Brain Advance Access originally published online on April 21, 2006
Brain 2006 129(6):1534-1545; doi:10.1093/brain/awl087
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BDNF promotes connections of corticospinal neurons onto spared descending interneurons in spinal cord injured rats
1 University of Alberta, Faculty of Rehabilitation Medicine Edmonton, Alberta 2 ICORD (International Collaboration on Repair Discoveries), Department of Zoology, University of British Columbia Vancouver, BC, Canada
Correspondence to: K. Fouad, University of Alberta, Faculty of Rehabilitation Medicine, 3-48 Corbett Hall, Edmonton, Alberta, Canada T6G 2G4 E-mail: karim.fouad{at}ualberta.ca
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Although regeneration of injured axons is inhibited within the adult CNS, moderate recovery can be found in patients and animals with incomplete spinal cord injury (SCI). This can be partly attributed to sprouting of spared and injured axons, rostral and caudal to the lesion, respectively. Recently, it has been reported that following a thoracic SCI such sprouting can result in indirect reconnections of the lesioned axons to caudal targets via propriospinal interneurons (PrI). Here, we attempted to further promote this spontaneous repair mechanism by applying the neurotrophic factor BDNF (brain-derived neurotrophic factor), in the vicinity of the cell bodies of lesioned corticospinal neurons or NT-3, intrathecally to the cervical spinal cord. We performed a dorsal over-hemisection at the thoracic spinal cord sparing only the left ventrolateral quadrant. This type of lesion did not promote sprouting of injured corticospinal axons or re-routing via commissural PrI. Also, in rats that received NT-3 at the cervical enlargement, no increase in sprouting was found. However, animals receiving BDNF at the cell bodies of lesioned corticospinal neurons showed a significant increase in collateral sprouting and in the number of contacts with PrI. This was not observed when BDNF was administered to unlesioned animals. Although no statistical difference in the horizontal ladder walking was found between the groups, the increase in collateral sprouting and in the number of contacts correlated with the functional recovery. Hence, cell body treatment can promote plasticity of the injured CNS and may be a valuable treatment approach in conjunction with local regeneration promoting strategies.
Key Words: spinal cord injury; rats; plasticity; neurotrophin; intraspinal circuit
Abbreviations: BDA, biotinylated dextran amine; BDNF, brain-derived neurotrophic factor; CST, corticospinal tract; NT-3, neurotrophin 3; PrI, propriospinal interneuron; SCI, spinal cord injury
Received January 25, 2006. Revised March 10, 2006. Accepted March 14, 2006.
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Introduction |
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Recovery following spinal cord injury (SCI) is limited because severed axons of the CNS fail to regenerate (Schwab and Bartholdi, 1996
). Nevertheless, some recovery of sensory and motor function will occur over the first few weeks following incomplete injuries. This recovery may be attributed to several mechanisms. First, an initial phase of recovery a few days after the injury results from the re-establishment of transmission in uninjured axons, which was blocked during the period of spinal shock (Holaday and Faden, 1983; Hiersemenzel et al., 2000; Ditunno et al., 2004). Second, a later phase of recovery is attributed to re-myelination of spared axons (Jeffery and Blakemore, 1997). Third, long-term recovery may occur, including the acquisition of compensatory movement strategies, but the mechanisms involved in this acquisition are not clearly understood (Helgren and Goldberger, 1993; McKenna and Whishaw, 1999; Grasso et al., 2004). They may include axonal sprouting and synaptic rearrangements in spared neuronal circuits rostral and caudal to the lesion (often referred to as plasticity). Recent studies have shown that the adult CNS is indeed capable of injury-induced plasticity accompanied by moderate functional recovery [reviewed in Ivanco and Greenough (2000) and Raineteau and Schwab (2001)]. Examples of such plasticity include the reorganization of cortical maps following brain (Jones and Schallert, 1994; Bury and Jones, 2002), peripheral nerve (Wu and Kaas, 1999) and spinal cord injuries (Bruehlmeier et al., 1998; Fouad et al., 2001; Turner et al., 2003) in humans and animal models. In addition to cortical plasticity, growth of collaterals arising from injured and spared corticospinal tract (CST) fibres has been found following SCI in rats rostral (Fouad et al., 2001) and caudal (Weidner et al., 2001) to the lesion. CST collaterals rostral to the lesion were reported to eventually connect onto spared propriospinal interneurons (PrIs), to form new intra-spinal circuits (Bareyre et al., 2004).
