Brain Cell Biology Volume 36, 213–227, 2008 DOI: 10.1007/s11068-009-9040-4
Differences in c-jun and nNOS expression levels in motoneurons following different kinds of axonal injury in adult rats Li-Hua Zhou1, Shu Han1, Yuan-Yun Xie1,2, Lin-Lin Wang1 and Zhi-Bin Yao1,* 1 Department of Anatomy, Zhong Shan School of Medicine, Sun Yat-sen University, Guangzhou 510080, P.R. China (*author for correspondence; e-mail:
[email protected]) 2 Department of Medical Genetics, Center for Molecular Medicine and Therapeutics, The University of British Columbia, Vancouver, BC, Canada Received 28 July 2008; Revised 13 December 2008; Accepted 18 December 2008 Published online 24 February 2009 Springer Science+Business Media, LLC 2009 In the peripheral nervous system (PNS), root avulsion causes motoneuron degeneration, but the majority of motoneurons can survive axotomy. In order to study the mechanism of motoneuron degeneration, we compared the expression patterns of c-jun and neuronal nitric oxide synthase (nNOS), the well-known molecular players in PNS regeneration and degeneration, among adult rats having undergone axotomy (Ax), avulsion (Av), or pre-axotomy plus secondary avulsion (Ax 1 Av) of the brachial plexus. Our results showed that the highest and longest-lasting c-jun activation occurred in Ax, which was much stronger than those in Av and Ax 1 Av. The time course and intensity of c-jun expression in Ax 1 Av were similar to those in Av except on day 1, while the pre-axotomy condition resulted in a transient up-regulation of c-jun to a level comparable to that in Ax. Axotomy alone did not induce nNOS expression in motoneurons. Pre-axotomy left-shifted the time course of nNOS induction in Ax 1 Av compared to that in Av. Motoneuron loss was not evident in Ax, while it was 70% in Av and more than 85% in Ax 1 Av at 8 weeks postinjury. The survival of motoneurons was positively correlated with c-jun induction, but not with nNOS expression in motoneurons. Moreover, c-jun induction was negatively correlated with nNOS induction in injured motoneurons. Our results indicate that functional crosstalk between c-jun and nNOS might play an important role in avulsion-induced motoneuron degeneration, while c-jun might act as a prerequisite survival factor and nNOS might act as a predictor for the onset of motoneuron degeneration.
Introduction
1993; Wu et al., 1994a). In clinical studies, functional recovery is poor because motoneuron death is irreversible. Compared to avulsion injury, axonal injury-axotomy is usually considered a less severe injury because the majority of motoneurons can survive axotomy. The mechanisms of the different outcomes of these two different axonal injuries are not yet fully understood. Studying the transcriptional responses of motoneurons after nerve injury is a current prevailing area of research. We think it is very important to compare changes in gene
Injury to the brachial plexus causes deficits in the motor, sensory, and autonomic functions of the upper limbs. It is well known that root avulsion causes motoneuron degeneration in adult rats (Wu,
Electronic supplementary material The online version of this article (doi:10.1007/s11068-009-9040-4) contains supplementary material, which is available to authorized users.
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Results
expressions following axotomy and avulsion because this may contribute to the development of treatments for nerve injury in humans. Previous studies have demonstrated that brachial root avulsion induced de novo expression of neuronal nitric oxide synthase (nNOS) in motoneurons, which is considered to be closely related to motoneuron degeneration (Liu and Martin, 2001; Martin et al., 1999, 2005; Novikov et al., 1995; Wu, 1996). However, Li et al. (1995) found that there was no nNOS induction, but rather motoneuron degeneration in adult mice following roots avulsion. Our recent study further revealed that down-regulation of nNOS by antisense oligos could not decrease, but instead increased avulsion-induced motoneuron death (Zhou and Wu, 2006a). In brief, the role of nNOS in motoneuron degeneration remains to be further elucidated. Compared to root avulsion, axotomy does not cause nNOS expression in injured motoneurons (Wu, 1996). Axotomyinduced, long-lasting c-jun induction in motoneurons has been regarded as a prerequisite factor for motoneuron regeneration (IKeda et al., 2005; Piehl et al., 1998; Ugolini et al., 2003; Wu et al., 1993; Wu, 1996). Based on previous studies of the potential beneficial effect of c-jun in motoneurons induced by axotomy (Wu et al., 1994b; Herdegen et al., 1997; Casanovas et al., 2001), we hypothesized that the transcriptional responses of motoneu ron s to avu lsion, inclu ding dramatic motoneuron loss as well as nNOS induction, might be attenuated by a pre-conditioning axotomy, and that this might be associated with c-jun expression. In the present study, we set up three kinds of axonal injury models in adult rats, including spinal nerve distal axotomy alone (Ax), spinal root avulsion alone (Av), and a pre-axotomy plus secondary root avulsion (Ax + Av). In an attempt to prevent avulsion-induced cell death, we performed axotomy 1 week prior to avulsion. We compared the expression patterns of c-jun induction, nNOS induction in motoneurons, and motoneuron survival among these three kinds of axonal injuries on days 1, 3, 5, 7, 14, 21, 28, 42, and 56 postinjury. Our results indicate that higher c-jun induction leads to greater motoneuron survival. The earlier nNOS induction leads to faster death of the motoneurons. The pre-conditioning axotomy not only induced strong c-jun expression, but also led to a quicker induction of nNOS and more motoneuron death than avulsion alone.
