Introduction 1 2 3 3 10 11 12 13 11 14 15 16 17 18 21 4 22 25 Although exogenous GDNF is available partially to promote propriospinal axonal regeneration and locomotion functional recovery, the role of intrinsic GDNF in injured spinal cords is largely unknown. Therefore, the present study investigated the possible role of GDNF at various time intervals following spinal cord transection injury in adult rats, so as to gain insights into the contribution of endogenous GDNF in spinal neuroplasticity. Materials and methods Characterization of antibodies To localize GDNF protein in the spinal cord, an affinity-purified rabbit polyclonal antibody (D-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used in this study. The specificity of antibody for GDNF was confirmed by Western blots using rat spinal cord homogenates. Preparation of probe for in situ hybridization For detection of GDNF cellular expression in situ, we used Digoxigenin-labeled oligonucleotide probe designed by Primer premier 5.0 package which was complementary to the rats GDNF gene sequence (33 mer, 5′-GCCCTACTTTGTCAC–TCACCAGCCTTCTATTTC-3′). This antisense DNA single-stranded oligonucleotide probe was synthesized by Takara Biotechnology Company. Animal grouping and surgery n n 7 8 9 10 9  10 Behavior tests 26 26 Measurement of somatosensory evoked potentials (SEP) The right peroneal nerve of the rats was dissected and a stimulation electrode was placed on it while a hole (3–4 mm in diameter) was made on the skull (2 mm to the left of the midline and 2 mm in front of the fonticulus posterior). An electrode was placed on the dura of brain cortex to record the SEP. The reference electrode was inserted at the nose epithelium. A ground electrode was inserted at the tail. The stimulus intensity was set high enough to produce a marked muscle twitch in the hind limb, about 1.1 mA, the amplitude was 0.2 ms and the frequency 3 Hz. The SEP Tracings represented the average of 200 responses. Immunohistochemistry 9 10 2 2 2 2  In situ hybridization Sections from sham-operated rats of 25 μm thickness were also cut on a freezing microtome and used for in situ hybridization. Sections were fixed in 4% paraformaldehyde in 0.1 M PBS, pH 7.2 (all treatments were performed at room temperature unless otherwise indicated), and further treated with 0.3% TritonX-100 solution for 10 min and proteinase K (5 μg/ml) at 37°C for 25 min, refixed with 4% paraformaldehyde for 5 min, repeatedly immersed in 0.1 M PBS, then acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) to prevent non-specific binding of the probes. The sections were washed with 2× SSC (pH 7.0) and then prehybridized in a hybridization solution (50% formamide, 10% dextran sulfate, 1× Denhardt’s solution, 0.2 mg/ml Herring sperm DNA, and 10 mM dithiothreitol) without probes at 37°C for 2 h before hybridization, then hybridized in 100 μl hybridization solution containing 1 μl probes at 37°C for 12–16 h in a moist chamber. This was followed by washing in decreasing concentrations of SSC, from 4× SSC (pH 7.0) at 37°C for 20 min, 2× SSC (pH 7.0) at 42°C for 20 min, 1× SSC (pH 7.0) at 48°C for 20 min and ending with 0.5× SSC (pH 7.0) at 50°C for 20 min. Then sections were incubated at 37°C in 1% blocking buffer (Roche) for 1 h, subsequently reacted in 1:1,000 sheep anti-digoxygenin-alkaline phosphatase (AP) antibody in 1% blocking buffer at 4°C overnight. Lastly, AP activity was detected using nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate (Roche). The sections were visualized with blue and purple sedimentation then observed with a light microscope. GFAP/GDNF double-label immunohistochemistry