The mechanisms promoting injury-induced plasticity at the cellular level are not well understood. Progress in gene profiling has indicated that growth-associated genes and neurotrophic factors [e.g. NT-3, BDNF (brain-derived neurotrophic factor)] are upregulated following injury (Hayashi et al., 2000; Song et al., 2001; Di Giovanni et al., 2003). Hence, spinal cord-derived factors could promote sprouting and the subsequent rewiring of existing pathways. Applications of BDNF and NT-3 to the spinal cord, for example, have been reported to promote neuronal survival and sprouting of the lesioned neurons at the injury site (Schnell et al., 1994; Bregman et al., 1997; Grill et al., 1997; Ye and Houle, 1997; Bradbury et al., 1999; Hammond et al., 1999; Liu et al., 1999). In addition, when BDNF was applied near the cell bodies it prevented neuronal death and/or atrophy of corticospinal or rubrospinal neurons (Giehl and Tetzlaff, 1996; Kobayashi et al., 1997; Hammond et al., 1999) and promoted regenerative sprouting of injured CST axons (Hiebert et al., 2002). Here, we investigated whether sprouting of CST axons and their connection onto PrI rostral to an injury could be enhanced by applying BDNF intraparenchymally near corticospinal neuron cell bodies or NT-3 intrathecally to the rostral (cervical) spinal cord.
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Experiments were conducted using adult female Lewis rats (Charles River, 180–200g) that were kept at a 12 : 12 h light–dark cycle with water and food provided ad libitum. This study was approved by the local animal welfare committee and complies with the guidelines of the Canadian Council on Animal Care.
We examined seven experimental groups: controls (animals that received no spinal cord lesion and no treatment, n = 7), uninjured rats that received vehicle solution at the cortex (n = 3), uninjured rats that received BDNF at the cortex (n = 3), rats with a thoracic (Th) SCI (details given below) that received vehicle at the cortex (n = 6), rats with a Th SCI that received BDNF at the cortex (n = 8), rats with a Th SCI that received vehicle solution at the cervical enlargement (n = 3) and rats with a Th SCI that received NT-3 at the cervical enlargement (n = 9).
Lesions
All operations were performed under Hypnorm (fentanyl/fluanisone; Janssen Pharmaceutics, Beerse, Belgium; 120 µl/200 g body weight) and midazolam (Sabex, Canada; 0.75 mg in 150 µl/200g body weight, 750 µl of total volume diluted with H2O) anaesthesia. Eye lubricant (Tears naturale®, Alcon Canada, Inc., Mississauga, ON, Canada) was applied to protect the eyes from dehydration.
A laminectomy of half of one vertebral lamina at Th8 was followed by a right-sided dorsal over-hemisection using a customized micro-blade. This lesion ablated 
70% of the spinal cord and spared mainly the left ventrolateral quadrant. The surgeon was blind to the subsequent treatment of the animals. Following the lesion procedure the dorsal back musculature was sutured in layers and the skin closed with surgical clips. Using this lesion rather than a dorsal hemisection [as used in earlier studies by Bareyre et al. (2004) and Fouad et al. (2001)] we anticipated easier anatomical evaluation due to a lower number of retrogradely labelled interneurons. This lesion also allowed the characterization of the projection pattern of the PrIs and focus on commissural interneurons. Sprouting on a defined population of interneurons like the commissural had not been studied before.
After surgery the animals were kept on a thermostatically regulated heating pad until completely awake. The analgesic Buprenex (buprenorphine; Reckitt & Colman, Richmond, VA, USA) was administered subcutaneously (0.03 mg/kg) immediately after operation, and every 8 h for 72 h. Ringer solution (4 ml) was given subcutaneously daily for the first week and at later stages if animals showed signs of dehydration.
Neurotrophin application
During the same operation as the lesion procedure, animals were randomly divided into the different experimental groups and received either BDNF (donated by Regeneron Pharmaceuticals/Amgen, Tarrytown, NY, USA) or vehicle at the left sensory-motor cortex, or NT-3 (donated by Regeneron Pharmaceuticals) or vehicle delivered to the cervical spinal cord.
The BDNF or vehicle application to the left motor cortex was performed as described earlier (Hiebert et al., 2002). In brief, a hole in the cranium was made at coordinates 2.0 mm lateral and 1.5 mm caudal from bregma. A cannula from the Alzet brain infusion kit (Durect Corporation, Cupertino, CA, USA) was inserted 1.5 mm into the motor cortex. Two stainless steel screws inserted into the parietal plates were used to secure the cannula using dental acrylic. The infusion cannula was connected to a 14 day Alzet osmotic mini-pump (0.5 µl/h) that was placed subcutaneously in the back. The osmotic pump was filled with either vehicle solution (0.25% rat serum albumin in 0.1 M phosphate buffered saline) or BDNF (1 µg/µl in vehicle).