Differences in c-jun induction in motoneurons following different kinds of axonal injury Following all axonal injuries, the non-lesioned motoneurons on the contralateral sides were ChAT-positive (Fig. 1A), but c-jun negative (Fig. 1B, C, J), while the lesioned motoneurons on the ipsilateral sides did not express ChAT (Fig. 1D) yet showed a remarkable c-jun induction in the nuclei of cells in injured C7 sections (Fig. 1E, F). There were different patterns of c-jun induction in motoneurons following different kinds of axonal injury. In Ax, the peak of c-jun expression occurred on day 1 postinjury (Fig. 1G), then declined gradually. According to the location, the morphology, and the size of the cells counterstained by neutral red, we found that almost all c-jun-positive cells in the ventral horns were motoneurons (Fig. 1K), a result coincident with a previous study that reported that about 98% of surviving motoneurons were cjun-positive by 3 days following axotomy (Wu, 1996). In Av, the peak of c-jun expression occurred on day 3, with a level only half that in Ax, then declined gradually (Fig. 1H). In Ax + Av, the peak of c-jun expression was on day 1, with a level almost the same as that in Ax (Fig. 1I). This upregulation of c-jun quickly decreased to half of the peak on day 3. From days 3 to 56, the c-jun expression pattern in Ax + Av was similar to that in Av (Fig. 1L). Statistical analysis showed that variations from animal to animal within groups were not significant. However, the differences in the number of c-jun-positive motoneurons among Ax, Av, and Ax + Av groups were significant (F = 353.796, P < 0.05). In Ax, for each time point from day 1 to 56 postinjury, the numbers of c-jun-positive motoneurons were 155 ± 3.2, 113.23 ± 3.2, 99.9 ± 1.0, 93.93 ± 1.2, 86.65 ± 1.2, 79.38 ± 1.0, 50.36 ± 3.3, 40.96 ± 1.2, and 31.61 ± 1.5. In Av, these numbers were only 67.33 ± 1.8, 82.96 ± 1.4, 69.83 ± 2.1, 60.00 ± 1.7, 52.89 ± 1.4, 48.68 ± 2.1, 25.18 ± 1.9, 14.00 ± 1.2, and 3.15 ± 0.3, significantly less than those in AX at every survival time point, with all P < 0.05 (Fig. 1L). In Ax + Av, this number was 141.50 ± 4.5 on day 1, which was significantly more than that in Av (P < 0.05) but less than that in Ax (P < 0.05) at the same time point. For the time points from day 3 through day 56 in Ax + Av, the numbers decreased to 68.23 ± 4.5, 61.43 ± 2.3, 214
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Fig. 1. Representative microphotographs of immunofluorescent double-labelling of c-jun and ChAT in the C7 ventral horn of adult rats at 24 h postinjury (A–F, scale bar = 300 lm). (A) Motoneurons in the contralateral nonlesioned ventral horn were ChAT-positive (green). (B) There was no c-jun induction (red), and (C) no double-labelling of c-jun and ChAT in contralateral motoneurons. (D) Avulsion resulted in the disappearance of ChAT (green), but (E) resulted in c-jun induction (red) in the nuclei of ipsilateral motoneurons. (F) There was no doublelabelling of ChAT and c-jun in ipsilateral motoneurons. Representative microphotographs of c-jun immuno-positive motoneurons (G–J, scale bar = 150 lm). Note the levels of c-jun immuno-positive motoneurons in axotomy (G), avulsion (H), and pre-axotomy plus avulsion (I), and the lack in normal control (J). Neutral red counter stain showing that c-jun-positive reactions occurred in the nuclei of motoneurons (K, scale bar = 300 lm). Pre-axotomy plus avulsion induced the same high level of c-jun expression as that of axotomy immediately after injury, but the levels declined quickly to those of animals with avulsion on postinjury day 3 and thereafter. #P < 0.01, axotomy plus avulsion versus avulsion alone; *P < 0.01, axotomy plus avulsion or axotomy alone versus avulsion alone (L).
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61.54 ± 1.8, 50.90 ± 1.4, 36.83 ± 2.0, 30.56 ± 1.18, 17.46 ± 0.9, and 3.74 ± 0.3, less than those in Ax at each time point (P < 0.05) and less than those in Av on day 3, day 5, and day 21 (P < 0.05). The present results showed a strong expression pattern for c-jun in motoneurons both in time course and in intensity in Ax compared to those in Av and in Ax + Av. Pre-axotomy only resulted in a transient up-regulation of c-jun on day 1 in motoneurons in Ax + Av, and then it quickly adopted and maintained an expression pattern similar to that in Av (Fig. 1L).
14, 28, 42, and 56 postinjury. About 98% of surviving motoneurons in the ipsilateral ventral horn were NADPH-d-positive by 21 days following avulsion. Statistical analysis showed that variations from animal to animal within groups were not significant, but that there was a significant difference between Av and Ax + Av (F = 151.59, P < 0.01). In the first 2 weeks postinjury, there were significantly more nNOS-positive motoneurons in Ax + Av than in Av. However, in the last 4 weeks postinjury, there were more nNOS-positive motoneurons in Av than in Ax + Av (P < 0.05, Fig. 2J). The pre-axotomy left-shifted the time course of secondary avulsion-induced de novo nNOS expression in motoneurons in Ax + Av relative to that in Av (Fig. 2J).
