Experimental Neurology 261 (2014) 451–461

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Sprouting of axonal collaterals after spinal cord injury is prevented by delayed axonal degeneration E. Collyer a, A. Catenaccio a, D. Lemaitre a, P. Diaz a, V. Valenzuela a, F. Bronfman a, F.A. Court a,b,⁎ a b

Millenium Nucleus for Regenerative Biology, Faculty of Biology, Pontificia Universidad Catolica de Chile, Santiago, Chile Neurounion Biomedical Foundation, Santiago, Chile

a r t i c l e

i n f o

Article history: Received 29 January 2014 Revised 2 July 2014 Accepted 20 July 2014 Available online 28 July 2014 Keywords: Spinal cord injury Wallerian degeneration Collateral sprouting Corticospinal neurons WldS Hemisection

a b s t r a c t After an incomplete spinal cord injury (SCI), partial recovery of locomotion is accomplished with time. Previous studies have established a functional link between extension of axon collaterals from spared spinal tracts and locomotor recovery after SCI, but the tissular signals triggering collateral sprouting have not been identified. Here, we investigated whether axonal degeneration after SCI contributes to the sprouting of collaterals from axons spared after injury. To this end, we evaluated collateral sprouting from BDA-labeled uninjured corticospinal axons after spinal cord hemisection (SCIH) in wild type (WT) mouse and WldS mouse strains, which shows a significant delay in Wallerian degeneration after injury. After SCIH, spared fibers of WT mice extend collateral sprouts to both intact and denervated sides of the spinal cord distant from the injury site. On the contrary, in the WldS mice collateral sprouting from spared fibers was greatly reduced after SCIH. Consistent with a role for collateral sprouting in functional recovery after SCI, locomotor recovery after SCIH was impaired in WldS mice compared to WT animals. In conclusion, our results identify axonal degeneration as one of the triggers for collateral sprouting from the contralesional uninjured fibers after an SCIH. These results open the path for identifying molecular signals associated with tissular changes after SCI that promotes collateral sprouting and functional recovery. © 2014 Elsevier Inc. All rights reserved.

Introduction Injuries to the spinal cord represent a significant clinical problem leading to lifelong disabilities with an enormous social and economical impact (Ackery et al., 2004; Hendriks et al., 2006; Thuret et al., 2006). Despite the poor regenerative capabilities of central nervous system (CNS) neurons, spontaneous functional recovery has been reported after incomplete spinal lesions in various organisms, including rats (Ballermann and Fouad, 2006; Bareyre et al., 2004; van den Brand et al., 2012), mice (Courtine et al., 2008; Liu et al., 2010; Valenzuela et al., 2012; Yip et al., 2010), cats (Martinez et al., 2011; Rossignol et al., 2002), monkeys (Babu and Namasivayam, 2008; Courtine et al., 2005) and humans (Raineteau and Schwab, 2001). A functional association between locomotor recovery after spinal cord injury (SCI) and collateral sprouting from intact spinal cord axons has been proposed. In Abbreviations: SCI, spinal cord injury; SCIH, spinal cord hemisection; WldS, Wallerian degeneration slow mutant mice; IL, ipsilateral to the injury; CL, contralateral to the injury; VST, vestibulospinal tract; VLAT, ventrolateral spinal tract. ⁎ Corresponding author at: Department of Physiology, Faculty of Biology, Pontificia Universidad Católica de Chile, Av. B. O'Higgins 340, Santiago 8331150, Chile. E-mail addresses: [email protected] (E. Collyer), [email protected] (A. Catenaccio), [email protected] (D. Lemaitre), [email protected] (P. Diaz), [email protected] (V. Valenzuela), [email protected] (F. Bronfman), [email protected] (F.A. Court).

http://dx.doi.org/10.1016/j.expneurol.2014.07.014 0014-4886/© 2014 Elsevier Inc. All rights reserved.

this respect, it has been shown that axon collaterals generate a new intraspinal circuit that bypass the injury zone and promotes locomotor recovery (Ballermann and Fouad, 2006; Bareyre et al., 2004; Courtine et al., 2008; Rossignol and Frigon, 2011). Collateral sprouting is not restricted to spinal cord levels close to the lesion (Ballermann and Fouad, 2006), suggesting that signals that propagates along the spine trigger axonal extension from intact neurons. Several studies have been carried out in order to identify activators of collateral extension after spinal cord and brain damage. Among the interventions proved to enhance collateral sprouting are the upregulation of neurotrophic factors such as NT3 and BDNF (Fouad et al., 2010; Ueno et al., 2012; Zhou et al., 2003), inhibition of myelin associated proteins (Bareyre et al., 2002; Lee et al., 2010), enzymatic digestion of the glial scar (Barrit et al., 2006; Wang et al., 2011), activation of the immune response and release of cytokines (Chen et al., 2008; Oshima et al., 2009), and activation of different molecular pathways such as Rho/ROCK (Chan et al., 2005), mTOR (Liu et al., 2010), STAT3 (Bareyre et al., 2011) and NCS1 (Yip et al., 2010). Although these tissular manipulations represent attractive therapeutic strategies to enhance collateral sprouting, the endogenous mechanism underlying the spontaneous extension of collateral after nervous system damage is still unknown. SCI elicit a plethora of cellular, tissular and systemic reactions with a defined temporal and spatial sequence, including the degeneration of axons severed from their cell body by a mechanism known as Wallerian