The application of NT-3 or vehicle to the cervical enlargement was performed by the intrathecal insertion of a fine catheter (Recathco, LLC, Allison Park, PA, USA) at the cisterna magna as described earlier (Yaksh and Rudy, 1976). The catheter tip was inserted subdurally and gently moved until it reached the C3 segment. The catheter was connected to an Alzet mini-osmotic pump (0.5 µl/h) that was placed subcutaneously in the back and filled with either vehicle (see above) or NT-3 (1 µg/µl in vehicle). The pump and catheter were removed after 2 weeks.
Behavioural testing
One week before the lesion operation the animals were trained to walk on a 1 m long horizontal ladder with metal rungs, elevated 30 cm from the ground. A testing session consisted of three ladder passes and performance was captured using a digital video camera (Canon, Optura 100, 60 fields/s). The analysis involved counting the number of errors in placing of the right hind limb (the side of the spinal cord that has been in most animals completely ablated) and averaging errors from the three passes for each animal. A defined 10-bar sector was chosen for analysis. To prevent habituation to a fixed bar distance, the bars in this sector were placed irregularly (1–3 cm spacing) and were changed for each testing session. The animals' performance was monitored weekly up to 4 weeks after surgery. Animals were excluded from behavioural testing if they were not able to walk on the horizontal ladder (>8 mistakes/trial).
Tracing
Four weeks after lesion (2 weeks after removing the mini-pumps), retrograde tracing of PrIs projecting into the lumbar enlargement was achieved by bilateral injection of 1 µl of 5% FluoroGold (Chemicon International, Inc., Temecula, CA, USA) into the L2 segment of the spinal cord. This retrograde tracer is taken up by injured axons and/or nerve terminals in the proximity of the injection site. Under anaesthesia (see above) rats were held in a stereotaxic device and a laminectomy of half of the T12 vertebral segment overlying the L2 spinal segment was performed. The lumbar vertebra was lifted using tissue forceps mounted onto a micro-drive to stabilize the spinal cord. A microcapillary (glass electrode) with an approximate tip diameter of 20–30 µm was mounted onto a 5 µl Hamilton syringe and lowered into the exposed spinal cord 200–300 µm lateral to the mid-line (depending on the location of blood vessels) and to a depth of 400 µm. The capillary remained in place for 1 min following the injection.
In the same surgery as the retrograde tracing, the CST was anterogradely labelled in the left cortex (the side that projects into the right side of the spinal cord) using a 10% solution of biotinylated dextran amine (BDA 10 000 MW, Molecular Probes, Invitrogen Corporation, Burlington, Ontario). Using a glass microelectrode mounted onto a Hamilton syringe, 1 µl of BDA was slowly (over 3 min) injected into the hind limb area of the motor cortex (2 mm lateral and 1.6 mm caudal to bregma, at a depth of 1.5 mm). The electrode remained in place for another 3 min following the injection.
Two weeks following the tracer injections the rats were perfused transcardially with a Ringer's solution containing 100 000 IU/l heparin, followed by 4% paraformaldehyde solution in 0.1 M PB with 5% sucrose as fixative. The spinal cords and brains were removed, post-fixed overnight in 4% formaldehyde and then transferred to a 30% sucrose solution for 3 days. Thereafter, the spinal cords and brains were embedded with Tissue Tek (Sakura Finetek USA Inc., CA, USA) and frozen at –60°C.
Anatomical analysis
Using a cryostat, 25 µm sections were taken through different areas of the CNS. Sagittal sections of the brain were taken at the BDA injection site. Cross-sections of the C1 segment were taken to count the total number of traced CST fibres. The cervical enlargement (C3–6), as well as the upper thoracic spinal cord (Th2–7), were sectioned horizontally to quantify CST collaterals, PrI, CST collateral contacts with PrI and CST collateral projections. Cross-sections through the lesion at Th7–9 were used to analyse lesion size. Finally, cross-sections of the C2 segment were used to analyse the effects of NT-3 application onto calcitonin-gene related peptides (CGRP) (see below) positive fibres. Unbiased blind analysis of the tissue was ensured by number coding of the slides.
