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Manipulations of the mouse femoral nerve influence the accuracy of pathway reinnervation by motor neurons
Experimental Neurology 192 (2005) 39 – 45 www.elsevier.com/locate/yexnr
Manipulations of the mouse femoral nerve influence the accuracy of pathway reinnervation by motor neurons Grant A. Robinsona, Roger D. Madisona,b,c,* a Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA c Research Service of the Veterans Affairs Medical Center, Building 16, Room 38, 508 Fulton Street, Durham, NC 27705, USA b
Received 27 August 2004; revised 11 October 2004; accepted 20 October 2004 Available online 12 January 2005
Abstract Previous studies using the femoral nerve model in both mice and rats have shown that regenerating motor axons prefer to reinnervate the terminal nerve branch to muscle versus a terminal nerve branch to skin, a process that has been termed preferential motor reinnervation (PMR). If end organ contact with muscle and skin is prevented, this preferential motor reinnervation still occurs in the rat. To better understand the process of preferential motor reinnervation in the mouse, we examined motor neuron reinnervation of muscle and cutaneous pathways without any end organ contact as well as with only cutaneous end organ contact. Surprisingly, there was no preferential motor reinnervation: Motor neurons preferred the cutaneous pathway over the muscle pathway when all end organ contact was prevented and showed an even greater preference for the cutaneous pathway when it was attached to skin. D 2004 Elsevier Inc. All rights reserved. Keywords: Axonal regrowth; Reinnervation accuracy; Preferential motor reinnervation; Regeneration; Femoral nerve
Introduction The peripheral nervous system is capable of robust regeneration after a nerve lesion. However, since functional recovery is primarily dependent upon the accurate regeneration of axons to their original end organs, poor regeneration accuracy often results in poor functional recovery. A better understanding of how regenerating axons select their terminal pathways is of fundamental interest to basic and clinical neuroscience. Preferential motor reinnervation (PMR) refers to the disproportionate reinnervation by regenerating motor axons of motor pathways with muscle contact compared to cutaneous pathways with skin contact (Brushart, 1988).
* Corresponding author. Research Service of the Veterans Affairs Medical Center, Building 16, Room 38, 508 Fulton Street, Durham, NC 27705, USA. Fax: +1 919 286 6811. E-mail address: [email protected] (R.D. Madison). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.10.013
The understanding of PMR comes mainly from rat femoral nerve studies, where motor and sensory axons within the parent nerve become anatomically segregated into a muscle branch innervating quadriceps muscle and a cutaneous branch innervating hindlimb skin (the saphenous nerve). Regenerating motor and sensory axons reach the end organs of muscle and skin using only these two branches. The cutaneous branch normally contains no motor axons, so its reinnervation by motor neurons represents a specificity failure of regeneration. In the bclassicalQ femoral nerve preparation where there is access to both branches and their end organs, regenerating motor axons prefer to reinnervate the muscle pathway by 4–8 weeks (Brushart, 1988; Madison et al., 1996), with prior pruning of motor axon collaterals from the inappropriate (cutaneous) pathway (Brushart, 1990). Even without the influence of end organ contact, studies in the rat have shown that regenerating motor neurons preferentially reinnervate the muscle pathway over the cutaneous pathway (Brushart, 1993; Brushart et al., 1998).
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PMR has also been demonstrated in the mouse using the classical end organ preparation (Mears et al., 2003; Robinson and Madison, 2003), although it required a manipulation of the nerve repair technique (Robinson and Madison, 2003) or pharmacological intervention during axon regeneration (Mears et al., 2003). In addition, it has been reported that mouse motor axons grow more vigorously into the cutaneous rather than the muscle pathway (Martini et al., 1992). These recent studies in the mouse suggest that there may be fundamental differences between PMR in the rat and mouse. We therefore decided to investigate two specific aspects of PMR in the mouse. First, we examined whether PMR was evident in the absence of any end organ contact by capping each of the terminal pathways distal to the femoral nerve bifurcation. Surprisingly, we found the opposite result as reported for the rat under these conditions: Significantly more motor neurons projected to the cutaneous pathway. Secondly, we examined whether in the absence of muscle contact the length of the cutaneous pathway influenced the number of motor neurons projecting to the cutaneous pathway. The results showed again that more motor neurons projected to the cutaneous pathway. In fact, significantly more motor neurons projected to the cutaneous pathway when it was left long and intact to skin than when it was capped. These findings raise the possibility of significant differences between rat and mouse femoral nerves within the context of PMR and also suggest that there may be a hierarchy of trophic support for regenerating motor axons with muscle contact being the highest, followed by the length of the terminal nerve branch and/or contact with skin.
Materials and methods Surgical procedures All procedures were approved by the Veterans Affairs Medical Center animal use committee. General mouse surgical procedures were carried out as previously described in detail (Robinson and Madison, 2003) with the following modifications. C57BL/6 mice (male and female, 20–22 g) were deeply anesthetized for all surgical procedures with a mixture of ketamine, xylazine, and acepromazine (100, 6, and 1 mg/kg, respectively) in normal (0.9%) saline. In all groups, the femoral nerve was exposed using an inguinal approach and the parent nerve was transected with microscissors ~5 mm proximal to the bifurcation of the nerve into its muscle and cutaneous branches (Fig. 1A). The nerve stumps were reapposed and repaired with fibrin sealant (Baxter Healthcare Products, Glendale, CA, USA). Three different repair groups were prepared that varied in terms of whether the distal nerve branches remained in continuity with their respective end organs of muscle and skin (Fig. 1A, gray box).