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degeneration (Profyris et al., 2004; Willerth and Sakiyama-Elbert, 2008), changes in neurotrophin expression and its receptors (Hajebrahimi et al., 2008), inflammatory reactions along the spinal cord (David and Kroner, 2011; Donnelly and Popovich, 2007), apoptotic death of neurons and oligodendrocytes (Beattie et al., 2002) and formation of a glial scar at the injury region (Yiu and He, 2006). Wallerian degeneration has been shown to evoke growth responses from damaged as well as intact neurons (Court and Alvarez, 2000; Diaz and Pecot-Dechavassine, 1990; Kerschensteiner et al., 2004; Ramer et al., 1997). Axonal degeneration takes place by mechanisms associated to mitochondrial dysfunction (Barrientos et al., 2011), intra-axonal calcium rise and calpain activation (Schlaepfer and Bunge, 1973). In wildtype mice, axonal injury triggers degeneration of severed axons by two to three days (Court and Coleman, 2012). Nevertheless, in the WldS (Wallerian degeneration-slow) mice, Wallerian degeneration after injury is delayed by weeks in both the CNS and the PNS (Coleman, 2005). It has been reported that after an SCI, the WldS mice show delayed locomotor recovery (Zhang et al., 1998) and diminished autonomic dysreflexia (Jacob et al., 2003). Interestingly, changes in the extent of collateral sprouting from spared fibers after SCI might be underlying those behavioral responses in the WldS mice. Here we asked if following SCI, Wallerian degeneration of axons triggers collateral sprouting from contralesional uninjured axons and the functional association between this growth response and locomotor recovery. By using a genetic model of delayed axonal degeneration (WldS) we demonstrate that after partial spinal damage, collateral sprouting of uninjured corticospinal neurons and locomotor recovery is dependent on axonal degeneration, defining for the first time an intrinsic signal triggering plastic responses in the spinal cord after damage. Materials and methods

were allowed to survive for 7 or 35 days. For behavioral analysis, the animals were allowed to survive for 35 days.

Corticospinal tract (CST) tracing To anterogradely label the corticospinal tract (CST) fibers mice were injected with BDA MW 10.000 (Invitrogen, Carlsbad, CA, USA) into the right hindlimb sensorimotor cortex, as previously described (Tysseling-Mattiace et al., 2008). Briefly, mice were anesthetized with 2-2-2 Tribromoethanol (Sigma, St. Louis, MO, USA), placed in a stereotaxic frame and injected in three points as follows: − 1.0 mm lateral to the midline, at 0.5 mm, − 0.5 mm and − 1.0 mm from Bregma, and at a depth of 0.7 mm ventral to the dura, using a 5 μl Hamilton syringe fitted with a 34 G needle. Each injection delivered 0.5 μl of the tracer over a 2 min period and the syringe was left in the site for 4 min before removal. The tracer was injected fourteen days before sacrificing the animals.

Tissue preparation Animals were transcardially perfused with ice-cold 4% paraformaldehyde in 0.1 M PBS, pH 7.2–7.4. The spinal cords were dissected and post-fixed for 3 h in 4% paraformaldehyde, subjected to a sucrose gradient (10, 20 and 30% sucrose in PBS) and fast frozen in OCT (Tissue-Tek, Alphen aan den Rijn, The Netherlands) using liquid nitrogen. A 4 mm block containing the cervical enlargement (C3–C5) region or the lumbar enlargement (L3–L5) region was transversally sectioned at 20 μm using a cryostat microtome (Leica, Nussloch, Germany) and direct mounted in Superfrost slides (Fisherbrand Superfrost Plus, Thermo Fischer Scientific, USA).

Animals Adult (10–12 weeks) female and male mice were obtained from the Animal Facility of the Biological Sciences Faculty of the P. Catholic University of Chile. The mouse strains used were C57BL/6 (WT) and Wallerian degeneration slow mutant mice (C57BL/OlaHsd-WldS; Lunn et al., 1989). The experiments were approved by the P. Catholic University bioethics committee (DFCB-078/2008). The total number of animals used in this study was 42 WT (23 for anatomy, 3 for in vitro studies, 8 for locomotion and 8 excluded) and 50 WldS (24 for anatomy, 3 for in vitro studies, 10 for locomotion and 13 excluded). The animals excluded from this study matched one of these criteria: complete paralysis of hindlimbs or movement of the right hindlimb one day after SCIH surgery (WT: 8 WldS: 4) and animals who died before end of study due to natural causes or euthanasia due to severe distress (WT:0; WldS: 9). Surgical procedures Mice were anesthetized with a single dose of 330 mg/kg I.P. of 2-2-2 Tribromoethanol (Sigma, St. Louis, MO, USA). Animals received laminectomies of the dorsal half of the thirteenth thoracic vertebra (T13) corresponding to L3 spinal level. This was followed by a lateral dorso-ventral hemisection of the right side of the spine (SCIH) using a pair of vannas microscissors (RS-5658, ROBOZ, Gaithersburg, MD, USA) as previously described (Valenzuela et al., 2012). The bone removed by laminectomy was replaced with Gelita-Spon (Gelita Medical, Ederbach, Germany), and the wound was closed by suturing the muscle with 4-0 silk and the skin with mice wound clips. Sham-operated animals include the removal of the vertebra without spinal hemisection. Control animals underwent only stereotaxic surgery but no laminectomy intervention. For post-operative recovery, mice were placed in a temperaturecontrolled chamber until fully awake. The animals did not show evidence of bladder malfunction. For collateral quantification, the animals