Differences in nNOS induction in motoneurons following different kinds of axonal injury Root avulsion also resulted in the disappearance of ChAT expression and nNOS induction in motoneurons in the ventral horns on the lesion sides of C7 segments (Fig. 2). There were differences in the pattern of nNOS induction in motoneurons following different kinds of axonal injury. There was no nNOS induction in motoneurons in Ax (Fig. 2A), in agreement with previous studies (Wu et al., 1994a; Yuan et al., 2006). While the time course of nNOS induction was different, the density was not different between Av and Ax + Av. In Av, evident nNOS expression was first observed on day 5 and the maximum was reached on day 21 (Fig. 2B), after which expression declined until the end of the study (Fig. 2D). In Ax + Av (Fig. 2C), evident nNOS expression appeared earlier, on day 3, and the maximum was reached on day 14, 7 days prior to that in Av. At the end of study, the expression level of nNOS was sustained (Fig. 2E). There was no nNOS induction in the non-lesion sides of C7 sections (Fig. 2F). Since avulsion induced not only nNOS but also c-jun expression in motoneurons, we checked whether c-jun and nNOS were colocalized by double immunofluorescent labelling. The results showed that most of the c-jun-positive motoneurons were also nNOS-positive, but only a portion of the nNOS-positive motoneurons were cjun-positive (Fig. 2G–I). Quantitative analysis showed that the number of nNOS-positive motoneurons in Av were 0.56 ± 0.12, 0.61 ± 0.2, 6.06 ± 0.5, 13.1 ± 1.1, 46.8 ± 1.6, 63.72 ± 2.6, 47.63 ± 1.7, 27.12 ± 1.8, and 15.58 ± 1.7 on days 1, 3, 5, 7, 14, 21, 28, 42, and 56 postinjury. In Ax + Av, these numbers were 0.61 ± 0.11, 18.37 ± 2.1, 32.03 ± 1.7, 46.1 ± 2.4, 58.93 ± 3.4, 44.55 ± 1.5, 37.68 ± 1.6, 15.48 ± 1.3, and 9.36 ± 1.5 on days 1, 3, 5, 7,
Differential outcomes of motoneuron degeneration following different kinds of axonal injury Axotomy did not induce any evident loss of motoneurons from day 1 to day 56 postinjury, which was consistent with previous studies (Wu et al., 1994a; Yuan et al., 2006). At the end of 8 weeks postinjury, the ipsilateral ventral horns were remarkably reduced in size in both Av (Fig. 2D) and Ax + Av (Fig. 2E) compared to the contralateral ventral horns (Fig. 2F). The reason for the atrophy of the injured ventral horns was the evident reduction of the size of the cell bodies as well as the total number of motoneurons, a result further confirming our previous studies (Zhou and Wu, 2006a, b). In Av, the dramatic motoneuron loss occurred 4 weeks postinjury. The numbers of surviving motoneurons in Av were 99.4 ± 0.3, 92.22 ± 0.5, 92.1 ± 1.2, 87.14 ± 1.5, 76.96 ± 1.2, 65.2 ± 2.3, 52.91 ± 2.0, 32.92 ± 1.7, and 28.07 ± 1.7 on days 1, 3, 5, 7, 14, 28, 42, and 56 postinjury. In Ax + Av, more than half of the motoneurons were lost by day 21 postinjury, which was 7 days prior to that in Av (Fig. 2K). The numbers of surviving motoneurons in Ax + Av were 98.69 ± 0.3, 87.82 ± 1.4, 85.63 ± 1.6, and 84.68 ± 2.1 on days 1, 3, 5, and 7 postinjury, without any significant difference from that in Av at the same time point. On days 14, 21, 28, 42, and 56 postinjury, these numbers decreased to 61.85 ± 3.3, 48.10 ± 1.7, 42.71 ± 1.8, 21.13 ± 1.3, and 14.90 ± 2.5 in Ax + Av, significantly lesser than those in Av at the same time points (P < 0.05, Fig. 2K). In order to study the effects of c-jun and nNOS induction in 216
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Fig. 2. Representative microphotographs of C7 ventral horns 21 days postinjury with NADPH-d plus neutral red stain (A–F, scale bar = 150 lm). No nNOS expression and neuronal degeneration were observed in motoneurons following axotomy (A); more than 85% of the motoneurons expressed nNOS (dark blue), with extensive expression in cell bodies as well as neurites of motoneurons following injury of avulsion (B); axotomy plus avulsion injury also induced evident nNOS expression in motoneurons (dark blue) (C); note the evident motoneuron degeneration with decreased cell body size, reduced total number of surviving motoneurons, and lighter Neutral red staining at the end of 8 weeks following injury both in avulsion (D) and in pre-axotomy plus avulsion injury (E); no nNOS expression and neuronal degeneration were observed in the control on the contralateral side of the spinal cord (F). Representative microphotographs of the C7 ventral horn 21 days postinjury with immunofluorescent double-labelling of c-jun and nNOS (G–I, scale bar = 300 lm). c-jun induction (green) in motoneurons was weak (G), but nNOS induction (red) was at its peak (H) at this time in avulsion injury. Asterisks indicate that most c-jun-positive motoneurons were co-localized with nNOS-positive motoneurons (I). A comparison of nNOS-positive motoneurons following avulsion alone and axotomy plus avulsion injury (J). Pre-axotomy up-regulated nNOS expression in avulsed motoneurons. Compared to avulsion alone, the total number of surviving nNOS-positive motoneurons increased significantly starting 3 days postinjury. The highest level of nNOS expression in avulsed motoneurons with pre-axotomy was observed 14 days after injury, which was one week earlier than that in animals with avulsion alone. *P < 0.05, axotomy plus avulsion versus avulsion alone. A comparison of surviving motoneurons following avulsion alone and axotomy plus avulsion injury (K). The percentage of surviving motoneurons significantly declined in both groups one week after avulsion injury. Twenty-one days after injury, the percentage of surviving motoneurons in animals with axotomy was significantly less than that of animals without axotomy injury. *P < 0.05, axotomy plus avulsion versus avulsion alone. Experimental schedule and animal survival time points. Three axonal injury groups were established, both axotomy and avulsion were injured on day 0, while the third group received an axotomy injury 7 days before avulsion. Then, the animals (n = 8) were allowed to survive post-injury for 1 day through 56 days (L).