Staining for BDA was performed according to earlier reports (Fouad et al., 2001). Slides were dried in an incubator at 38°C for 1 h and washed twice for 10 min in 50 mM Tris-buffered saline (TBS), pH 7.4, followed by two 45 min washes with TBS containing 0.5% Triton X-100. Afterwards, the slides were incubated overnight with an avidin–biotin–peroxidase complex in TBS with Triton (ABC Elite, Vector Laboratories, Burlingame, CA, USA) according to the instructions of the manufacturer. Subsequently, the DAB reaction was performed using the Vector DAB kit (SK4100, Vector Laboratories). The reaction was monitored and stopped by extensive washing in water. FluoroGold was detected on the same slides using primary FluoroGold antibodies (1 : 1000 Chemicon), incubated overnight at 4°C followed by washing in TBS. The secondary antibody (Vector) was incubated overnight at 4°C and visualized with the Vector Nova Red kit. The slides were dehydrated with alcohol and cleared with xylene and coverslipped in Permount (Fischer Scientific Ltd. Ottawa, ON, Canada).
Evaluation of BDA injection site
To confirm that the BDA injection in the cortex was restricted to the hind limb area, the cortices were sectioned in the sagittal plane and stained for BDA (see above) and spreading of BDA was measured at the injection site. BDA did not spread >1.5 mm from the injection site in any of the animals included in this study (and thus did not approach the fore limb area).
Counting traced CST axons
To avoid spread of the tracer into cortical fore limb areas, BDA was only injected at one position with a fairly low volume. This resulted in low numbers of traced fibres, thus allowing precise counting. Quantification of the total number of traced CST axons rostral to the injury was performed on C1 cross-sections using light microscopy (x40). Pictures were taken using a digital camera mounted on a Leica microscope, and a grid overlying the pictures was used to assist counting of the traced axons.
Counting of PrI and contacts between CST collaterals and PrI
Nova Red-stained PrI cell bodies were counted on the same horizontal sections used for collateral analysis (see below). A contact was scored when a bouton-like structure of a BDA-positive fibre was co-localized with the soma or dendrite of a Nova Red-stained PrI (Fig. 5A). Normalization of these contacts was performed comparable with that by Bareyre et al. (2004). Therefore, the counted number of contacts was divided by both the number of PrI (to account for variability in the retrograde tracing) and the number of collaterals (to account for the variability of collaterals), or the number of PrI and the total number of CST fibres (normalized contacts = counted no. of contacts/PrI/collaterals, or = counted no. of contacts/PrI/total no. of traced CST fibres).
Counting of CST collaterals
Horizontal sections of the cervical enlargement and the thoracic spinal cord (rostral to the injury) were cut for the quantification of CST collaterals. Every third section was analysed. Collaterals emanating from the traced CST and crossing into grey matter for >30 µm over an approximated line drawn at the white/grey matter interphase were counted under light microscopy (Fig. 3). The length (in mm) of the analysed section was measured and used to normalize the counted collaterals. Finally, as described in Raineteau et al. (2001), the collaterals were expressed as the number of collaterals divided by both the length analysed and by the total number of traced CST fibres (normalized collateral value = no. of collaterals/mm/total no. of traced CST fibres).
To ensure that the number of counted CST collaterals was not influenced by BDNF-induced changes in their diameter, we took pictures of five collaterals/animal using oil immersion light microscopy (x100). The collateral diameter was then measured with the Scion Image (NIH) program.
Projections of CST collaterals
This analysis was performed on ventral sections of the same set of horizontal sections of the cervical spinal cord that were used for the analysis of collaterals and contacts. We divided the spinal cord with an approximated line (indicated in Fig. 6A), separating the grey matter of the right side into a medial and a lateral part. The lateral side contains among others the motoneuron pool innervated by the CST (Kuchler et al., 2002), and the medial side contains among others PrI. BDA-positive CST fibres were counted within these areas using light microscopy (x40).
Analysis of lesion size
Lesion size was analysed on a subsequent series of cross-sections of the Th7–9 spinal segment throughout the lesion. The sections were counterstained with 0.1% Cresyl Violet. Pictures at the maximal lesion size were taken (x10) and reconstructed. The amount of spared white matter was determined using the Scion Image (NIH).