Two-color immunohistochemical staining for simultaneous detection of glial fibrillary acidic protein (GFAP)/GDNF expression was performed as described above. Briefly, sections were stained with anti-GDNF rabbit polyclonal antibody (1:1,000), and using DAB solutions as a substrate. This was followed by second incubation with anti-GFAP rabbit polyclonal antibody (1:1,000, AB5804, Chemicon), and finally, reacted with TMB solutions. Double labeling showed a combination of brown and blue product. Western blotting g BDA anterograde tracing At 14 dpo, the animals for this part were anesthetized and fixed in a David Kopf Instruments (Tujunga, CA) stereotaxic head-holder device. Burr holes were made in the dorsal cranium, and biotinylated dextran amine (BDA) (10% BDA solution, Molecular Probes) was microinjected into eight sites at a depth of 0.7 mm from the cortical surface (0.5 μl/site) to cover the hindlimb region. Animals were then sacrificed 2 weeks later to allow sufficient time for axonal transport of BDA in corticospinal tract. The spinal cords were removed and postfixed at 3 days in cold 4% paraformaldehyde in 0.1 M PBS (pH 7.2). Transverse sections (30 μm) of spinal cord at the injury site and neighboring rostral and caudal parts to the injury site were processed for the presence of BDA-labeled axons by incubation in avidin-HRP (Molecular Probes). Lastly, DAB stain was performed to visualize the positive fiber, as brown color staining. GDNF antibody neutralization After 14 dpo, each rat was intraperitoneally injected with 0.5 ml (30 mg/ml, 30 mg of anti-GDNF diluted in 1 ml of distilled water) anti-GDNF solution once every 2 days until 21 dpo. GDNF antibody was the distilled water replace as control in another five rats. The locomotion in hindlimbs by BBB score was evaluated at 3, 7, 14, and 21 dpo. Statistical analysis P Results Behavior tests P 1 Table 1 Mean values of BBB scores in cord transected rats (mean ± S.E.M) Group 3 Days 7 Days 14 Days 21 Days Sham-operation group 5 ± 0.6 21 ± 0 21 ± 0 21 ± 0 Transection group 0 ± 0 0.8 ± 0.3 2.4 ± 0.7 3.6 ± 0.5 Distilled water group 0 ± 0 0.6 ± 0.7 2.0 ± 0.4 3.3 ± 0.3 GDNF-antibody treated group 0 ± 0 0.2 ± 0.4 0.6 ± 0.3 0.9 ± 0.5 P P P Somatosensory evoked potentials (SEP) 1 1 2  1 Fig. 1 1 1 2  1  1  2 1  1  2   The GDNF immunostained intensity changes 3 3 2 2 Fig. 2 a b  3 2 3 4 2 3 4 2 Fig. 3 a b c e g, d, f, h  Table 2 GDNF immunostaining intensity in neurons and astrocytes   Normal  3 Days 7 Days 14 Days 21  Days  Neurons + +++ +++ ++∼+++ ++∼+++ Rostral part  Astrocytes  ++ +++ +++ +++ +++ Caudal part  Neurons + +∼++ ++ +++ ++  Astrocytes ++ ++ ++ +++ +++ Intensity: + weak, ++ moderate, +++ strong Neurons in the ventral horn of the rostral stump was strongly stained for GDNF at 3 and 7 dpo, and in the caudal stump at 14 dpo. The GDNF positive staining in astrocytes was also stronger at all time-points after transection injury than seen in control group Fig. 4 a b c d e g f h  4 2 4 4 2 Localization of GDNF mRNA in spinal cord of rats 5 5 5 5 Fig. 5 a b c  Western blotting A single positive band was observed at a molecular weight of about 34,000, which corresponds to the molecular weight of GDNF. P P 6 7 P 6 7 Fig. 6 P P a P b  Fig. 7 P P  BDA tracing 8 8 8 Fig. 8 a b c  Discussion 4 23 25 2 16 27 23 15 28 4 7 29 32 2 15 30 33 36 37 38 39 40 41 30 42 36 43 44 2 The present study provides new evidence to understand the role of intrinsic GDNF in the rostral and caudal stumps of injured spinal cords. It suggests that GDNF plays an essential role in the neuroplasticity of the local circuitry, especially in the caudal stump of the transected spinal cords.