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Role of c-jun in the survival of injured motoneurons c-Jun in the mammalian peripheral nervous system (PNS) and CNS has been thought to have dual functions in both neuronal death and survival (Herdegen et al., 1997; Raivich et al., 2004). However, in the PNS, c-jun induction in axotomized sensory and motor neurons has long been regarded as a prerequisite for successful nerve regeneration, and is involved in the early signalling of the regenerative process (Casanovas et al., 2001; Herdegen and Leah, 1998; Tsujino et al., 2000). Additionally, cjun induction in avulsed motoneurons has been considered necessary for nerve regeneration (IKeda et al., 2005; Piehl et al., 1998; Wu, 1996). Our present data further demonstrate that prolonged activation of c-jun exists both in axotomized and in avulsed motoneurons. Moreover, our present study showed that c-jun induction in axotomy was much stronger than that in avulsion, and that a preconditioning axotomy did not significantly change the intensity or the expression pattern of c-jun induction in the secondary avulsion. The positive correlation between c-jun induction and the number of surviving motoneurons further indicated that the intensity of c-jun induction in injured motoneurons might be used to predict the regenerative capacity of injured motoneurons in peripheral axonal injuries. This result also indicates that the characteristics of the axonal injury decide the expression patterns of c-jun induction in motoneurons. The mechanism for injury-induced c-jun expression in motoneurons is still not clear. The trigger signals for c-jun up-regulation following axotomy have been extensively studied (Herdegen et al., 1992; Jenkins and Hunt, 1991; Leah et al., 1991). It has been thought that the signal is negative; some examples include interruption of the retrograde flow of unidentified molecule(s) (Doya et al., 2006; Leah et al., 1991; Tsujino et al., 2000; Wu, 1993) and deprivation of target factors (Defelipe et al., 1993; Kenney and Kocsis, 1998; Leah et al., 1991). Other studies have proposed that a distance-dependent signalling mechanism is involved because a difference in the time course of c-jun induction was observed in a sciatic nerve cut compared to a spinal nerve cut (Easton et al., 1997; Kenney and Kocsis, 1997). Our results support this hypothesis because the distance from the site of axonal injury to the cell body of the motoneurons was apparently longer in axotomy than that in avulsion in the present study.
motoneuron degeneration in Av and Ax + Av injuries, a Pearson correlation was made between the number of surviving motoneurons and the number of c-jun-positive motoneurons or between the number of surviving motoneurons and the number of nNOS-positive motoneurons in all animals suffering from Av and Ax + Av injuries. The results showed that there was a significant positive correlation between the number of surviving motoneurons and the number of c-junpositive motoneurons (r = 0.085, P < 0.01). Additionally, there was no significant correlation between the number of surviving motoneurons and the number of nNOS-positive motoneurons (r = -0.086, P > 0.05). Furthermore, there was a significant negative correlation between the number of c-jun-positive motoneurons and the number of nNOS-positive motoneurons (r = -0.223, P < 0.05).
