurons do not preferentially reinnervate the quadriceps femoris muscle when cross-reinnervated with the femoral nerve. This lack of selectivity was not simply due to cross-reinnervation per se because cross-reinnervated obturator motoneurons exhibited PMR when electrically stimulated. Because it seems likely that the expression of guidance molecules in the distal targets does not vary between experimental conditions (femoral nerve reinnervation versus cross-reinnervation), these results indicate that neuronal properties that are intrinsic to distinct motor pools dictate whether they elicit PMR.
Molecular distinctions in individual motor pools arise during early spinal cord development (Landmesser, 2001). Differential expression of homeodomain transcription factors (Livet et al., 2002), as well as Hox genes (Song and Pfaff, 2005), are responsible for instructing motor pool development. These genes, in combination with epigenetic factors such as neuronal activity (Hanson and Landmesser, 2004, 2006), regulate the expression of axonal guidance factors that ultimately guide their axons to their appropriate targets. Guidance molecules differentially expressed by distinct groups of developing motoneurons include type II cadherins (Price et al., 2002), EphA4 (Helmbacher et al., 2000) and PSA (Tang et al., 1992; Allan and Greer, 1998). In the developing mouse (Fig. 2), PSA is expressed by dorsal (including femoral neurons) and ventral (including obturator neurons) projecting hindlimb motoneurons, but is nearly absent on ventral flank neurons (including genitofemoral neurons). Whether individual pools of motoneurons retain and/or re-express intrinsic molecular differences during regeneration is poorly understood. Motoneurons innervating the extensor digitorm longus and soleus muscles differentially express tyrosine kinase C (trkC) after axotomy (Simon et al., 2002). However, it is not known whether these two motor pools differentially express trkC during development. Thus, our finding that PSA is differentially re-expressed by regenerating mouse femoral, obturator and genitofemoral motoneurons represents the first example where distinct molecular differences, which were likely established during development, are recapitulated after injury. In other words, the capacity to up-regulate PSA after injury appears to be an intrinsic neuronal property that is determined during embryogenesis. Furthermore, because PSA expression is essential for PMR, these results suggest that neuronal properties that are established during development have a profound influence on the neuron's capacity to respond to instructive guidance cues after peripheral nerve injury.
Potential mechanisms underlying PMR
Potential regulators of PMR include the (1) distal nerve pathways, (2) end organs and (3) motoneurons (reviewed by Madison et al., 2007). Transected femoral motoneurons preferentially reinnervate the muscle pathway in the absence of end organs (muscle and skin) in young rats (Brushart, 1993) indicating that molecular differences must exist between the two pathways at this stage in development. Indeed, HNK-1 is at least one axon guidance molecule that is expressed to a greater extent in the muscle pathway compared to the cutaneous pathway in rats (Martini et al., 1992) and mice (Eberhardt et al., 2006). HNK-1 enhances neurite outgrowth from motor, but not sensory neurons in vitro (Martini et al., 1992) and thus it is a potential guidance molecule regulating PMR. This assumption is supported by the fact that BDNF/trkB-dependent up-regulation of HNK-1 appears to be required for enhanced PMR after brief electrical stimulation (Eberhardt et al., 2006). Pleiotrophin and glial cell-line-derived neurotrophic factor are up-regulated to a greater extent by Schwann cells in denervated ventral roots compared to dorsal roots. Consequently, these neurotrophins could be differentially expressed guidance molecules in the denervated femoral nerve muscle pathway.
Selective recognition of the muscle pathway by regenerating femoral motor axons has recently been challenged as a possible mechanism promoting PMR (reviewed by Madison et al., 2007). In an elegant series of experiments, Robinson, Madison and colleagues (Robinson and Madison, 2004, 2005; Uschold et al., 2007) showed that regenerating femoral motoneurons in adult rats preferentially reinnervate the cutaneous pathway provided the muscle target is removed. Furthermore, they propose that the length of the distal pathway is a greater determinant of PMR than the actual identity of the pathway. Taken together, the authors propose a hierarchy of axon guidance cues with muscle contact being the highest, followed by the length of the nerve pathway and/or contact with the skin (Madison et al., 2007).