In the classical PMR preparation (C-PMR, n = 10, data from Robinson and Madison, 2003), the muscle branch and the cutaneous branch remained intact. In the short muscle and short cutaneous branch group (SM-SC, n = 9), the muscle branch was transected at the quadriceps muscle, ligated, and placed in a blind-ended silicone tube to prevent end organ contact of regenerating axons. The cutaneous branch was transected at the same length as the muscle branch and was also ligated and placed in a silicone tube. In the short muscle and long cutaneous group (SM-LC, n = 5), only the muscle branch was transected, ligated, and capped as above. The cutaneous branch remained intact to skin. The surgical site was then closed in layers and each animal allowed to recover. Animals were maintained in approved housing with controlled lighting and free access to food and water. Retrograde labeling and counting of motor neurons Eight weeks after nerve repair, the femoral nerve was reexposed. The muscle and cutaneous branches were separated by silicone grease dams, trimmed to ~1 mm distal to the bifurcation, and randomly assigned to receive application of crystals of either fluorescein dextran (FD, D3306; Molecular Probes, Eugene, OR, USA) or tetramethylrhodamine dextran (TD, D-3308; Molecular Probes). After crystal application, each branch was sealed with silicone grease and separated from each other by plastic food wrap. Three days later, the animal was perfused through the heart with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS. The lumbar spinal cord was removed, postfixed for several hours, and sucrose protected. The cord was frozen on dry ice and stored at 808C until being sectioned with a cryostat. Serial 25-Am frozen longitudinal sections were thawed in PBS, mounted onto glass slides, air dried, and coverslipped with Prolong (P7481, Molecular Probes) according to the manufacturer’s instructions. All serial sections were examined, and all retrogradely labeled motor neurons containing a nucleus were identified using a composite filter set (#51006; Chroma Technology, Brattleboro, VT, USA) in a fluorescenceequipped Zeiss Axiophot microscope at 250 magnification. Motor neuron counts were carried out by blinded independent observers and scored as either single labeled (fluorescein or tetramethylrhodamine only) or double labeled (both fluorescein and tetramethylrhodamine). Counting variation among the observers was ~2%. Motor neurons labeled from the muscle, the cutaneous, or both branches were tabulated and their counts corrected for split cells (Abercrombie, 1946). Control data from previously unlesioned quadriceps nerves (Robinson and Madison, 2003) labeled 161 F 4 motor neurons (n = 4, mean F SE). Statistical analysis Student’s t tests for paired data were used to compare muscle and cutaneous branch motor neuron counts
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Fig. 1. (A) The three experimental femoral nerve models. In the classical preferential motor reinnervation model (C-PMR), only the parent nerve was transected and repaired. In addition to repair of the parent nerve, the second model also transected, ligated, and capped both nerve branches (short muscle, short cutaneous; SM-SC). The parent nerve was also repaired in the third model, but only the muscle branch was transected, ligated, and capped (short muscle, long cutaneous; SM-LC). In all models, motor neurons reinnervating each branch were retrogradely labeled 8 weeks after the initial nerve repair by application of tracers just distal to the bifurcation of the nerve. (B) Average motor neuron counts for the three femoral nerve models. Comparisons within the C-PMR group showed that significantly more motor neurons were labeled from the muscle branch compared to the cutaneous branch (D). For groups SM-SC and SM-LC, the results were reversed: significantly more motor neurons were labeled from the cutaneous branch compared to the muscle branch (D). Across groups, the number of motor neurons projecting to the cutaneous branch differed significantly from each other in the order SM-LC N SM-SC N C-PMR. Conversely, the number of motor neurons projecting to the muscle branch was significantly greater for the C-PMR group compared to the other two models. In addition, only group SM-SC showed a significant reduction (§) in the total number of labeled motor neurons compared to normal (dashed lines). Bars = SE.
within groups. Differences were considered statistically significant when P b 0.05. Analysis of variance (using Student–Newman–Keuls post hoc comparisons) was
used to determine differences among groups. DiffeQ rences were considered statistically significant when P b 0.05.
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Results Eight weeks after the parent femoral nerve was transected and repaired, the classical PMR (C-PMR) group showed that motor neurons preferentially reinnervated the motor pathway and that the total number of retrogradely labeled motor neurons was not different from normal (Fig. 1B, left side; data from Robinson and Madison, 2003). In contrast, when contact with both end organs was prevented (SM-SC), the relationship between the two branches was reversed: Significantly more motor neurons reinnervated the cutaneous pathway compared to the muscle pathway with a ratio of ~2:1 (Fig. 1B, middle). Total motor neuron counts were significantly reduced from normal. In the short muscle and long cutaneous group (SM-LC), where the influence of muscle on axon regrowth was prevented, significant preferential cutaneous pathway reinnervation was also evident, with a cutaneous–muscle ratio of ~4:1(Fig. 1B, right side). The total number of motor neurons in this group was not significantly reduced from normal. Among the three preparations, a significant progression was evident in terms of the number of motor neurons projecting to the cutaneous branch: When contact with both end organs was allowed (C-PMR), the fewest motor neurons projected to the cutaneous branch; when contact with both end organs was prevented (SM-SC), an intermediary number was seen; and when only skin contact was maintained (SM-LC), the most motor neurons projected to the cutaneous branch. In terms of the muscle branch, there was a significant reduction in the number of motor neurons projecting to it when muscle contact was prevented (SM-SC and SM-LC) compared to when muscle contact was allowed (C-PMR).