Discussion The goal of the present study was to evaluate the significance of c-jun and nNOS expression patterns in degenerative motoneurons following axonal injury. We also wanted to know how pre-axotomyinduced c-jun up-regulation influences avulsioninduced c-jun and nNOS expression patterns and motoneuron death. Our results showed that root avulsion, either alone or secondary to axotomy, was fatal to motoneurons, but axotomy was nonfatal within 2 months. If the injured motoneurons had no chance to regenerate, the pre-conditioning axotomy was not beneficial to motoneuron survival; moreover, it accelerated secondary avulsioninduced motoneuron death. c-jun induction in motoneurons was much weaker in fatal avulsion than that in nonfatal axotomy. Additionally, nNOS induction in motoneurons only occurred in avulsion, and not in axotomy. Pre-axotomy did not reduce, but pre-headed avulsion-induced nNOS induction in motoneurons. At the peak of nNOS induction in avulsion, most of the c-jun-positive motoneurons co-localized with nNOS-positive motoneurons, but only some of the nNOS-positive motoneurons colocalized with c-jun-positive motoneurons. The present study suggests that the crosstalk between c-jun and nNOS might play an important role in avulsion-induced motoneuron degeneration. 218
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In other words, the longer the length of the remaining nerve stems, the stronger was the c-jun induction in motoneurons. From the present data, we could not determine the difference in trigger signals for c-jun induction between axotomy and avulsion injuries. However, we know that the injured motoneurons in Ax can get the remaining target factors from the remaining proximal nerve stems, nerve roots, and collateral projections (such as the branches of the roots of the brachial plexus), while the motoneurons in injured Av or Ax + Av can get the remaining target factors only from the remaining proximal nerve roots. Thus, we theorized that differences in the amounts of the target factors may contribute to the different expression patterns of c-jun induction in different kinds of axonal injury. If this is true, then changing the remaining target factors would change the expression pattern of c-jun. This hypothesis is further supported by the presented data in Ax + Av, which show a c-jun expression pattern similar to that in Ax on day 1 that quickly changed to an expression pattern similar to that in Av. Increasing the target factors by PNS graft transplantation to avulsed nerve roots may also have changed the c-jun expression pattern, where only the regenerative motoneurons were c-jun-positive while non-regenerative motoneurons were c-jun-negative (Wu, 1996). From the present study, we could not absolutely exclude another possibility, that the differences in the number of c-jun-positive motoneurons were due to different numbers of the surviving motoneurons. However, quantitative data did not support this. In Av, there was about an 8% motoneuron loss on day 3 compared to day 1, but the number of c-junpositive motoneurons increased at the same time. The role of c-jun in regenerative capacity has not been yet identified. The positive correlation of c-jun induction and motoneuron survival presented here further supports the survival role of c-jun in motoneurons of PNS. It has been thought that the c-jun transcription factor may play a role in the transcription of mRNAs that code for proteins important for neuronal structure and/or synapse function reestablishment, as well as the principal constituents of the cytoskeleton (Mikucki and Oblinger, 1991; Tetzlaff et al., 1991). Up-regulation of ATF3 (Averill et al., 2004; Huang et al., 2006; Shortland et al., 2006; Takeda et al., 2000; Tsujino et al., 2000) or down-regulation of ATF2 (Lindwall and Kanje, 2005; Martin-Villalba et al., 1998) have
been demonstrated to be coincident with c-jun upregulation. However, it was also found that strong nuclear Jun immunolabelling in motoneurons is always associated with intense cytoplasmic Bax staining in axotomy (Gillardon et al., 1996). Our present data suggest that there may be a functional crosstalk between c-jun and nNOS in motoneurons, and this might play a role in motoneuron degeneration. Firstly, our data showed that avulsion-induced c-jun expression occurred about one week earlier than avulsion-induced nNOS expression in injured motoneurons. Secondly, doublelabelling of c-jun and nNOS was present in injured motoneurons. Thirdly, there was a negative correlation between the number of c-jun-positive motoneurons and the number of nNOS-positive motoneurons in avulsion and in axotomy plus avulsion injuries. Finally, the number of c-junpositive motoneurons was positively correlated with the number of surviving motoneurons. It will be very important to explore how c-jun and nNOS crosstalk inside motoneurons in the future. Role of nNOS in injured motoneuron degeneration In the present study, axotomy (at the point 10 mm distal to the vertebra) itself did not induce nNOS expression, but root avulsion induced strong and prolonged nNOS expression in motoneurons, which is similar to the results of previous studies (Wu et al., 1994; Gu et al., 1997; Yuan et al., 2006). Pre-axotomy before avulsion resulted in a left-shift of nNOS induction compared to that in avulsion alone. Since the outcome of Ax + Av was to increase motoneuron death in the present study, the appearance of nNOS seemed to predict the start of motoneuron death. Thus, our present results support the idea that nNOS could be used as a molecular marker for failure of regeneration of motoneurons (Wu et al., 2004). Regarding the role of nNOS in avulsion-induced neuronal death, previous reports are controversial (Keilhoff et al., 2002, 2004). Some studies proposed that avulsion-induced nNOS might play a neurotoxic role (Denninger and Marletta, 1999; Li et al., 1995; Martin et al., 2005; Novikov et al., 1995; Yu, 2002) since nNOS expression is always linked to oxidative stress. Excessive NO might react with superoxide to form peroxynitrite, which itself is toxic and could cause tyrosine residue nitrosylation in proteins and subsequently worsen the status of injured 219
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neurons (Chan et al., 2001, 2003; Liu and Martin, 2001; Martin et al., 1999). A previous study also suggested that NO/nNOS might act as a replacement for neurotrophic factors, representing an attempt of avulsed motoneurons to resist the directly lethal effects of an unknown molecule (Wu et al., 2004). Recently, we down-regulated the existing avulsion-induced nNOS gene expression in motoneurons via antisense oligos, but apparently aggravated avulsion-induced motoneuron death (Zhou and Wu, 2006a). Further studies found that exogenous GDNF used 4 weeks after avulsion could inhibit nNOS expression, but could not rescue avulsed motoneurons (Zhou and Wu, 2006b). The present results demonstrate that there is no significant correlation between the number of nNOS-positive motoneurons and the number of surviving motoneurons. All of these data suggest that nNOS induction in avulsion either plays a role in a survival signalling pathway or represents a response of injured motoneurons to an unknown, lethal molecule. Considering that there was a negative correlation between nNOS induction and c-jun induction, we also consider another possibility, that crosstalk might occur between nNOS and key molecules responsible for the survival or regeneration (such as c-jun) of motoneurons. Recent studies showed that protein–protein interactions represent an important mechanism in the control of NOS activity (Alderton et al., 2001; Dedio et al., 2001; Zimmermann et al., 2002). The carboxy-terminal PDZ ligand of nNOS (CAPON), nNOS-interacting proteins (Jaffrey et al., 2002), and Dexras1, a physiologic target of nNOS (Fang et al., 2000), were upregulated in the neurons of lumbar DRG or the spinal cord in an adult rat following sciatic axotomy. These two proteins were considered to play an important role in different pathological conditions, including nerve regeneration or neuron death, and possibly pain (Shen et al., 2008). Our present results showed down-regulation of c-jun whenever nNOS appeared in avulsed motoneurons. Future research must focus on where and when the functional link between c-jun and nNOS is established in the degenerative and regenerative processes of avulsed motoneurons.