Regardless of the source, the capacity to respond to distal guidance cues ultimately rests with the reinnervating motoneurons. This fact is shown in the present study where regenerating obturator and genitofemoral motoneurons failed to preferentially reinnervate the muscle pathway even though they had access to the same guidance molecules as the regenerating femoral neurons. Furthermore, the capacity to respond to guidance molecules was directly related to the motoneuron's capacity to up-regulate PSA because PMR was absent in wild-type mice treated with Endo-N, NCAM –/– mice and NCAMlox/lox/Hb9cre/+ mice. The latter mice were used to definitively show that PSA must be up-regulated on regenerating motor axons and not cells in the distal pathways or end organs.
PSA is required for proper motor axon targeting in the developing chick (Tang et al., 1992, 1994) because it enables individual axons to respond to distant guidance cues by decreasing axon–axon adhesion (Landmesser, 2001). A similar phenomenon may also occur during regeneration whereby PSA permits individual motor axons to respond to distant guidance cues emanating from the distal pathways and/or end organs. In addition, PSA expression promotes the formation of collateral sprouts. The presence of supernumerary collaterals increases the probability that at least one sprout will contact the appropriate muscle pathway (Brushart, 1993; Brushart et al., 1998; Franz et al., 2005; Redett et al., 2005) that will guide it back to its correct distal target (Sanes et al., 1978; Nguyen et al., 2002). Collaterals extending into the cutaneous pathway are selectively pruned provided a single sprout correctly innervates the muscle (Brushart, 1993; Brushart et al., 1998; Franz et al., 2005; Redett et al., 2005).
The exact mechanism by which PSA promotes sprouting is not known. Sprouting is initiated through the stabilization of individual filopodia that normally form and collapse within the growth cone. By attenuating cell–cell adhesion, PSA may allow filopodia to explore the environment for positive signals which, when encountered, results in the formation of a stabilized sprout (El Maarouf and Rutishauser, 2003). PSA expression may also attenuate neuronal contact with growth inhibiting molecules such as myelin associated glycoprotein (MAG). Mice lacking MAG have significantly more axonal sprouts distal to the injury site (Schafer et al., 1996), and daily treatment of regenerating femoral motor axons with antibodies to MAG enhances PMR (Mears et al., 2003). Finally, PSA expression modulates BDNF signalling in neurons (Vutskits et al., 2001, 2003; Gascon et al., 2007). This modulation may explain the correlation between BDNF/trkB signalling and enhanced motor axon regeneration/PMR after electrical stimulation (Eberhardt et al., 2006).
Electrical stimulation, PMR and PSA
Brief electrical stimulation of the proximal femoral nerve stump immediately after nerve transection hastens the onset of PMR and recovery of motor function (Al-Majed et al., 2000a; English et al., 2007). Electrical stimulation itself promotes axon collateral sprouting (Al-Majed et al., 2000b; Eberhardt et al., 2006), increases axon defasciculation (Maehlen and Nja, 1982; Manivannan and Terakawa, 1994), and accelerates axonal growth (Itoh et al., 1995). The effects of electrical stimulation on PMR have been linked to axon regeneration associated molecules including BDNF, neurotrophin-4/5 (NT-4/5), neurotrophin receptor trkB and the HNK-1 carbohydrate (Nix and Hopf, 1983; Pockett and Gavin, 1985; Al-Majed et al., 2000b; Brushart et al., 2002; English, 2005; English et al., 2007; Geremia et al., 2007). The present results extend these findings to show that brief electrical stimulation enhances PMR in motoneuron pools capable of increasing PSA expression. Furthermore, the capacity to up-regulate PSA appears to be limited to motor pools that normally express PSA during neuromuscular development. Finally, improved reinnervation accuracy requires a PSA-dependent increase in the number of collateral sprouts and arborization size.
Taken together, these results indicate that therapeutics aimed at increasing PSA levels on regenerating motor axons could lead to superior functional outcomes following nerve injury. However, clinical treatments must also consider that motor pools have different intrinsic neuronal properties that may limit the use of a global treatment strategy.
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Acknowledgements |
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This work was supported by a grant from the Canadian Institutes for Health Research (V.F.R.). C.K.F. was funded by a Natural Sciences and Engineering Research Council of Canada graduate student scholarship award. The NCAM-floxed mice were kindly provided by Melitta Schachner (Professor of spinal cord research, M. Keck Center for Collaborative Neuroscience, Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ). We like to acknowledge the excellent technical assistance of Alexandra Nelson, Lindsay Fisher, Crystal Milligan and Simone Laforest. The PSA antibody was obtained from the Developmental Studies Hybridoma Bank under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology. Finally, we would like to thank Dr Lynn Landmesser for her helpful comments during the preparation of this manuscript.
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