Discussion There are three primary findings from these mouse experiments: (1) unlike the rat, significantly more motor neurons project to the cutaneous compared to the muscle branch when contact with both end organs is prevented; (2) there is a significant reduction in the number of motor neurons that regenerate into the femoral nerve when contact with both end organs is prevented; and (3) in the absence of muscle contact, the length of the cutaneous branch and/or contact with skin significantly increases the number of motor neurons projecting to the cutaneous branch. Previous work in the rat femoral nerve model, including some from this laboratory, has shown that regenerating motor neurons preferentially regenerate into their original muscle branch rather than into the branch to skin (Al-Majed et al., 2000; Brushart, 1990, 1993; Brushart and Seiler, 1987; Madison et al., 1996, 1999); a phenomenon that has been termed preferential motor reinnervation (PMR). Virtually all of this previous work involved the classical preparation for PMR where regenerating motor axons are
free to contact the end organs of muscle and skin. The current findings in the mouse are in contrast to these previous studies. Our results show that in the absence of muscle contact, significantly more motor neurons project to the foreign cutaneous pathway as opposed to their original muscle pathway. To understand the significance of these contradictory results, we must examine some of the nuances of the mouse and rat femoral nerve models. Anatomical differences The rat femoral nerve is considered a useful model to follow the regeneration accuracy of motor neurons because both terminal nerve branches are nearly equal in size and are thus well matched to compete for regenerating axons. The muscle pathway has a 24–30% larger cross-sectional area than the cutaneous branch and is composed of fewer, but larger, myelinated axons. On average, there are ~35% more myelinated axons in the cutaneous branch than in the muscle branch (Brushart and Seiler, 1987). In the mouse, the cross-sectional area difference between the two branches is slightly less (the muscle branch is only ~21% larger), but the difference between branches in the number of myelinated axons is greater (~50% more myelinated axons in the cutaneous branch; Frei et al., 1999; Martini et al., 1992; Sancho et al., 1999). These differences between the rat and mouse may be due to the fact that in the rat ~340 motor neurons innervate the quadriceps muscle (Brushart, 1990; Le et al., 2001), while in the mouse that number is reduced to ~170 (Mears et al., 2003; Robinson and Madison, 2003). These anatomical differences may account for recently reported differences in PMR between the two species. Using the same repair methods as for the rat (direct suture), we (Robinson and Madison, 2003) and other investigators (Mears et al., 2003) could not demonstrate PMR in the mouse. Given the smaller size of the mouse and its more challenging repair site, we investigated the use of a fibrin sealant for the nerve repair for its ease of use in anatomically confined areas and successfully demonstrated PMR (Robinson and Madison, 2003). It should be noted that in the rat model, both suture-based (Al-Majed et al., 2000; Brushart, 1990, 1993; Brushart and Seiler, 1987; Madison et al., 1996, 1999) and fibrin sealant repairs (Robinson and Madison, unpublished observations) of the femoral nerve with end organ contact have demonstrated PMR. In the present studies, we repaired the femoral nerve by the fibrin sealant method in order to investigate specific aspects of PMR in the mouse. Projections of regenerating motor neurons without end organ contact A study to determine the influence of the two femoral nerve branches on rat PMR was carried out by Brushart (1993). The parent femoral nerve was transected and
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repaired, but the muscle and cutaneous pathways were each sutured within blind-ended tubes (i.e., the present SM-SC group). Compared to the classical PMR preparation, this paradigm completely removed the influence of end organs (muscle or skin) on pathway choice, but the outcome was similar: By 8 weeks, approximately two- to threefold more motor neurons were labeled from the muscle pathway than from the cutaneous pathway. Our current results in the mouse in terms of motor neuron regeneration in the absence of end organ contact (SM-SC) are the opposite of that reported for the rat: By 8 weeks, significantly more motor neurons are labeled from the cutaneous branch. The underlying mechanism for this potential species difference is unknown but may relate to the anatomical differences noted above. There were also significantly fewer motor neurons labeled in the absence of end organ contact compared to normal numbers, suggesting that by themselves the pathways are not capable of supporting the full complement of motor neurons. More motor neurons project to the cutaneous branch due to increased length and/or skin contact It is well known that motor neurons receive significant trophic support from muscle once reinnervation has occurred (Vrbova et al., 1995). It is also well known that a denervated pathway provides trophic support to regenerating neurons prior to end organ contact (Bunge, 1993; Fu and Gordon, 1997; Jessen and Mirsky, 1999; Hall, 2001; Riethmacher et al., 1997). We found a significant difference between the number of motor neurons projecting to a long cutaneous branch with skin contact (SM-LC) compared to a shortened cutaneous branch without skin contact (SM-SC). This finding suggests that the length (or volume) of the cutaneous branch and/or contact with skin significantly increases the number of motor neurons innervating this branch, a finding that has also been recently demonstrated in the rat (Robinson and Madison, 2004). Each of these possibilities will be considered separately. Although not directly comparable to the present