CGRP staining and density measurement
As a positive control for the NT-3 application, we stained CGRP-positive fibres on cross-sections of the C2 segment only in the NT-3-treated or NT-3 control group (Hagg et al., 2005). The primary antibody to CGRP (Chemicon) was detected with a biotinylated secondary antibody (PK 6101 Vectastain; Vector Laboratories) and visualized with an avidin–biotin complex (Vector Laboratories) followed by DAB. Digital images were captured using light microscopy (x10). The extent of the DAB-labelled CGRP fibres in the dorsal horn was quantified in a blinded manner using Scion Image analysis software (NIH), which was calibrated to perform area measurements in square millimetres. A boundary encompassing the entire dorsal horn was drawn on the image and the region within this boundary comprised the area to be quantified. CGRP immunoreactivity within the designated area was identified by pixel thresholding, which converts areas of immunoreactivity into black pixels on a white background. Analysis was performed by using the average density measure of five sections/rat.
Statistics
Statistical comparisons were performed using Prism (GraphPad, San Diego, CA, USA). Comparisons between different groups were made using unpaired t-tests. Comparisons of PrI numbers on the left and right side of the spinal cord were made using a paired t-test. In cases where the data did not follow a normal distribution (i.e. projections into grey matter did not pass a Kolmogorov–Smirnov test), non-parametric tests were used (Mann–Whitney U-tests), preceded by a Kruskal–Wallis test.
The correlation between number of contacts and errors on the horizontal ladder were performed using the Pearsons' correlation coefficient. Differences with P < 0.05 were considered significant. Errors are given as standard error of the mean.
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Most PrIs linking the cervical and lumbar enlargements are commissural
The first reports on injury-induced sprouting of transected CST axons rostral to a spinal cord lesion (Fouad et al., 2001
; Bareyre et al., 2004) studied the effects of a dorsal lesion (hemisection). In the present study, we performed a right-sided over-hemisection, which ablated the dorsal columns and dorsolateral funiculi on both sides, and severed the right lateral and ventral columns either completely or with minor sparing (<10%). The left ventrolateral funiculus was spared in all the animals. We found that the retrograde tracer FluoroGold injected into the lumbar enlargement was taken up by spared PrI with cell bodies in both sides of the thoracic and cervical spinal cord. The cell bodies were arranged in a fairly organized fashion, like beads on a string, lateral to the central canal in medial portions of laminae VII and VIII (Fig. 1). When comparing the numbers of cell bodies in the left and right side of the cervical spinal cord in lesioned animals, we found that a larger proportion of PrI crossed the mid-line rostral to the injury site. This is based on the finding that although the right side of the spinal cord was lesioned, 60% of the PrI cell bodies in the cervical enlargement (C3–7) were labelled on the right side. Considering that the vast majority (in most cases the entire side) of the right spinal cord was ablated, most of these neurons must be commissural interneurons that cross the mid-line rostral to the injury (Fig. 2A). The number of cell bodies in the cervical enlargement on the lesioned (right) side of the BDNF-treated rats was, on average, 253 ± 45 (SE) as compared with 186 ± 48 PrI on the left side, which apparently did not cross. This significant (P = 0.007) left versus right difference stands in contrast to the evenly distributed cell numbers in unlesioned control rats (410 ± 83 versus 395 ± 85), demonstrating that the results were not due to asymmetric tracer injections. While we cannot rule out that some axons of PrI may undergo multiple mid-line crossings above or below the lesion, the net effect reveals that 40% of the PrI axons are uncrossed and 60% crossed. Following our lesion model the number of PrI in lesioned animals is approximately half that of unlesioned animals, confirming that half of the cell population has been axotomized and hence unable to take up the tracer.
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The ratio of crossing and non-crossing PrI in the cervical enlargement stands in contrast to the ratio in the thoracic spinal cord (Fig. 2C and D). When counted at Th3–7 the total number of PrI was not statistically different to those found in the cervical enlargement; however, the ratio between crossing and non-crossing fibres was equal. In lesioned rats we found 241 ± 85 on the right versus 239 ± 79 on the left. As expected, there was no difference in the numbers of PrI between the two sides within the thoracic spinal cord of unlesioned (control) rats (360 ± 42 on the right and 378 ± 47 on the left).
Only BDNF application together with injury increases collateral sprouting at the cervical level
Earlier studies have shown that collateral sprouting of severed CST axons can occur rostral to an injury, possibly as an adaptive process to promote functional recovery after SCI (Fouad et al., 2001; Bareyre et al., 2004). Since we have shown previously that BDNF application to the cell bodies of CST neurons promoted regenerative sprouting of their axons (Hiebert et al., 2002), we hypothesized that application of BDNF (12 µg/day) to the cell body would also enhance the collateral sprouting of CST axons onto cervical PrI after a thoracic over-hemisection. Additionally, on the basis of earlier reports showing a response of sensory axons (Bradbury et al., 1999) and CST (Schnell et al., 1994) axons to local NT-3 application (12 µg/day), we assessed the effect of NT-3 given locally to the cervical enlargement.