Collateral sprouting quantification To visualize the BDA-labeled CST collateral sprouting axons, sections were washed 3 times in 0.1 M PBS, incubated for 2 h in Alexa-488 conjugated Avidin (1:250, Invitrogen, Carlsbad, CA, USA) in 0.1% Triton X100, 5% gelatin from cold water fish (Sigma, St. Louis, MO, USA) in PBS, washed three times in 0.1 M PBS and covered with Fluoro-mount (Electron Microscopy Sciences, Hatfield, PA, USA). Slices were imaged using an OLYMPUS IX71 microscope fitted with a CCD camera. Three transverse sections per animal at either the cervical or lumbar spinal level were selected for analysis. In each section, two regions of interest were defined: contralateral to the injury (contralateral horn, CL), containing the fibers growing towards the uninjured side of the spinal cord (left in figures and images); and ipsilateral to the injury (ipsilateral horn, IL), containing the fibers growing towards the injured side of the spinal cord (right in figures and images) (Fig. 1A). To diminish the background induced error in the quantification of collaterals a hand-made digital outline of collaterals was drawn by an experimenter blind to the treatment and mice strain (Fig. 1B). The digital outline was drawn in Photoshop CS2 (Adobe 9.0.2) with a 4 pixel wide pencil at 120% magnification images. The collateral sprout profile was calculated using a custom made script for MATLAB software version 7.0.1 (MathWorks). Briefly, the procedure selects a 1-pixel (3.6 μm) wide column in the yaxis of the outline mask and sums the red pixels, generating the number of pixels per column, and repeats the procedure along the entire outline mask. The collateral sprout profile is the plot of the number of pixels per column along the x-axis. By doing this, we obtained a 2D profile with information about the magnitude and location of the collateral population (Fig. 1C). To correct for the inter-animal variation in the tracing efficiency, we normalized the collateral profile matrix in each slide by the number of traced fibers in the main CST of the same slide, obtaining the collateral pixels per stained CST fibers.

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Fig. 1. Collateral sprouting quantification method. (A) Schematic of BDA anterograde CST tracing (in red) and areas of analysis. Two regions of spinal cord were selected for analysis: cervical enlargement and lumbar enlargement. In each region, two zones were defined: contralateral to the SCIH (contralateral horn, CL, in green), containing the fibers growing towards the uninjured side; and contralateral to the tracer injection (contralateral, CL, in purple), containing the fibers growing towards the injured side of the spinal cord. (B) Low-magnification image of transverse sections of the spinal cord processed for BDA stain showing the CST main tract and sprouting fibers. (B′) Magnification of B left inset showing sprouting BDAlabeled fibers into the ipsilateral gray matter. (B″) Magnification of B right inset showing BDA-labeled CST main tract. Scale bar, 200 μm. (C) Hand-made digital outline (in red) of BDAlabeled axons from B. The collateral sprout profile was calculated using the previous outline. The procedure sums the pixels (red) in each 1-pixel (3.6 μm) wide column in the outline mask and plots the number of pixels per column along the x-axis. The collateral sprout profile (in blue) is a 2D representation of the fiber sprouting that provides information about the magnitude and location of the sprouting fiber population in the regions of analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Electron microscopy For EM analysis, spinal cord segments from cervical and lumbar regions of one animal perfused with 4% PFA were fixed overnight by immersion in EM fixative solution (2.5% glutaraldehyde, 0.01% picric acid and 0.1 M Cacodylate buffer pH 7.4). The tissue was rinsed in the same buffer, immersed in 1% OsO4 for 1 h followed by in block incubation with 2% uranyl acetate for 2 h. The tissue was dehydrated with a graded series of ethanol, propylene oxide and infiltrated with Epon (Ted Pella Inc). One-micron thick semithin sections of the spinal cord at the cervical and lumbar regions were stained with 1% toluidine blue for light microscopy. Ultrathin sections from the dorsal funiculus at the lumbar level were contrasted with 1% uranyl acetate and lead citrate. Grids were examined with a Phillips Tecnai 12 electron microscope operated at 80 kV.

heat-inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin–streptomycin (Gibco, Carlsbad, CA, USA). Neurons were allowed to grow for 2 days and then fixed with icecold 4% paraformaldehyde in 0.1 M PBS, pH 7.2–7.4 for 10 min, followed by three 5 min washes in PBS. Cells were blocked in 0.1% Triton X-100, 5% gelatin from cold water fish (Sigma, St. Louis, MO, USA) in PBS for 2 h and immunostained with polyclonal rabbit anti-MAP2 (1:1000, Abcam) in blocking solution overnight at 4 °C. Cells were incubated with fluorescent-labeled antibodies (1:1000, Invitrogen) for 2 h, washed three times in 0.1 M PBS and covered with Fluoro-mount (Electron Microscopy Sciences, Hatfield, PA, USA). For data analysis a microscope fitted for fluorescence and equipped with a computer-driven digital camera was used. The quantification of neurite length was performed blinded to the condition, from MAP2-stained images for a total of 30 images per animal per condition. Using the NIH ImageJ software, images were calibrated and the longest neurites were measured.

Conditioning lesion Behavioral analysis Mice were anesthetized with a single dose of 330 mg/kg I.P. of 2-2-2 Tribromoethanol (Sigma, St. Louis, MO, USA). Sciatic nerve was exposed and injured by compression (3 times 5 s) with fine Dumont no. 5 forceps near to the Notch. Dorsal root ganglia (DRG) from L3, L4 and L5 spinal levels were extracted seven days after injury and cultured.