work, previous studies from several laboratories using Y-shaped tubes have examined the influence of various tissues and nerve segments to act as attractants for regenerating axons. For example, regenerating axons are known to distinguish between a nerve and nonnerve target (e.g., tendon), suggesting tissue specificity in terms of being able to attract and retain regenerating axons (Lundborg et al., 1986; Mackinnon et al., 1986). Building on the idea of distal nerve as an attractive target for regenerating axons, several studies have implicated distal nerve volume as an influence on axonal outgrowth. In a study somewhat similar to the present one, Takahashi et al. (1999) investigated the influence of denervated nerve length to attract regenerating axons. Regenerating axons within a Y-shaped silicone tube were forced to reinnervate either a short nerve segment or a longer one. After 6 weeks, more myelinated axons were
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quantified in the longer distal nerve segment compared to the shorter one. In a related study, Iwabuchi et al. (1999) used a Y-shaped tube with the singular end containing the proximal stump of either the peroneal or tibial nerve. The bifurcated ends of the tube contained the distal stumps of either the peroneal or tibial nerve. In all cases, more axons projected to the larger tibial nerve compared to the smaller peroneal. Comparable findings have been reported by Zhao et al. (1992) also using the rat sciatic nerve. Perhaps most similar to the present experiments is a study by Mackinnon and Dellon (1992) utilizing the primate femoral nerve and a Y-shaped chamber. The singular end of the tube contained the proximal saphenous nerve, and the bifurcated ends of the tube contained either a long distal saphenous nerve still in continuity with skin or a short nerve graft. Eight months later, both distal targets contained myelinated axons; however, in all cases there were more axons in the saphenous nerve with skin contact, suggesting that the volume of the nerve target and/or contact with skin increased the number of regenerating axons projecting to that target. In relation to the present work with regenerating motor neurons, studies have shown that the neurotrophins brainderived neurotrophic factor (BDNF), nerve growth factor, and neurotrophin-3 are produced by skin (Acheson and Lindsay, 1996; Buchman and Davies, 1993; LeMaster et al., 1999). The demonstration of BDNF in skin is especially interesting since it is well known that BDNF acts as a neurotrophic factor for motor neurons (Henderson et al., 1993; Koliatsos et al., 1993; Oppenheim et al., 1992; Yan et al., 1992, 1994). Electrophysiological studies have shown that the abnormal electrical properties of axotomized motor neurons return to normal values following reinnervation of muscle (Mendell et al., 1995; Munson et al., 1997; Nishimura et al., 1991). If motor neurons instead reinnervate only a cutaneous pathway, their electrical properties also recover, albeit less so. If this regenerating axon contact with skin is prevented by distal ligation, motor neuron electrical properties resemble those of permanently axotomized motor neurons (Nishimura et al., 1991). Thus, by some poorly understood mechanism, skin is also capable of facilitating the physiological recovery of axotomized motor neurons. So in addition to differences in the amount of trophic support provided by increasing the volume of the cutaneous nerve branch, it is possible that motor neurons also receive trophic support from skin. This additional support from skin may account for the greater number of motor neurons projecting to the cutaneous pathway with end organ contact (SM-LC) compared to without end organ contact (SM-SC). Similarly, there is a significant reduction in the total number of regenerated motor neurons (compared to normal) when all end organ contact is prevented (SM-SC) that may relate to reduced trophic support. In the current study, we labeled regenerated motor axons just distal to the bifurcation of the parent femoral nerve into its muscle and cutaneous branches. We therefore have no direct evidence that
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regenerating motor axons in the cutaneous branch actually grew to skin, although the 8-week survival period should have been adequate for the axons to travel that distance. In summary, these data from the mouse femoral nerve model do not support the idea that Schwann cell tubes maintain a specific identity that can be recognized by regenerating motor axons; a possibility that has been suggested to explain PMR in the rat (Brushart, 1993). Instead, they show that in the absence of muscle, contact motor neurons preferentially reinnervate the functionally inappropriate pathway to skin as opposed to the functionally appropriate pathway to muscle. Moreover, they suggest that maintaining contact with skin further enhances this preferential cutaneous reinnervation. We suggest that motor neurons initially project randomly to both terminal nerve branches, assess the relative levels of trophic support in each, and preferentially remain in the branch that provides the greater amount of trophic support. The results of the present work suggest a possible hierarchy of support for regenerating motor neurons with muscle contact being the highest, followed by the volume of the terminal nerve branch and/or contact with skin. We also propose that manipulations to the relative levels of trophic support between the two nerve branches would thus have predictable outcomes in terms of motor neuron projections, with the parent neuron being able to compare the amount of trophic support received from sibling axons.
Acknowledgments We thank Baxter Healthcare for providing the Tisseel fibrin sealant. Supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (to RDM). RDM is a Research Career Scientist for the Medical Research Service, Department of Veterans Affairs.
