Glial-derived Nerve Growth Factor (gdnf) Mediates Neuronal Phenotypic Changes

Introduction Until recently, chronic nerve compression (CNC) injuries have been relatively understudied. Recent studies have shown that CNC injury induces Schwann cell proliferation and apoptosis with no morphologic evidence of axonal injury (1,2). It has also been shown that the axon undergoes a sprouting response within the zone of compression that is readily apparent by two weeks following injury. Early work has shown that this response may also be manifest within the corresponding dorsal root ganglia (DRG) and may actually initiate a phenotypic switch during the early months as a rise in the population of small diameter IB4 neurons has been detected (3). The molecule responsible for this phenotypic switch has not been determined. As glial derived nerve growth factor (GDNF) is integral to the development and survival of IB4 neurons in a dosedependent fashion (4), we sought to rigorously evaluate the DRG phenotypic change secondary to CNC injury and determine if there was any relationship with changes in GDNF expression. Materials and Methods Surgical Technique As previously described, we create the CNC injury model in adult male Sprague-Dawley rats (Simonsen Laboratories, Inc). Briefly, using a gluteal-splitting approach, the right sciatic nerve was exposed and atraumatically fitted with a 1 cm sample of inert tubing (Baxter Healthcare) with internal diameter of 1.3mm and outer diameter of 2.0mm. The left sciatic nerve was mobilized and returned to its muscular bed to serve as a comparative control. Animals were then sutured and returned to their cages until time of harvest. Experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. Tracing, Immunohistochemistry, Quantification Five days prior to harvest, fluorogold was injected distal to the site of compression. The animals were perfused with 4% paraformaldehyde at time of sacrifice. Ipsilateral and contralateral L4 and L5 DRG were fixed in paraformaldehyde and subjected to standard immunohistochemical procedures. Primary antibodies included mouse anti-neurofilament 200 (Sigma), rabbit anti-CGRP (Chemicon), and FITC-conjugated IB4. Secondary antibodies directed against anti-NF200 and anti-CGRP were FITC-conjugated goat anti-mouse and goat anti-rabbit (Chemicon) respectively. Six random sections for each DRG were pooled for counting of NF-200, CGRP, or IB4 profiles. Western Blot Contralateral and ipsilateral DRG for two week, one and eight month animals were harvested and homogenized in ice-cold BUST buffer (50mM Tris-HCl, 8 M urea, 2% B-mercaptoethanol, 0.5% SDS) supplemented with 1mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotonin, and 2 μg/ml pepstatin A and centrifuged at 14,000 rpm for 30 minutes. Fifty micrograms of protein were loaded and electrophoresed in a 10% Tris-HCl gel at 120 V for 1.5 hours after which the protein was transferred unto a PDVF membrane at 60V for 2 hours. The membrane was treated with rabbit polyclonal anti-GDNF (1:200, Santa Cruz Biotechnology) at 4oC overnight. Following thorough washing, blots were treated with biotin conjugated rabbit secondary antibody (1:200, Vector) for one hour. Visualization of protein was achieved by use of Vectastain ABC and VIP Substrate Kits (Vector Laboratories, Burlingame, CA) and as quantified by ImageQuant software. Results An example of immunohistochemical staining is shown in Figure 1. The number for IB4 and CGRP profiles from L4/L5 DRG neurons approximately doubled at the expense of NF200-positive neurons relative to the uninjured control DRG (Table 1). By eight months postcompression, populations of neurons reverted to normal distributions. In concordance with the reversion of neuronal phenotype by eight months post-injury, there was no appreciable difference in expression of GDNF protein in both contralateral and ipsilateral L4 and L5 DRG (Table 2). Quantification of the protein in one month animals, however, indicated that GDNF in the injured L4 and L5 DRG increased by 100 percent relative to contralateral control DRGS. Table 1: Numerical comparison of characteristic neurons in ipsilateral and contralateral DRG at one month.