To evaluate the locomotor recovery of the animals we used two behavioral paradigms, the Basso-Mouse Scale and Grid Walking. Animal handling for locomotor recovery began 7 days before the SCI surgery. Basso-Mouse-Scale

Adult DRG neuron culture and neurite growth assay Primary DRG cultures were prepared from control and preinjured (conditional lesion) adult mice (WT and WldS) as previously described (Scott, 1977). Briefly, ganglia were removed from L3, L4 and L5 spinal levels and chemically dissociated in 3% collagenase type I (Gibco, Carlsbad, CA, USA) for 1.5 h at 37 °C, followed by incubation in 0.25% trypsin (Gibco, Carlsbad, CA, USA) for 25 min at 37 °C and finally mechanically dissociated by gentle trituration through a Pasteur pipette. Dissociated cells were plated on poly-L -lysine (0.1 mg/ml, Sigma, St. Louis, MO, USA) and laminin (1 μg/ml, Millipore, Billerica, MA, USA) coated covers. The growth medium was DMEM/F12 (Gibco, Carlsbad, CA, USA) supplemented with 10%

Hindlimb motor function was assessed in an open-field test, using the nine-point Basso-Mouse-Scale (BMS) locomotor rating scale described previously (Basso et al., 2006; Valenzuela et al., 2012). The gait of the mouse was observed for 4 min and scored according to the BMS by two independent investigators blinded to the experimental condition. The animals were tested 1–3 days before the surgery and at days 1, 3, 7, 10, 14, 21, 28 and 35 after SCI. Our laboratory was certified during the “2009 Spinal Cord Injury Research Training Program” at Ohio State University, USA, to perform BMS assays. Two exclusion criteria were used to validate accuracy of hemisection SCI: (i) movement in the right hindlimb or paralysis in the left hindlimb at day 1 postinjury; and (ii) paralysis in the right hindlimb at day 7 post-injury.

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The animals that presented any of these criteria were excluded from the study. To assess finer locomotor abilities, the BMS subscore was calculated based on previously described parameters (Basso et al., 2006). To complement the subscore information, each subscore parameter was isolated and plotted as percentage of animals displaying the top performance of each parameter. The parameters evaluated were weight support (present), plantar stepping (consistent), plantar placing (parallel–parallel), coordination (mostly), trunk stability (normal) and tail position (up). Grid walk The mouse was placed on an elevated wire grid as described elsewhere (Ma et al., 2001). Performance on the grid was videotaped for 3 min during which a minimum of 30 s of walking was required. The number of footfalls and the total walking time were counted from videotapes and expressed as number of footfalls per second. A footfall was defined as hindlimb slip between the wires as it moves along the grid with a weight-bearing step. Only the animals that demonstrated consistent plantar stepping in the BMS at 14 days post-injury were tested on the grid. The grid walk test was performed 1 to 3 days prior the surgery and at days 14, 21, 28 and 35 after the injury. Statistical analysis Data are shown as mean ± SEM. Statistical evaluation was tested using Prism 5.0 software for Windows (GraphPad Software Inc.). Tracing efficiency comparisons between strains was analyzed using a Student's t-test. To simplify the statistical analysis of the collateral sprout profile, the profile matrix was averaged every 100 μm. Anatomical data comparing inter-strain collateral sprout profile at single time points (main text figures) was analyzed using two-way ANOVA test, followed by Bonferroni post hoc test for multiple comparisons. Anatomical data comparing intra-strain collateral sprout profile at different time points (supplementary figures) and In vitro neurite growth assay data were analyzed using one-way ANOVA test, followed by Bonferroni post hoc test for multiple comparisons. The behavioral data was analyzed using two-way repeated-measures ANOVA, followed by a Bonferroni post hoc test for multiple comparisons. The significance thresholds were *p b 0.05, **p b0.01 and ***p b0.001. Results Wallerian degeneration of spinal cord axons after SCIH is delayed in WldS mice After partial SCI, both ascending and descending axonal tracts are usually injured. Unilateral lesion of the right side of the spinal cord at T13 level (lateral hemisection, SCIH) interrupts all ascending and descending projections of the right side, but left intact axonal tracts on the left side (Figs. 1A, 2A). This injury model leads to a Brown–Séquard syndrome that consists in loss of sensation and motor function ipsilaterally and below the lesion level. Due to the surgical difficulty in performing a perfect hemisection, we defined our hemisection as successful according to the behavioral outcome. Any animal showing incomplete motor deficit in the right hindlimb, due to incomplete lesion, or paralysis in the left hindlimb, due to over lesion, was excluded from the study. To complement the injury extention estimation, we evaluated the number of BDA-labeled CST fibers in the contralateral CST main tract. We found no differences in the number of BDA-labeled CST fibers between animals at the cervical or lumbar level or in the cervical to lumbar ratio between the mouse strains (Figs. 2C–D). Also, no statistical differences were found between control and injured animals in both strains (data not shown). This suggests that the hemisection performed does not interrupt the contralesional CST axons in the two mouse strains used in this study.