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G.A. Robinson, R.D. Madison / Experimental Neurology 192 (2005) 39–45 Oppenheim, R.W., Qin-Wei, Y., Prevette, D., Yan, Q., 1992. Brain-derived neurotrophic factor rescues developing avian motoneurons from cell death. Nature 360, 755 – 757. Riethmacher, D., Sonnenberg-Riethmacher, E., Brinkmann, V., Yamaai, T., Lewin, G.R., Birchmeier, C., 1997. Severe neuropathies in mice with targeted mutations of the erbB3 receptor. Nature 389, 725 – 729. Robinson, G.A., Madison, R.D., 2003. Preferential motor reinnervation in the mouse: comparison of femoral nerve repair using a fibrin sealant or suture. Muscle Nerve 28, 227 – 231. Robinson, G.A., Madison, R.D., 2004. Motor neurons can preferentially reinnervate cutaneous pathways. Exp. Neurol. 190, 407 – 413. Sancho, S., Magyar, J.P., Aguzzi, A., Suter, U., 1999. Distal axonopathy in peripheral nerves of PMP22-mutant mice. Brain 122, 1563 – 1577. Takahashi, Y., Maki, Y., Yoshizu, T., Tajima, T., 1999. Both stump area and volume of distal sensory nerve segments influence the regener-
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Manipulations of the mouse femoral nerve influence the accuracy of pathway reinnervation by motor neurons Grant A. Robinsona, Roger D. Madisona,b,c,* a Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA c Research Service of the Veterans Affairs Medical Center, Building 16, Room 38, 508 Fulton Street, Durham, NC 27705, USA b
Received 27 August 2004; revised 11 October 2004; accepted 20 October 2004 Available online 12 January 2005
Abstract Previous studies using the femoral nerve model in both mice and rats have shown that regenerating motor axons prefer to reinnervate the terminal nerve branch to muscle versus a terminal nerve branch to skin, a process that has been termed preferential motor reinnervation (PMR). If end organ contact with muscle and skin is prevented, this preferential motor reinnervation still occurs in the rat. To better understand the process of preferential motor reinnervation in the mouse, we examined motor neuron reinnervation of muscle and cutaneous pathways without any end organ contact as well as with only cutaneous end organ contact. Surprisingly, there was no preferential motor reinnervation: Motor neurons preferred the cutaneous pathway over the muscle pathway when all end organ contact was prevented and showed an even greater preference for the cutaneous pathway when it was attached to skin. D 2004 Elsevier Inc. All rights reserved. Keywords: Axonal regrowth; Reinnervation accuracy; Preferential motor reinnervation; Regeneration; Femoral nerve
Introduction The peripheral nervous system is capable of robust regeneration after a nerve lesion. However, since functional recovery is primarily dependent upon the accurate regeneration of axons to their original end organs, poor regeneration accuracy often results in poor functional recovery. A better understanding of how regenerating axons select their terminal pathways is of fundamental interest to basic and clinical neuroscience. Preferential motor reinnervation (PMR) refers to the disproportionate reinnervation by regenerating motor axons of motor pathways with muscle contact compared to cutaneous pathways with skin contact (Brushart, 1988).
* Corresponding author. Research Service of the Veterans Affairs Medical Center, Building 16, Room 38, 508 Fulton Street, Durham, NC 27705, USA. Fax: +1 919 286 6811. E-mail address: [email protected] (R.D. Madison). 0014-4886/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2004.10.013
The understanding of PMR comes mainly from rat femoral nerve studies, where motor and sensory axons within the parent nerve become anatomically segregated into a muscle branch innervating quadriceps muscle and a cutaneous branch innervating hindlimb skin (the saphenous nerve). Regenerating motor and sensory axons reach the end organs of muscle and skin using only these two branches. The cutaneous branch normally contains no motor axons, so its reinnervation by motor neurons represents a specificity failure of regeneration. In the bclassicalQ femoral nerve preparation where there is access to both branches and their end organs, regenerating motor axons prefer to reinnervate the muscle pathway by 4–8 weeks (Brushart, 1988; Madison et al., 1996), with prior pruning of motor axon collaterals from the inappropriate (cutaneous) pathway (Brushart, 1990). Even without the influence of end organ contact, studies in the rat have shown that regenerating motor neurons preferentially reinnervate the muscle pathway over the cutaneous pathway (Brushart, 1993; Brushart et al., 1998).
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PMR has also been demonstrated in the mouse using the classical end organ preparation (Mears et al., 2003; Robinson and Madison, 2003), although it required a manipulation of the nerve repair technique (Robinson and Madison, 2003) or pharmacological intervention during axon regeneration (Mears et al., 2003). In addition, it has been reported that mouse motor axons grow more vigorously into the cutaneous rather than the muscle pathway (Martini et al., 1992). These recent studies in the mouse suggest that there may be fundamental differences between PMR in the rat and mouse. We therefore decided to investigate two specific aspects of PMR in the mouse. First, we examined whether PMR was evident in the absence of any end organ contact by capping each of the terminal pathways distal to the femoral nerve bifurcation. Surprisingly, we found the opposite result as reported for the rat under these conditions: Significantly more motor neurons projected to the cutaneous pathway. Secondly, we examined whether in the absence of muscle contact the length of the cutaneous pathway influenced the number of motor neurons projecting to the cutaneous pathway. The results showed again that more motor neurons projected to the cutaneous pathway. In fact, significantly more motor neurons projected to the cutaneous pathway when it was left long and intact to skin than when it was capped. These findings raise the possibility of significant differences between rat and mouse femoral nerves within the context of PMR and also suggest that there may be a hierarchy of trophic support for regenerating motor axons with muscle contact being the highest, followed by the length of the terminal nerve branch and/or contact with skin.