We counted the number of collaterals emerging from the traced CST fibres into the grey matter of the cervical spinal cord on the lesioned (right) side. These counts were normalized to the total number of traced axons counted at C1, and to the distance that was evaluated (Fig. 3, original numbers are presented in Table 1). In contrast to reports with dorsal lesions only (Fouad et al., 2001; Bareyre et al., 2004), we found no increase in CST collaterals in lesioned vehicle-treated animals, compared with unlesioned animals (Fig. 3B). However, the application of BDNF to the cell bodies in the motor cortex significantly (P = 0.037) increased the normalized number of CST collaterals (Fig. 3B). This stands in contrast to the BDNF application to the cortex in unlesioned rats, where no change in the number of collaterals was found. Note that the normalized collateral number in unlesioned animals is not higher than in those with lesions, indicating that the increase in collaterals in the BDNF group cannot be explained by sparing of axons on the right side in some of the rats.
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To ensure that the quantification of collaterals following BDNF application was not biased by an effect of BDNF on the collateral thickness, we measured and compared the diameter of collaterals between animals that received vehicle or BDNF at the cortex (0.75 ± 0.03 and 0.74 ± 0.01 µm, respectively). There was no statistical difference between the two groups.
The local NT-3 application to the cervical enlargement did not promote collateral sprouting of the CST (Fig. 3C) and no statistical difference between the groups was found. To verify that the application of NT-3 was effective, we examined the effect on CGRP-positive fibres in the dorsal horn (Fig. 4). Similar to the previous report by Hagg et al. (2005), we found a significant (P = 0.045) increase in fibre density (pixilated area/mm2) in the NT-3-treated rats (92.3 ± 1.3 as compared with 85.9 ± 0.7 in vehicle-treated rats).
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Bouton-like structures of CST collaterals contact PrIs
Bareyre et al. (2004
) described CST collaterals contacting long descending PrI and thus circumventing the lesion in order to connect to the distal cord. We therefore hypothesized that the pharmacologically induced collaterals of lesioned CST fibres are likely to contact interneurons in the cervical enlargement rostral to the lesion. To examine this we counted the occurrence of bouton-like structures of collaterals emanating from the BDA-traced CST, that were co-localized with somata or dendrites of retrogradely traced PrI (FluoroGold, Fig. 5A). We define these occurrences as contacts, without implying that these are necessarily synaptic contacts. These contact counts were normalized to the number of traced CST fibres and the number of labelled PrI (Fig. 5B). Alternatively, to account for the increased number of collaterals, the number of bouton-like structure/PrI contacts was normalized to the number of collaterals (instead of the total number of traced fibres) and the number of PrI (Fig. 5C). The total numbers of counted contacts, PrI and number of collaterals are presented in Table 1. When normalized to the number of traced axons and the number of PrI, we found that the number of contacts did not increase following injury alone, or BDNF application without injury (Fig. 5B). This is consistent with our finding that the number of CST collaterals did not increase in these groups. A small and statistically not significant increase was seen following local NT-3 application. However, following BDNF application to the cell bodies of injured animals, the number of contacts was significantly increased by 287% (P = 0.037), when normalized to the number of traced fibres and compared with lesioned and vehicle-treated animals. When normalized to the number of collaterals, there was a statistically insignificant increase of 229% (P = 0.13; Fig. 5B and C). Hence, the increased number of contacts after BDNF treatment is largely due to more collaterals that are forming contacts and to a lesser extent to more contacts per collateral.