Fig. 2. Spinal cord hemisection and CST anterograde tracing. (A) Representative mouse spinal cord transverse sections taken at the T13 vertebral level from WT and WldS animals at 7 and 35 dpi stained for BDA (green), GFAP (red) and DAPI (blue). Scale bar, 100 μm (B) Left: Representation of BDA injection coordinate of the sensorimotor cortex taken from Frankling and Paxinos (2007). Red inset highlights the specific injection region. Middle: BDA-injected mouse brain coronal section, a clear staining is found in the boxed area. Scale bar, 100 μm. Right: Magnification of the injection site in the sensorimotor cortex. Scale bar, 200 μm. (C) Total CST fibers traced at the cervical and lumbar level of the spinal cord in WT and WldS mice, no difference in the mean of labeled fibers is observed. (D) Cervical to lumbar ratio (C/L) in WT and WldS mice, no differences in the tracing efficiency is observed. Values represent means ± SEM. Student's t-test between strains at cervical level, lumbar level and C/L ratio comparisons. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The ascending and descending axonal tracts degenerating after SCIH can be defined by neuroanatomical studies (Anderson et al., 2009). After a thoracic SCIH, the main descending motor tract affected includes the corticospinal tract (CST), the reticulospinal tract (ReST) and the vestibulospinal tract (VST). On the other hand, the main ascending tracts undergoing Wallerian degeneration after SCIH, include the dorsal column ascending tract (DCAT) and the ventrolateral ascending tract (VLAT) (Fig. 3A). To determine the progression of Wallerian degeneration in WT and WldS mice after SCIH, we analyzed white tract morphology at cervical and lumbar regions in uninjured mice and at 7, 14 and 35 days after SCIH (dpi) in semithin sections. No morphological differences were observed in sham operated WT and WldS mice in both ascending VLAT and descending VST (Fig. 3B). After SCIH, a temporal progression of degenerative changes in ascending and descending white matter tracts was observed in WT mice (Fig. 3B). From 7 dpi onward, WT animals presented

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Fig. 3. Wallerian degeneration of ascending and descending tracts after SCIH in WT and WldS mice. (A) Schematic showing the major ascending and descending axonal tracts undergoing Wallerian degeneration after SCIH, which is denoted by a black triangle. The figure highlights the ventrolateral ascending tract (VLAT) at the cervical level and the vestibulospinal tract (VST) at the lumbar level. The animals were subjected to an SCIH and Wallerian degeneration of the affected tracts was evaluated at 7, 14 and 35 dpi by semithin sections or electron microscopy. (B) Toluidine blue staining of semithin sections of the VLAT and VST at the cervical level in WT and WldS mouse strains. Following SCIH a progression in the pathological appearance of white matter is appreciated and characterized by the presence of collapsed myelin (arrowheads). Scale bar, 10 μm. (C) Electron microscopy of the CST at the lumbar level after SCIH in WT and WldS mouse strains at 7, 14 and 28 dpi. At the ultrastructural level, progressive myelin sheet disorganization, axoplasm vacuolization and mitochondria enlargement are observed after SCIH. Scale bar, 1 μm. WT; control (n = 1), 7 dpi (n = 1) and 35 dpi (n = 1). WldS; control (n = 1), 7 dpi (n = 1) and 35 dpi (n = 1).

collapsed axons characterized by dense myelin debris and disorganized myelin sheets at both VLAT and VST (Fig. 3B). On the other hand, the WldS mice exhibit delayed axonal degeneration in both VLAT and VST axonal tracts (Fig. 3B). At 35 dpi, WldS mice exhibited the first signs of structural disorganization with collapsed axons and disrupted myelin sheets (Fig. 3B). We further evaluate the dynamics of axonal degeneration of CST fibers by electron microscopy. The CST tract originates in layer V of the primary motor cortex and project to all spinal cord levels through a crossed dorsal component that contains 95% of all descending axons (Oudega and Perez, 2012). In sham-operated animals, myelinated axons display normal appearances in both mouse strains (Fig. 3C). One week after SCIH, CST fibers of WT animals presented morphological changes associated with axonal degeneration, including vacuolization of the axoplasm, myelin sheet disorganization, loss of cytoskeletal components and enlarged mitochondria (Fig. 3C). However, axotomized WldS axons in the CST still presented an intact appearance (Fig. 3C). Two weeks after injury, axotomized WldS axons start to exhibit subtle signs of degenerations such as enlarged mitochondria and loss of cytoskeletal organization in the axoplasm. Finally, 28 days after SCIH, axonal degeneration of the CST was extensive in both mouse strains (Fig. 3C).

Taken together, these results demonstrate that unilateral spinal cord hemisection in the WldS mice represents an excellent model to study the influence of axonal degeneration in spinal changes after SCI, as Wallerian degeneration is delayed between 1 and 2 weeks in both ascending and descending tracts.

Axonal degeneration is required for collateral sprouting after SCIH After SCI, non-injured axons form collateral extensions that invade gray matter regions at spinal levels distant from the injury site (Ballermann and Fouad, 2006; Maier and Schwab, 2006). To assess collateral sprouting from CST axons, the anterograde tracer BDA was injected unilaterally into the right motor cortex projecting to lumbar regions (Fig. 2B) (see Materials and methods for specific injection coordinates). Fourteen days postinjection, in both WT and WldS mice, the extent of CST labeling was comparable at both cervical and lumbar levels (Figs. 2C–D). After a right SCIH at the T13 spinal level, collateral sprouting from intact BDA-labeled CST fibers was determined at cervical levels at different times after damage. Distributions of collateral sproutings from