Materials and methods Surgical procedures All procedures were approved by the Veterans Affairs Medical Center animal use committee. General mouse surgical procedures were carried out as previously described in detail (Robinson and Madison, 2003) with the following modifications. C57BL/6 mice (male and female, 20–22 g) were deeply anesthetized for all surgical procedures with a mixture of ketamine, xylazine, and acepromazine (100, 6, and 1 mg/kg, respectively) in normal (0.9%) saline. In all groups, the femoral nerve was exposed using an inguinal approach and the parent nerve was transected with microscissors ~5 mm proximal to the bifurcation of the nerve into its muscle and cutaneous branches (Fig. 1A). The nerve stumps were reapposed and repaired with fibrin sealant (Baxter Healthcare Products, Glendale, CA, USA). Three different repair groups were prepared that varied in terms of whether the distal nerve branches remained in continuity with their respective end organs of muscle and skin (Fig. 1A, gray box).
In the classical PMR preparation (C-PMR, n = 10, data from Robinson and Madison, 2003), the muscle branch and the cutaneous branch remained intact. In the short muscle and short cutaneous branch group (SM-SC, n = 9), the muscle branch was transected at the quadriceps muscle, ligated, and placed in a blind-ended silicone tube to prevent end organ contact of regenerating axons. The cutaneous branch was transected at the same length as the muscle branch and was also ligated and placed in a silicone tube. In the short muscle and long cutaneous group (SM-LC, n = 5), only the muscle branch was transected, ligated, and capped as above. The cutaneous branch remained intact to skin. The surgical site was then closed in layers and each animal allowed to recover. Animals were maintained in approved housing with controlled lighting and free access to food and water. Retrograde labeling and counting of motor neurons Eight weeks after nerve repair, the femoral nerve was reexposed. The muscle and cutaneous branches were separated by silicone grease dams, trimmed to ~1 mm distal to the bifurcation, and randomly assigned to receive application of crystals of either fluorescein dextran (FD, D3306; Molecular Probes, Eugene, OR, USA) or tetramethylrhodamine dextran (TD, D-3308; Molecular Probes). After crystal application, each branch was sealed with silicone grease and separated from each other by plastic food wrap. Three days later, the animal was perfused through the heart with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS. The lumbar spinal cord was removed, postfixed for several hours, and sucrose protected. The cord was frozen on dry ice and stored at 808C until being sectioned with a cryostat. Serial 25-Am frozen longitudinal sections were thawed in PBS, mounted onto glass slides, air dried, and coverslipped with Prolong (P7481, Molecular Probes) according to the manufacturer’s instructions. All serial sections were examined, and all retrogradely labeled motor neurons containing a nucleus were identified using a composite filter set (#51006; Chroma Technology, Brattleboro, VT, USA) in a fluorescenceequipped Zeiss Axiophot microscope at 250 magnification. Motor neuron counts were carried out by blinded independent observers and scored as either single labeled (fluorescein or tetramethylrhodamine only) or double labeled (both fluorescein and tetramethylrhodamine). Counting variation among the observers was ~2%. Motor neurons labeled from the muscle, the cutaneous, or both branches were tabulated and their counts corrected for split cells (Abercrombie, 1946). Control data from previously unlesioned quadriceps nerves (Robinson and Madison, 2003) labeled 161 F 4 motor neurons (n = 4, mean F SE). Statistical analysis Student’s t tests for paired data were used to compare muscle and cutaneous branch motor neuron counts
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Fig. 1. (A) The three experimental femoral nerve models. In the classical preferential motor reinnervation model (C-PMR), only the parent nerve was transected and repaired. In addition to repair of the parent nerve, the second model also transected, ligated, and capped both nerve branches (short muscle, short cutaneous; SM-SC). The parent nerve was also repaired in the third model, but only the muscle branch was transected, ligated, and capped (short muscle, long cutaneous; SM-LC). In all models, motor neurons reinnervating each branch were retrogradely labeled 8 weeks after the initial nerve repair by application of tracers just distal to the bifurcation of the nerve. (B) Average motor neuron counts for the three femoral nerve models. Comparisons within the C-PMR group showed that significantly more motor neurons were labeled from the muscle branch compared to the cutaneous branch (D). For groups SM-SC and SM-LC, the results were reversed: significantly more motor neurons were labeled from the cutaneous branch compared to the muscle branch (D). Across groups, the number of motor neurons projecting to the cutaneous branch differed significantly from each other in the order SM-LC N SM-SC N C-PMR. Conversely, the number of motor neurons projecting to the muscle branch was significantly greater for the C-PMR group compared to the other two models. In addition, only group SM-SC showed a significant reduction (§) in the total number of labeled motor neurons compared to normal (dashed lines). Bars = SE.
within groups. Differences were considered statistically significant when P b 0.05. Analysis of variance (using Student–Newman–Keuls post hoc comparisons) was
used to determine differences among groups. DiffeQ rences were considered statistically significant when P b 0.05.