ries, but we did not compare the responses of different subpopulations within the same ventral horn motoneurons to different axonal injuries quantitatively. In terms of c-jun induction in motoneurons, almost all subpopulations of ventral horn motoneurons were c-jun positive, but the time course was different among different axonal injuries. Since the peak of c-jun induction was detected at the very beginning (24 h postinjury), while all subpopulations of motoneurons were c-jun positive, it is hard for us to evaluate which subpopulations of motoneurons show differential responses to the different injuries. In terms of nNOS induction, previous studies have shown that avulsion-induced nNOS expression appears first in the central group, then in the ventral, ventrolateral and dorsolateral groups, and finally in the dorsomedial and ventromedial groups of ipsilateral ventral horn motoneurons (Wu, 1993; Wu et al., 1994). The loss of motoneurons also showed this subpopulation difference, but occurred 2 weeks later (Wu, 1993; Wu et al., 1994). Many previous studies have confirmed that avulsion-injured motoneurons die from apoptosis (Chan et al., 2001, 2003; Gu et al., 1997; Martin et al., 2005; Novikov et al., 1995; Wu et al., 1994; Wu, 2000; Yang et al., 2006). Our present results confirm these previous studies (Fig. 2). All of these data indicate that the central and ventral subpopulations of ventral horn motoneurons respond quickly to avulsion injury. From the present and previous studies, we can say that all subpopulations within the ventral horn motoneurons do respond to all axonal injuries, but with different time courses, in terms of c-jun and nNOS induction and cell death. The outcome of the pre-conditioning axotomy was to increase motoneuron death in the present study, which was different from the results of preconditioning injury in ischemia in hippocampal and spinal neurons (Cafferty et al., 2004; Gardiner et al., 2007; Neumann and Woolf, 1999; Trendelenburg and Dirnagl, 2005). We should emphasize that the purpose of the present study was not to increase the regenerative capacity of injured motoneurons, but to observe the expression patterns of c-jun and nNOS in motoneuron degeneration. Thus, we did not allow for avulsed motoneurons to regenerate. We would rather state that our preaxotomy plus secondary avulsion was a combined axonal injury to motoneurons. Thus, the functions of motoneurons in the Av and Ax + Av rats were seriously damaged because the injured motoneu-
Different outcomes of motoneurons following different types of axonal injury In the present study, we compared the motoneuron response in terms of c-jun and nNOS induction and motoneuron death following different axonal inju220
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rons did not express ChAT, a motoneuron marker. The outcome of combined axonal injury to increase neuron degeneration has been reported before. Repeated injuries at the same site in the hypoglossal nerve in adult rats (Arvidsson and Aldskogius, 1982) and combined axonal injury with a secondary crush carried out contralaterally to the facial nerve in adult mice (Sakamoto et al., 2000) were reported to increase motoneuron death. Previous studies proposed that the compound signal from repeated injury to a site may jeopardize the survival of injured motoneurons (Kashihara et al., 1987; Lowrie and Vrbova´, 2001). It was suggested that in axotomized central neurons, the threshold for apoptosis may be lowered until regeneration is completed (Gillardon et al., 1996). This hypothesis is supported by the present finding of more serious motoneuron death in pre-conditioning axotomy plus secondary avulsion rats compared to in avulsion alone. Many previous studies have demonstrated that the length of the remaining nerve stem, which offers trophic support for injured motoneurons, decides the fate of injured motoneurons in axonal injuries (Gu et al., 1997; Wu et al., 2004). Our present data support this hypothesis: more serious motoneuron death occurred in root avulsion compared to that in stem axotomy of the brachial plexus. Recent studies proposed that different intracellular molecular events in motoneurons occurring early after injury may determine the different outcomes of axonal injury (Nagasao et al., 2008; Wu et al., 2003; Yang et al., 2006). Our present results further demonstrate that different expression patterns of c-jun and nNOS in motoneurons and the different levels of motoneuron death occur following different axonal injuries. In addition to intracellular molecular events in motoneurons, the environment around the injured motoneurons has also been shown to affect motoneuron degeneration. Recently, microglial cell reaction, T cell infiltration, and T cell memory were found in injured spinal segments following pre-conditioning crush injury, which may contribute to decreasing the threshold of injured motoneuron apoptosis following secondary injuries in facial nerves of adult rats (Raivich et al., 1998; Moran and Graeber, 2004; Brown and Sawchenko, 2007). This might explain why more motoneurons were lost in the pre-axotomy plus secondary avulsion injury compared to avulsion injury alone.