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Fig. 4. Collateral sprouting from CST at cervical level is delayed in WldS mice after SCIH. (A, C and E) Low-magnification images of transverse sections of the spinal cord in a gray matter region close to the left and right sides of the CST. In blue, drawings of BDA-labeled CST axons. Scale bar, 100 μm. In the right side spinal cord schematic representation and labeled CST axons that originate from the uninjured fibers at the C3–C5 level in WT (blue) and WldS (red) mice at control (A), 7 dpi (C) and 35 dpi (E). Scale bar, 500 μm. (B, D and F) Collateral sprouting profile of the contralateral and ipsilateral horn, comparing WT and WldS mice at control (B), 7 dpi (D) and 35 dpi (F). WT animals exhibit progressive growth of collateral sprouts after SCIH compared to the WldS mice. Values represent means ± SEM. Two-way ANOVA followed by Bonferroni's post-hoc test. Significance is plotted as colored scale on the top of each graph. WT; control (n = 8), 7 dpi (n = 8) and 35 dpi (n = 6). WldS; control (n = 6), 7 dpi (n = 11) and 35 dpi (n = 4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

intact CST were measured in the gray matter ipsilateral (IL) and contralateral (CL) to the hemisection. In non-injured adult WT mice, axons arising from the hindlimb motor cortex send collaterals into the gray matter of the cervical spinal cord (Fig. 4A). Compared to the IL horn, the collateral distribution was much higher in the CL horn, corresponding to the side of the labeled CSTs. Similar values and distribution of basal collaterals were observed between non-damaged WT and WldS mouse strains (Fig. 4B). Seven days after SCIH, WT animals showed a nearly two-fold increase in collaterals sprouting to the CL and IL sides of the gray matter compared to control condition (Figs. 4C–D, Fig. S1B). Thirty-five days after SCIH, collateral sprouting in WT animals stabilizes with little further increase towards the CL (Figs. 4E–F and S1B). However, collateral sprouting towards the IL increases reaching significant differences at 200 μm from the midline. On the other hand, the WldS mice exhibit no significant increase in collateral sprouting at 7 or 35 days after SCIH compared to control conditions (Figs. 4A–F and S1C). Significative differences in collateral sprouting towards IL and CL horn are appreciated at 7 (Figs. 4C–D) and 35 (Figs. 4E–F) days after SCIH in WT animals compared to WldS mice.

To assess if the lack of axonal sprouting observed in the WldS mice was due to an intrinsic impairment of axon growth capacity, we evaluated the growth potential of adult DRG neurons in vitro. DRG neurons were obtained from both uninjured mice and after a sciatic nerve conditional lesion (CL) in both WT and WldS mouse strains. We found no significative differences in neurite length in either control or CL conditions between WT and WldS mice (Figs. 5A–B). After a conditional lesion of the sciatic nerve, WldS DRG neurons present a two-fold increase in neurite length that was comparable to the length attained by WT DRG after a sciatic nerve CL (Fig. 5B). These results suggest that the intrinsic axonal growth capabilities are not impaired in the WldS mice and that the lag in axonal sprouting observed may be mainly due to the delay in the axonal degeneration process. Axonal degeneration is required for collateral sprouting after SCIH distal to the injury site Considering the differences in sprouting response between WT and WldS mice observed at the cervical level after SCIH, we wanted to evaluate collateral extension of spared fibers below the lesion, at lumbar

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Fig. 5. In vitro adult DRG neurite growth is not affected in the WldS mice. (A) Low magnification images of adult WT and WldS DRG neurons stained with MAP2 in control and after a sciatic nerve conditional lesion. Scale bar, 100 μm. (B) Neurite length quantification in control and after a conditional lesion. The conditional lesion enhances neurite growth in both WT and WldS DRG neurons. No statistical differences between strains are observed at control conditions or after a conditional lesion. Values represent means ± SEM. One-way ANOVA followed by Bonferroni's post-hoc test. *p b 0.05; **p b 0.01 compared to control conditions. WT; control (n = 3), CL (n = 3). WldS; control (n = 3), CL (n = 3).

regions L3 to L5. Related to similar measurement at cervical levels, the number of collaterals was higher at lumbar regions, which is expected as the BDA-injection site was performed in the motor region corresponding to the hindlimb representation. In control non-injured WT or WldS mice, comparable density of collaterals extends to both IL and CL sides of the gray matter (Figs. 6A–B). No significant differences in collateral sprouting profile distribution along the gray matter was observed at 7 days postinjury of both WT and WldS mice compared to control (Figs. S2B–C). However, 35 days after the SCIH, a nearly two-fold increase in collateral sprouting to the IL side was observed in WT animals compared to control condition (Figs. 6E–F and S2B). As in cervical level, the WldS strain shows no collateral sprouting at the lumbar level after an SCIH at any time point evaluated. Taken together, these results suggest that Wallerian degeneration, which propagates to both cervical and lumbar regions after SCIH, is a pivotal event for triggering collateral sprouting from non-injured CST fibers at the cervical level. Locomotor recovery after SCIH is delayed in WldS mice Previous reports had shown that collateral sprouting enhances locomotor recovery after SCI through formation of new intraspinal circuits bypassing the injured zone (Bareyre et al., 2004; Courtine et al., 2008; Weidner et al., 2001). Therefore we assessed locomotion recovery after T13 hemisection using the BMS open-field score (Basso et al., 2006) in WT and WldS mice. One day after SCIH, WT and WldS mice showed complete paralysis of the right hindlimb and no functional changes in the left hindlimb (Fig. 7A and data not shown). After SCIH, WT mice partially recovers the locomotor ability with an initial phase of rapid increase in the BMS score in the first 2 weeks after injury and a more stable plateau the following 3 weeks (Fig. 7A). On the other hand, the WldS mice present a delayed recovery of locomotion compared to WT animals, reaching the plateau 1 week later than the wild type mice (Fig. 7A). The recovery curves of both animals separate from each other at 7 dpi. In that time point, the WldS mice exhibited a significant lower BMS score than WT mice (Fig. 7A). This difference was maintained until 14 dpi (Fig. 7A). At 21 dpi, both mouse strains already reached the plateau and no statistical differences were observed. The BMS score does not discriminates differences in fine locomotion capabilities after animals reached frequent stepping (Basso et al., 2006), therefore