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Results Eight weeks after the parent femoral nerve was transected and repaired, the classical PMR (C-PMR) group showed that motor neurons preferentially reinnervated the motor pathway and that the total number of retrogradely labeled motor neurons was not different from normal (Fig. 1B, left side; data from Robinson and Madison, 2003). In contrast, when contact with both end organs was prevented (SM-SC), the relationship between the two branches was reversed: Significantly more motor neurons reinnervated the cutaneous pathway compared to the muscle pathway with a ratio of ~2:1 (Fig. 1B, middle). Total motor neuron counts were significantly reduced from normal. In the short muscle and long cutaneous group (SM-LC), where the influence of muscle on axon regrowth was prevented, significant preferential cutaneous pathway reinnervation was also evident, with a cutaneous–muscle ratio of ~4:1(Fig. 1B, right side). The total number of motor neurons in this group was not significantly reduced from normal. Among the three preparations, a significant progression was evident in terms of the number of motor neurons projecting to the cutaneous branch: When contact with both end organs was allowed (C-PMR), the fewest motor neurons projected to the cutaneous branch; when contact with both end organs was prevented (SM-SC), an intermediary number was seen; and when only skin contact was maintained (SM-LC), the most motor neurons projected to the cutaneous branch. In terms of the muscle branch, there was a significant reduction in the number of motor neurons projecting to it when muscle contact was prevented (SM-SC and SM-LC) compared to when muscle contact was allowed (C-PMR).
Discussion There are three primary findings from these mouse experiments: (1) unlike the rat, significantly more motor neurons project to the cutaneous compared to the muscle branch when contact with both end organs is prevented; (2) there is a significant reduction in the number of motor neurons that regenerate into the femoral nerve when contact with both end organs is prevented; and (3) in the absence of muscle contact, the length of the cutaneous branch and/or contact with skin significantly increases the number of motor neurons projecting to the cutaneous branch. Previous work in the rat femoral nerve model, including some from this laboratory, has shown that regenerating motor neurons preferentially regenerate into their original muscle branch rather than into the branch to skin (Al-Majed et al., 2000; Brushart, 1990, 1993; Brushart and Seiler, 1987; Madison et al., 1996, 1999); a phenomenon that has been termed preferential motor reinnervation (PMR). Virtually all of this previous work involved the classical preparation for PMR where regenerating motor axons are
free to contact the end organs of muscle and skin. The current findings in the mouse are in contrast to these previous studies. Our results show that in the absence of muscle contact, significantly more motor neurons project to the foreign cutaneous pathway as opposed to their original muscle pathway. To understand the significance of these contradictory results, we must examine some of the nuances of the mouse and rat femoral nerve models. Anatomical differences The rat femoral nerve is considered a useful model to follow the regeneration accuracy of motor neurons because both terminal nerve branches are nearly equal in size and are thus well matched to compete for regenerating axons. The muscle pathway has a 24–30% larger cross-sectional area than the cutaneous branch and is composed of fewer, but larger, myelinated axons. On average, there are ~35% more myelinated axons in the cutaneous branch than in the muscle branch (Brushart and Seiler, 1987). In the mouse, the cross-sectional area difference between the two branches is slightly less (the muscle branch is only ~21% larger), but the difference between branches in the number of myelinated axons is greater (~50% more myelinated axons in the cutaneous branch; Frei et al., 1999; Martini et al., 1992; Sancho et al., 1999). These differences between the rat and mouse may be due to the fact that in the rat ~340 motor neurons innervate the quadriceps muscle (Brushart, 1990; Le et al., 2001), while in the mouse that number is reduced to ~170 (Mears et al., 2003; Robinson and Madison, 2003). These anatomical differences may account for recently reported differences in PMR between the two species. Using the same repair methods as for the rat (direct suture), we (Robinson and Madison, 2003) and other investigators (Mears et al., 2003) could not demonstrate PMR in the mouse. Given the smaller size of the mouse and its more challenging repair site, we investigated the use of a fibrin sealant for the nerve repair for its ease of use in anatomically confined areas and successfully demonstrated PMR (Robinson and Madison, 2003). It should be noted that in the rat model, both suture-based (Al-Majed et al., 2000; Brushart, 1990, 1993; Brushart and Seiler, 1987; Madison et al., 1996, 1999) and fibrin sealant repairs (Robinson and Madison, unpublished observations) of the femoral nerve with end organ contact have demonstrated PMR. In the present studies, we repaired the femoral nerve by the fibrin sealant method in order to investigate specific aspects of PMR in the mouse. Projections of regenerating motor neurons without end organ contact A study to determine the influence of the two femoral nerve branches on rat PMR was carried out by Brushart (1993). The parent femoral nerve was transected and
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repaired, but the muscle and cutaneous pathways were each sutured within blind-ended tubes (i.e., the present SM-SC group). Compared to the classical PMR preparation, this paradigm completely removed the influence of end organs (muscle or skin) on pathway choice, but the outcome was similar: By 8 weeks, approximately two- to threefold more motor neurons were labeled from the muscle pathway than from the cutaneous pathway. Our current results in the mouse in terms of motor neuron regeneration in the absence of end organ contact (SM-SC) are the opposite of that reported for the rat: By 8 weeks, significantly more motor neurons are labeled from the cutaneous branch. The underlying mechanism for this potential species difference is unknown but may relate to the anatomical differences noted above. There were also significantly fewer motor neurons labeled in the absence of end organ contact compared to normal numbers, suggesting that by themselves the pathways are not capable of supporting the full complement of motor neurons. More motor neurons project to the cutaneous branch due to increased length and/or skin contact It is well known that motor neurons receive significant trophic support from muscle once reinnervation