In summary, the present results suggest that cjun may promote nerve regeneration only if there is a chance for injured motoneurons to regenerate. Otherwise, c-jun may not overcome the avulsioninduced stress. The present study also showed that avulsion-induced c-jun activation occurs before nNOS reaction. Thus, it is necessary to further study whether nNOS is a downstream or upstream molecule of c-jun, and whether the JNK/c-jun pathway (Goldstein, 2001; Karin and Gallagher, 2005; Lindwall and Kanje, 2005) is involved in the initiation of avulsion-induced motoneuron apoptosis.
Methods Animal models All animals were obtained from the Laboratory Animal Center of Sun Yat-sen University, and all procedures are approved by the Committee for the Use of Live Animals in Teaching and Research at Sun Yat-sen University. Adult female Sprague– Dawley rats (250–280 g) were randomly divided into three groups: axotomy (Ax), avulsion (Av), and combined axonal injury (pre-axotomy plus secondary avulsion) (Ax + Av). The experimental design and all animal surviving time points after injury are shown in Fig. 2L. All rats were anesthetized with intraperitoneal injections of 1% Nembutal (40 mg/ kg). Under a surgical microscope, a 20 mm incision was made along the lower border of the right clavicle. A layered dissection was then performed to expose the right brachial plexus in the axillary cavity to the intervertebral foramen. In Ax, all stumps, including the superior, middle, and inferior trunks of the right brachial plexus, approximately 14 mm distal to the spinal cord, were individually crushed with microhemostatic forceps for 3 min. In Av, all nerve roots, including C5, C6, C7, C8, and T1 of the right brachial plexus, were separated. A small hemostat was used to hold the spinal nerve, and both the dorsal and ventral roots of every spinal nerve were pulled away from the spinal cord one by one. The exposed and avulsed dorsal and ventral roots were cut away from the peripheral nerve and examined under a microscope to confirm the success of the lesion. In Ax + Av, axotomy and avulsion were carried out on the same brachial plexus as described above, but axotomy was performed 7 days prior to avulsion. Then, the wounds of animals were sutured. After the surgical 221
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procedure, all animals were given 60 mg s.c. of benzylpenicillin. The animals were kept in a warm chamber and allowed to recover from the anaesthesia before returning to their home cages. The survival time points were 1, 3, 5, 7, 14, 21, 28, 42, and 56 days postinjury (n = 8 in each survival time point of each group). All rats were housed under a 12 h light/dark cycle with free access to food and water. The experimental design and all animal survival time points after injury are shown in Fig. 2L.
10 min and incubated in 0.3% peroxide in methanol (100%) at room temperature for 20 min to quench endogenous peroxidase activity. Sections were then incubated in 2% BSA and 0.2% Triton X-100 in 0.1 M PBS at room temperature for 1 h, followed by incubation in mouse monoclonal anti-c-jun (sc822, 1:200; Santa Cruz Biotechnology) for 48 h at 4C. After washing in PBS, biotinylated anti-mouse IgG (BA-2000, 1:200; Vector Labs) was added to the sections and incubated at room temperature for 1 h. Then, ABC reagents (the avidin biotin-peroxidase system, Vector Labs) were added to these sections and incubated at room temperature for 60 min. Sections were then washed thoroughly and incubated in 0.05% DAB and 0.03% H2O2 for 3– 5 min until a brown reaction product was observed. c-jun induction by avulsion has been shown to be expressed in motoneurons in the ventral horn of the spinal cord (Wu et al., 1994b, 1996).
Animal perfusion and tissue preparation After being anesthetized with a lethal dose of Nembutal, the animals were perfused intracardially with saline, followed by 300–400 ml of cold fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M PB (pH 7.4). The injured C5 to T1 spinal segments were harvested, post-fixed in fresh fixative for 4 h at 4C, and then cryoprotected with 30% phosphate buffered sucrose at 4C until the tissues dropped to the bottom. Frozen transverse spinal sections (40 lm) from C7 segments were cut and collected in 0.01 M PB. Every third section from each rat was used for c-jun immunohistochemistry, NADPH-d histochemistry, and neutral red counter staining, respectively.