to assess finer locomotion abilities, we calculated the BMS subscore (see Materials and methods). Using this scale, Wlds mice show decreased locomotor capabilities compared to WT mice at any time point evaluated (Fig. 7B). To complement the subscore information, we isolated the individual parameters evaluated by the BMS for the right hindlimb (see Materials and methods for details). In all evaluated parameters, except for trunk stability, WT animals achieved better performance before WldS animals (Fig. 7D). The most determinant parameters in the BMS subscore calculation are the plantar placing, coordination and trunk stability. The WT animals present better performance earlier than WldS mice in plantar placing and coordination. Neither of the mouse strains reaches good performance in trunk stability. Therefore, WT animals not only perform better and faster than WldS, but also improve the most relevant parameters of the BMS subscore. As the CST has been associated to the control of fine locomotor movements (Lemon and Griffiths 2005), we also determine the locomotor performance of our experimental mice using the grid walk, which assess finer aspects of locomotion. At 14 and 21 days after SCIH, WT mice exhibited less errors in this test WldS animals, confirming the BMS results (Fig. 7C). Taken together, these results demonstrate that locomotor recovery associated to partial spinal damage is hindered when axonal degeneration is delayed. Considering the reported association between collateral sprouting and locomotor recovery, these results suggest that the axonal degenerative mechanism initiates a cascade of events leading to regenerative processes that culminate in partial recovery of locomotor function. Discussion In the present study we investigated the role of the Wallerian degeneration in collateral sprouting of contralesional spared CST fibers after an SCIH. By using WT and WldS mice, we showed that collateral sprouting of undamaged CST fibers is severely reduced if Wallerian degeneration is delayed. Functional recovery after SCIH assessed by well characterized open field and skilled locomotor tests, demonstrates that delayed Wallerian degeneration response leads to diminished locomotor recovery, consistent with the demonstrated positive role of collateral sprouting in functional recovery after spinal cord injury. In the peripheral nervous system (PNS), Wallerian degeneration of axons is required for axonal regeneration (Court and Alvarez, 2000). In addition, a degenerating nerve tissue can elicit a sprouting response

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Fig. 6. Collateral sprouting from CST at lumbar level is delayed in WldS mice after SCIH. (A, C and E) Low-magnification images of transverse sections of the spinal cord in a gray matter region close to the left and right sides of the CST. In blue, drawings of BDA-labeled CST axons. Scale bar, 100 μm. In the right side spinal cord schematic representation and labeled CST axons that originate from the uninjured fibers at the L3–L5 level in WT (blue) and WldS (red) mice at control (A), 7 dpi (C) and 35 dpi (E). Scale bar 400 μm. (B, D and F) Collateral sprouting profile of the contra and ipsilateral horn, comparing WT and WldS mice at control (B), 7 dpi (D) and 35 dpi (F). A significant increase in the collateral sprouting towards the ipsilateral horn at 35 dpi is appreciated in the WT mice compared with the WldS animals. Values represent means ± SEM. Two-way ANOVA followed by Bonferroni's post-hoc test. Significance is plotted as colored scale. WT; control (n = 6), 7 dpi (n = 5) and 35 dpi (n = 5). WldS; control (n = 6), 7 dpi (n = 5) and 35 dpi (n = 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

in undamaged motor axon terminals (Diaz and Pecot-Dechavassine, 1990), suggesting that microenvironmental changes after axonal degeneration are sufficient to stimulate the sprouting of collateral from intact axons. The WldS mice constitute a well characterized model to study the functional role of axonal degeneration in tissular responses associated to mechanical injury or neurodegenerative conditions (Lingor et al., 2012; Zheng et al., 2006). The expression of the mutant Wallerian degeneration slow (WldS) protein strongly delays axonal degeneration after injury, both in the PNS and the CNS (Lunn et al., 1989; Perry et al., 1991). The delay in Wallerian degeneration in WldS mice is an intrinsic property of axons and does not depend on the genetic modification in glial cells (Glass et al. 1993) or macrophages (Perry et al. 1990). Nevertheless, delayed axonal degeneration leads to a delay in glial cell activation and a slower immune reaction (Fujiki et al., 1996). After SCIH in the WldS mice, oligodendrocytes near the injury zone remain viable until axon degeneration begins (Dong et al. 2003). Importantly, processes such as developmental pruning (Hoopfer et al. 2006) or peripheral nerve cell-autonomous axonal regeneration are not affected

in the WldS mice (Court and Alvarez, 2000). Our results show that WldS adult DRG neurons exhibit equivalent neurite extension capabilities in control and after a conditional lesion paradigm to WT neurons, confirming the notion that the intrinsic axonal growth capacity is not affected by the WldS protein. Also, our results demonstrate that axonal degeneration elicited after an SCIH is delayed in the WldS mice compared to the WT animals. We evaluated the axonal collateral sprouting at 7 and 35 days after injury. At the cervical level, we found collateral sprouting in WT mice as early as 7 days at both the IL and CL sides of the injury. But at the lumbar level, we only found a significant increase of collaterals at 35 days after injury and exclusively towards the IL horn. This difference in the normal sprouting response between cervical and lumbar levels could be due to a combination of events, including a differential rate of axonal degeneration between sensory and motor tracts or differential sprouting/branching ratio. It has been suggested that axonal degeneration occurs faster in the sensory ascending tracts than in the motor descending ones (Warden et al., 2001). Additionally, it has been demonstrated that after a focal EAE lesion to the CST, the axonal