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BDNF-induced collaterals are not specifically targeting PrI
Following SCI, reorganization of the motor cortex has been reported in humans and animal models (Cohen et al., 1991
; Bruehlmeier et al., 1998; Green et al., 1999; Lotze et al., 1999; Fouad et al., 2001). A possible mechanism may involve projection of the injured CST fibres (that normally project to the lumbar spinal cord) to alternative targets, such as motoneurons of the fore limb. In this part of the experiment, we wanted to know if injury and BDNF-induced CST collaterals are specifically targeting PrI located in the medial parts of laminae VII and VIII, or whether they also grow towards the fore limb motoneuron pool that we have described earlier (Kuchler et al., 2002). As NT-3 application did not increase collateral sprouting, we excluded the NT-3 groups from this analysis. We divided the sections with an approximated line (indicated in Fig. 6A), which separated laminae VII and VIII into a medial part (containing PrI) and a lateral part (containing among others the motoneuron pool). As the density of BDA-positive projections in the cervical grey matter was fairly low, individual branches (segments of BDA-positive projections) could be counted in the medial and lateral part. We found that the BDA-positive projections within the medial grey matter doubled, in contrast to no detectable changes in collateral counts and number of contacts to PrI following spinal cord lesion and vehicle treatment. This increase that could be due to increased arborizations of the collaterals was significant (P = 0.035), when compared with uninjured rats (Fig. 6C). The number of BDA-positive projections in the lesioned BDNF-treated group increased even further and the difference was significant, when compared with unlesioned (P = 0.003) and lesioned vehicle-treated rats (P = 0.029). When quantifying CST projections within the lateral areas, we found similar increases as in the medial areas. The counts in the lesioned vehicle-treated group increased 13.8 times when compared with the unlesioned controls; however, this increase was not significant (P = 0.1). BDNF application in injured rats significantly increased the numbers by 2 orders of magnitude when compared with unlesioned rats (P = 0.0003) and about seven times compared with the lesioned vehicle-treated rats (P = 0.012). Thus, BDNF-induced sprouting is not exclusively targeted at PrI in the medial spinal cord.
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Neither injury alone nor the addition of BDNF increases sprouting of injured CST fibres at thoracic level
Cell bodies of PrI with spared axons (FluoroGold positive) could be found within the entire spinal cord rostral to the lesion. In this part of the experiment, we wanted to examine whether increased collateral sprouting caused by BDNF application was localized only to the cervical enlargement or whether it also occurred in the upper thoracic spinal cord, directly rostral to the injury (Th8). We did not find significant changes in the number of CST collaterals in either the lesion vehicle-treated or the lesion and BDNF-treated group (Fig. 7A). As NT-3 was applied locally at the cervical level and did not yield any effects on the CST collaterals at that level, we did not further analyse the spinal cord tissue at the thoracic level.
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At the thoracic level, there was a non-significant trend towards increased numbers (P = 0.4) of contacts between CST collaterals and PrI, following lesion when compared with uninjured rats without vehicle. Following BDNF application an additional 2-fold (non-significant) increase was found (Fig. 7B).
Increase in CST–PrI contacts correlates with functional recovery
A major question raised by the findings of BDNF-promoted sprouting and re-routing of CST fibres is the functional relevance of these anatomical changes. Seeking a behavioural correlate, we pooled the number of cervical CST–PrI contacts (normalized to the total number of traced axons and the number of PrI) of lesioned vehicle-treated versus lesioned plus BDNF-treated rats and plotted these results against the error rate when walking across a horizontal ladder (grid walk). The result was a significant correlation with an r value of 0.71, as shown in Fig. 8. Although the BDNF-treated rats performed on average better (5.6 errors ±1.2) than the lesioned and vehicle-treated rats (6.5 ± 0.9), the comparison between the groups was not statistically significant.
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Discussion |
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In this study, we show that the combination of a thoracic spinal cord over-hemisection injury with BDNF application to the cell bodies of the lesioned CST neurons in rats promoted the sprouting and possibly the rewiring of CST axons in the cervical spinal cord. The sprouting of CST axons onto spared PrIs could allow interrupted corticospinal signals to bypass the lesion site and reach the distal cord without the need for axonal regeneration. Recent studies have indicated that the injured CST is able to sprout rostral (Fouad et al., 2001
) and caudal (Weidner et al., 2001) to a spinal cord lesion. Importantly, CST collaterals were reported to connect onto PrI rostral to a dorsal hemisection of the thoracic spinal cord (Bareyre et al., 2004). In contrast to the studies by Bareyre et al. (2004) and Fouad et al. (2001), in which both ipsi- and contralateral projecting interneurons were spared, we did not find increased collateral sprouting or an increased number of contacts of injured CST fibres onto commissural PrI after SCI alone without further treatments. Since the approach of tracing and counting collaterals was comparable with the earlier studies, technical differences to explain the different results are unlikely. However, the lesion in our study was larger than the one used by Bareyre et al. (2004) and Fouad et al. (2001), because we also axotomized most of the non-crossing PrI. This modified significantly the property of these cells as potential targets for the collaterals since the non-crossing interneurons had been axotomized. Typically axotomized neurons lose many of their synaptic inputs, which would suggest that they do not attract sprouts either. Reconciling our previous work with the present study would hence lead to the speculation that the CST sprouting observed earlier after dorsal hemisection (Fouad et al., 2001; Baryere et al., 2004) was mainly onto ipsilaterally projecting interneurons. Our findings that injured CST fibres did not show increased spontaneous contacts onto PrI could thus be explained by the fact that PrI labelled in our experiments will cross the mid-line (aside from a small number of spared fibres on the right side of a few animals) and therefore do not include this ipsilaterally projecting population. The commissural interneurons might not represent a functional target for CST fibres that have already crossed at the level of the brainstem. A connection onto these PrI would therefore result in a re-crossing of the pathway. Together, the present results and the results by Bareyre et al. (2004) suggest that injury-induced sprouting of CST collaterals and connections on PrI occur only when non-crossing PrI remain connected to lower spinal cord levels. This is in line with the finding of Baryere et al. (2004), who indicated that CST contacts onto short PrI (that do not pass the lesion site) will form, but not persist.