ChAT, c-jun, nNOS immunofluorescence reactions For double immunofluorescence of ChAT and cjun, sections were incubated with mouse anti-ChAT (1:800, Santa Cruz Biotechnology) and rabbit antic-jun (1:200, Cell Signaling Technology) at 4C for 72 h. After washing in PBS, the sections were incubated with fluorescein isothiocyanate (FITC)conjugated goat anti-mouse IgG (1:300, Sigma) and tetramethylrhodamine isothiocyanate (TRITC)conjugated goat anti-rabbit IgG (1:400, Sigma) at room temperature in the dark for 2 h. For double immunofluorescence of ChAT and nNOS, sections were incubated with mouse anti-ChAT (1:800, Santa Cruz Biotechnology) and rabbit anti-nNOS (1:2000, Santa Cruz Biotechnology) at 4C for 72 h. After washing in PBS, the sections were incubated with FITC-conjugated goat anti-mouse IgG (1:300, Sigma) and TRITC-conjugated goat anti-rabbit IgG (1:400, Sigma) at room temperature in dark for 2 h. For double immunofluorescence of nNOS and c-jun, sections were incubated with mouse anti-nNOS (1:3000, Santa Cruz Biotechnology) and rabbit anti-c-jun (1:200, Cell Signaling Technology) at 4C for 72 h. After washing in PBS, the sections were incubated with TRITC-conjugated goat anti-mouse IgG (1:400, Sigma) and FITC-conjugated goat anti-rabbit IgG (1:300, Sigma) at room temperature in dark for 2 h. Finally, the sections were mounted on gelatin-coated glass slides, coverslipped in 0.1 M PBS containing 50% glycerin, then examined via fluorescence micros-
NADPH-d histochemistry Neuronal NOS-containing neurons were stained with NADPH-diaphorase (NADPH-d) following our previous studies (Zhou and Wu, 2006a, b). Briefly, free-floating sections were incubated in 10 ml 0.1 M Tris–HCl (pH 8.0) containing 0.2% Triton X100, 10 mg NADPH (Sigma), 2.5 mg nitro-blue tetrazolium (NBT) at 37C for 1 h and then washed with 0.1 M PB three times. The stained sections were mounted onto slides and counterstained with 1% neutral red (Sigma). These sections were used to count the numbers of nNOS-positive and surviving motoneurons. The NADPH-d technique is reliable for expression of nNOS since the previous studies have demonstrated that NADPH-d stains exactly the same population of injured motoneurons visualized by nNOS ICC and nNOS in situ hybridization (Bredt et al., 1991; Wu, 1993; Zhou and Wu, 2006b). c-Jun immunohistochemistry The procedures were similar to previous studies (Wu et al., 1994b, 1996, 2003). Briefly, sections were first washed three times with 0.1 M PBS for 222
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copy (DFC350FX/DMIRB; Leica, USA). Control experiments included omission of the primary or secondary antibodies. Sham-operated and nonlesioned animals served as negative controls.
and data handling for c-jun (shown by c-jun ICC) and nNOS (shown by NADPH-d reaction) expressions and the survival of motoneurons (shown by neutral red stain) were performed using SPSS 11.0 version. All data are expressed as means ± standard errors of the mean (X ± SE). All variations from animal to animal within each group (n = 8) were analyzed and showed no statistically significant differences by univariate one-way ANOVA. Then, the effects of different axonal injuries on c-jun expression in motoneurons, nNOS expression in motoneurons, and motoneuron survival were analyzed and compared among the Ax, Av, and Ax + Av groups at the same survival time points by univariate one-way ANOVAs, followed by post-hoc Bonferroni’s tests. Statistical comparisons of the relationship between the number of surviving motoneurons and the number of c-jun-positive motoneurons, between the number of surviving motoneurons and the number of nNOS-positive motoneurons, and between the number of c-jun-positive motoneurons and the number of nNOS-positive motoneurons in the Av and Ax + Av groups were made by Pearson correlation coefficient. P values £0.05 were considered statistically significant for all analyses.
Cell counting of motoneurons Data quantification and analysis were performed by a person who was blind to animal injury according to the previous studies (Wu et al., 1993, 1994a, b, 1996, 2000; Zhou and Wu, 2006a, b). The C7 spinal segment was defined as the region between the uppermost root and lowermost root of the C7 nerve of the contralateral spinal cord. Every third section from each rat was used for c-jun immunohistochemistry, NADPH-d histochemistry, and neutral red counterstaining. Motoneurons in ten sections were counted in total for c-jun or NADPH-d reactions plus neutral red stain, respectively. All motoneurons showing visible nuclei were counted under a 209 objective lens. On the lesioned sides, motoneurons showing a nucleus stained with c-jun antibody were considered c-junpositive motoneurons, and the number of c-junpositive motoneurons in each rat is expressed as the total number of c-jun-positive motoneurons in 10 serial C7 sections (Wu, 1996). In NADPH-d plus neutral red-stained sections, the numbers of motoneurons were counted on both the intact side and the lesioned side of the C7 section. The number of motoneurons on the contralateral intact side served as a control and is expressed as 100%. On the lesioned side, the number of surviving motoneurons included both NADPH-d-positive and -negative motoneurons, and is expressed as a percentage of the number of motoneurons on the intact contralateral side of the same C7 section (Wu et al., 1994b). The number of nNOS-positive motoneurons included only NADPH-d-positive motoneurons and is expressed as a percentage of the number of motoneurons on the intact contralateral side of the same C7 section. The number of nNOS-positive or survival motoneurons of each rat is expressed as the mean number of nNOS-positive or survival motoneurons of the 10 serial C7 sections (Wu et al., 1994b; Zhou and Wu, 2006a, b).
Acknowledgments This study was supported by grants from the Research Grants Council of Guangdong (2004 B50301006, 5001760, 8151008901000028) and the National Science Foundation Council of China (30570119).
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