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Fig. 7. Locomotor recovery after SCIH is delayed in WldS mice. (A) BMS scores in WT (blue) and WldS (red) until 35 dpi. A significative difference in the BMS score is observed at 7, 10 and 14 dpi in WT mice compared to WldS animals. (B) BMS subscore of the same animals. A significative difference between both strains is observed at every time point evaluated. (C) Gridwalk test, measured as the number of footfalls per second. A significative decrease in footfalls is exhibited in WT mice compared to WldS animals. Figures represent the average score for the right hindlimb. Values represent means ± SEM. Two-way Repeated Measures ANOVA followed by Bonferroni's post-hoc test. *p b 0.05; **p b 0.01; ***p b 0.001. WT (n = 8) and WldS (n = 10). (D) Isolation of individual subscore parameters for the right hindlimb. In each case, values were calculated as percentage of animals exhibiting the maximum performance on one particular parameter: Weight support, plantar placing parallel at initial contact and lift off, plantar stepping, coordination, normal trunk and tail up. WT (n = 8) and WldS (n = 10). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

sprouting below the lesion was not augmented, but the axonal branching ratio increased significantly (Kerschensteiner et al., 2004). This result suggests that due to the highly dense collateral mass present in the lumbar level, the plastic response elicited by an injury is associated with an increase in the formation of new branches from pre-existing collaterals. Our results reinforce this idea, as no change was observed in the CL horn at any time point evaluated, but the IL crossing fibers were increased significantly 35 days postinjury. Our results revealed that collateral extension from spared axons after an SCIH is delayed in the WldS mice. The idea of degenerating axons inducing collateral growth from intact fibers has been previously suggested. Predegenerated nerve grafts induce sprouting from undamaged motoneuron terminals (Diaz and Pecot-Dechavassine, 1990). Also, the WldS mice exhibit less collateral sprouting of dentate gyrus fibers after a lesion to the perforant path (Shi and Stanfield, 1996), and less collateral sprouting of sympathetic fibers into DRG after sciatic nerve constriction (Ramer et al., 1997). Nevertheless, this is the first report of delayed collateral sprouting from spared axons after SCI in WldS mice. We suggest that axonal degeneration is necessary for the plasticity of neural circuits after injury. Nevertheless, collateral sprouting is not always beneficial to animal behavior. After sciatic nerve constriction, WT mice exhibit allodynia and thermal hyperalgesia due to sympathetic sprouting into DRGs and these symptoms are attenuated in WldS mice under the same experimental damage (Ramer et al., 1997). Likewise,

after spinal compression, the WldS mice do not develop autonomic dysreflexia, another consequence of SCI due to small-diameter afferent sprouting (Jacob et al., 2003). This suggests that any therapy focused on the enhancement of collateral sprouting must be focalized on specific axonal tracts (i.e. motor tracts) to avoid the adverse side effects of the sensory sprouting after a spinal trauma. By using the BMS rating scale to assess general locomotion and the grid walk, for skilled locomotion aspects, we showed a diminished locomotor recovery in Wlds mice, in agreement with a previous report (Zhang et al., 1998). Using a more specific assessment of individual locomotor parameters, we found that in the WldS mice, the locomotor recovery does not reach the levels of the WT strain, even though axons eventually degenerate (Fig. 4B). This suggests that after SCI, a temporal window for axonal degeneration-induced collateral sprouting exists, which contributes to the partial locomotor recovery. The identification of the axonal degeneration associated-events underlying the extent of this temporal window will provide an interesting therapeutic tool to enhance axonal collateral sprouting after a CNS injury. Conclusion Taken together, our results suggest that axonal degeneration is a determinant tissular event contributing to collateral sprouting from spared axonal tracts after an SCI and contributing to the spontaneous

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locomotor recovery observed. Also, our results bring up the notion of a temporal window of plastic axonal remodeling that depends on the initiation of the axonal degeneration. Unveiling the mechanisms that contribute to axonal sprouting of the spared fibers after an injury is essential to the generation of effective therapies after damage to the CNS. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.expneurol.2014.07.014. Acknowledgments This work was supported by grants from the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT, no. 1110987), Millennium Nucleus (no. RC-120003), Ring Initiative ACT1109, Fondef-Idea (CA 12I10120) and FONDECYT no. 1070444. We thank the Ohio State University Research Training Program 2009 (SCIRTP) for technical advice, Jaime Alvarez for experimental advice and comments and Monica Perez for outstanding EM processing. References Ackery, A., Tator, C., Krassioukov, A., 2004. 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Sprouting of axonal collaterals after spinal cord injury is prevented by delayed axonal degeneration.

After an incomplete spinal cord injury (SCI), partial recovery of locomotion is accomplished with time. Previous studies have established a functional...
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