has occurred (Vrbova et al., 1995). It is also well known that a denervated pathway provides trophic support to regenerating neurons prior to end organ contact (Bunge, 1993; Fu and Gordon, 1997; Jessen and Mirsky, 1999; Hall, 2001; Riethmacher et al., 1997). We found a significant difference between the number of motor neurons projecting to a long cutaneous branch with skin contact (SM-LC) compared to a shortened cutaneous branch without skin contact (SM-SC). This finding suggests that the length (or volume) of the cutaneous branch and/or contact with skin significantly increases the number of motor neurons innervating this branch, a finding that has also been recently demonstrated in the rat (Robinson and Madison, 2004). Each of these possibilities will be considered separately. Although not directly comparable to the present work, previous studies from several laboratories using Y-shaped tubes have examined the influence of various tissues and nerve segments to act as attractants for regenerating axons. For example, regenerating axons are known to distinguish between a nerve and nonnerve target (e.g., tendon), suggesting tissue specificity in terms of being able to attract and retain regenerating axons (Lundborg et al., 1986; Mackinnon et al., 1986). Building on the idea of distal nerve as an attractive target for regenerating axons, several studies have implicated distal nerve volume as an influence on axonal outgrowth. In a study somewhat similar to the present one, Takahashi et al. (1999) investigated the influence of denervated nerve length to attract regenerating axons. Regenerating axons within a Y-shaped silicone tube were forced to reinnervate either a short nerve segment or a longer one. After 6 weeks, more myelinated axons were
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quantified in the longer distal nerve segment compared to the shorter one. In a related study, Iwabuchi et al. (1999) used a Y-shaped tube with the singular end containing the proximal stump of either the peroneal or tibial nerve. The bifurcated ends of the tube contained the distal stumps of either the peroneal or tibial nerve. In all cases, more axons projected to the larger tibial nerve compared to the smaller peroneal. Comparable findings have been reported by Zhao et al. (1992) also using the rat sciatic nerve. Perhaps most similar to the present experiments is a study by Mackinnon and Dellon (1992) utilizing the primate femoral nerve and a Y-shaped chamber. The singular end of the tube contained the proximal saphenous nerve, and the bifurcated ends of the tube contained either a long distal saphenous nerve still in continuity with skin or a short nerve graft. Eight months later, both distal targets contained myelinated axons; however, in all cases there were more axons in the saphenous nerve with skin contact, suggesting that the volume of the nerve target and/or contact with skin increased the number of regenerating axons projecting to that target. In relation to the present work with regenerating motor neurons, studies have shown that the neurotrophins brainderived neurotrophic factor (BDNF), nerve growth factor, and neurotrophin-3 are produced by skin (Acheson and Lindsay, 1996; Buchman and Davies, 1993; LeMaster et al., 1999). The demonstration of BDNF in skin is especially interesting since it is well known that BDNF acts as a neurotrophic factor for motor neurons (Henderson et al., 1993; Koliatsos et al., 1993; Oppenheim et al., 1992; Yan et al., 1992, 1994). Electrophysiological studies have shown that the abnormal electrical properties of axotomized motor neurons return to normal values following reinnervation of muscle (Mendell et al., 1995; Munson et al., 1997; Nishimura et al., 1991). If motor neurons instead reinnervate only a cutaneous pathway, their electrical properties also recover, albeit less so. If this regenerating axon contact with skin is prevented by distal ligation, motor neuron electrical properties resemble those of permanently axotomized motor neurons (Nishimura et al., 1991). Thus, by some poorly understood mechanism, skin is also capable of facilitating the physiological recovery of axotomized motor neurons. So in addition to differences in the amount of trophic support provided by increasing the volume of the cutaneous nerve branch, it is possible that motor neurons also receive trophic support from skin. This additional support from skin may account for the greater number of motor neurons projecting to the cutaneous pathway with end organ contact (SM-LC) compared to without end organ contact (SM-SC). Similarly, there is a significant reduction in the total number of regenerated motor neurons (compared to normal) when all end organ contact is prevented (SM-SC) that may relate to reduced trophic support. In the current study, we labeled regenerated motor axons just distal to the bifurcation of the parent femoral nerve into its muscle and cutaneous branches. We therefore have no direct evidence that
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regenerating motor axons in the cutaneous branch actually grew to skin, although the 8-week survival period should have been adequate for the axons to travel that distance. In summary, these data from the mouse femoral nerve model do not support the idea that Schwann cell tubes maintain a specific identity that can be recognized by regenerating motor axons; a possibility that has been suggested to explain PMR in the rat (Brushart, 1993). Instead, they show that in the absence of muscle, contact motor neurons preferentially reinnervate the functionally inappropriate pathway to skin as opposed to the functionally appropriate pathway to muscle. Moreover, they suggest that maintaining contact with skin further enhances this preferential cutaneous reinnervation. We suggest that motor neurons initially project randomly to both terminal nerve branches, assess the relative levels of trophic support in each, and preferentially remain in the branch that provides the greater amount of trophic support. The results of the present work suggest a possible hierarchy of support for regenerating motor neurons with muscle contact being the highest, followed by the volume of the terminal nerve branch and/or contact with skin. We also propose that manipulations to the relative levels of trophic support between the two nerve branches would thus have predictable outcomes in terms of motor neuron projections, with the parent neuron being able to compare the amount of trophic support received from sibling axons.
Acknowledgments We thank Baxter Healthcare for providing the Tisseel fibrin sealant. Supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (to RDM). RDM is a Research Career Scientist for the Medical Research Service, Department of Veterans Affairs.
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