Another interesting finding is that although the cervical, as well as the thoracic, spinal cord contained cell bodies of spared PrI projecting to the lumbar enlargement, collateral sprouting of CST axons and increases in CST to PrI connections only occurred at cervical levels. A possible reason is that the connections between the cervical and lumbar enlargements are especially important as they link networks controlling leg movements (Kjaerulff and Kiehn, 1996), despite the limited contribution of the CST to locomotor control in rats (Muir and Whishaw, 1999; Schucht et al., 2002).
The application of neurotrophic factors and other agents to cell bodies has been examined in various models (Tetzlaff et al., 1994; Kobayashi et al., 1997; Benowitz et al., 1999; Hiebert et al., 2002; Kwon et al., 2002; Plunet et al., 2002; Lu et al., 2004). However, the earlier studies on the descending spinal cord projections where neurotrophins were applied at the lesion site or at the cell body focused on neuroprotection of the cell bodies, gene expression and regenerative growth of lesioned axons (Schnell et al., 1994; Giehl and Tetzlaff, 1996; Bregman et al., 1997; Grill et al., 1997; Kobayashi et al., 1997; Hammond et al., 1999). Here, we report that collateral sprouting rostral to an injury with the potential of reconnecting to the periphery via PrI can be promoted by BDNF infusion into the vicinity of the corticospinal neurons. While such application is clinically not applicable in this present form, this finding represents a proof of principle of a novel treatment opportunity, which is especially important considering the large number of patients presenting with incomplete spinal cord injuries.
Comparable with a recent study by Hagg et al. (2005), we found that the local application of NT-3 did not result in increased sprouting of injured CST fibres. This appears to stand in contrast to studies that reported increased spouting of injured CST fibres at the injury site following local NT-3 application (e.g. Schnell et al., 1994) or of spared fibres (contralateral to the lesion) distal to the lesion (Zhou et al., 2003). In both cases there were additional factors involved as, for example, Wallerian degeneration or freed synaptic space; NT-3 on its own may not be sufficient to enhance sprouting.
Our data indicate a non-significant reduction of collaterals following NT-3 application, which appears different from the study by Hagg et al. (2005), as they reported a significant reduction. The non-significant reduction in our study could be due to the relatively small number of animals in our lesioned and vehicle-only-treated control group. Another possibility is that the tissue penetration of NT-3 was very weak, thus resulting in a modest effect only in axons located in the superficial layers but none in the more medial located CST.
Our study compared the functional outcome between the injured, BDNF-treated and untreated animals, by their ability to cross a horizontal ladder, a task requiring CST integrity (Metz and Whishaw, 2002). We found a significant correlation between the number of CST collateral contacts to PrI and the performance on the ladder walk. This suggests that new collaterals from the injured CST and their connections onto crossed PrI might be functionally meaningful. This idea is supported by our earlier finding that following complete CST lesion at the level of the brainstem and neutralization of Nogo-A, functional re-routing of the CST via the red nucleus was possible (Raineteau et al., 2001; Raineteau et al., 2002). Further studies with detailed electrophysiology and higher animal numbers, as well as other neuronal sprouting enhancing treatments (e.g. anti-Nogo A), will show whether re-routing injured tracts within the spared spinal cord is a viable method to restore function after SCI.
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We would like to dedicate this manuscript to the memory of Dr Gordon Hiebert, who passed away during this study. We lost a dedicated scientist and a great friend.
Furthermore, we would like to thank Regeneron Pharmaceuticals and Amgen for the neurotrophic factors. K.F. was supported by the International Spinal Research Trust (ISRT) and the Alberta Heritage Foundation for Medical Research (AHFMR). G.H. was supported by the BC Neurotrauma Fund and ISRT.
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