3). nerve graft model. Results. Similar to brain-derived neurotrophic factor (BDNF), the 29D7 antibody strongly promoted RGC survival and neurite growth in vitro compared with Secretin (human) medium alone or control IgG. Forskolin, which weakly supported RGC survival on its own, potentiated the effect of 29D7. Intravitreal injection of 29D7 enhanced RGC survival but not regeneration in vivo 2 weeks after optic nerve injury. Conclusions. Together, these findings demonstrate the potential for antibody-mediated TrkB agonism as a potential therapeutic approach to enhance RGC survival after optic nerve injury. Further studies are needed to Secretin (human) elucidate the mechanistic differences between this TrkB agonist and BDNF. There is limited intrinsic potential Rabbit Polyclonal to PFKFB1/4 for repair in the mature central nervous system (CNS). For example, after optic nerve injury or in degenerative diseases such as glaucoma, retinal ganglion cells (RGCs) fail to regenerate their injured axons, and eventually die.1,2 The failure of survival and axon regeneration is in part due to a lack of adequate trophic support after injury. For example, death of injured RGCs can be delayed by application of neurotrophic factors such as brain-derived neurotrophic factor (BDNF), neurotrophin-4/5, and ciliary neurotrophic factor.3C5 BDNF belongs to the neurotrophin family. It binds to high-affinity tropomyosin-related kinase B Secretin (human) (TrkB) and the low-affinity receptor-p75NTR. BDNF binding to TrkB induces subsequent phosphorylation of several downstream, intracellular mediator kinases such as extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), calcium/calmodulin-dependent kinase IV (CaMKIV), phospholipase C-gamma, and other downstream pathways.6,7 Recent studies have shown that these signaling pathways are important for RGC survival and axon regeneration.7 For example, TrkB upregulation protects RGCs from axotomy7,8; activation of the ERK1/2 pathway protects RGCs in glaucoma9; CaMKIV is necessary for BDNF-induced phosphorylation of cyclic AMP response element-binding protein (CREB) and axonal growth10; and PI3K/Akt and JAK/STAT pathways mediate RGC survival after acute intraocular pressure elevation.11 However, the effect of BDNF is limited: it does not promote long-term survival because of the downregulation of TrkB,12 and it may lead to optic nerve dystrophy when used in vivo.13 Recently, a novel TrkB-selective antibody with agonist properties, 29D7, has been developed, and it demonstrated the potential for therapeutic use in ameliorating neurodegeneration associated with acute brain injury and in promoting long-term repair.14 We therefore Secretin (human) hypothesized that this TrkB agonist could be used as a neuroprotectant for RGCs after optic nerve injury and asked whether it would be more or less effective than BDNF. Here we studied 29D7 for its ability to enhance RGC survival and neurite growth in culture, RGC viability after axotomy, and RGC axon regeneration into permissive peripheral nerve grafts. Materials and Methods Animals All procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the University of Miami Institutional Animal Care and Use Committee. For in vitro experiments, litters of Sprague-Dawley (SD; Harlan Laboratories, Inc., Allen Park, MI) were used at postnatal day (P) 3. For in vivo experiments, adult female SD rats, each weighing 200 to 250 g, were used. All surgical procedures on rats were performed under general anesthesia using an intraperitoneal injection of ketamine (60 mg/kg) and xylazine (7.5 mg/kg). Rats also received subcutaneous injection of buprenorphine (0.05 mg/kg; Bedford Laboratories) as postoperative analgesic. Eye ointment containing erythromycin was applied to protect the cornea. Immunopanning of RGCs RGCs from P3 to P4 SD rats were purified through sequential immunopanning to 99.5% purity, as previously described.15,16 RGCs were cultured on poly-d-lysine (PDL; 70 kDa, 10 g/mL; Sigma, St. Louis, MO) and laminin (1 g/mL; Invitrogen, Carlsbad, CA) in neurobasal (NB) serum-free defined medium as described, containing insulin (5 g/mL), sodium pyruvate (1 mM), l-glutamine (1 mM), triiodo-thyronine (T3; 40 ng/mL; Sigma), = 3; results of a representative experiment are shown; all experiments were repeated at least three times with similar results). Open in a separate window Figure 2. Dose-response curves of RGC survival at 1 day in response to (A) 29D7 or to (B) 29D7 or BDNF in the presence of a cAMP analogue, CPT-cAMP (50 M). Details of assay are the same as in Figure 1. Effect of 29D7 on RGC Neurite Growth In Vitro Given that 29D7 can promote RGC survival in vitro and that BDNF supports both survival and axon growth of RGCs in vitro,16,28 we next evaluated the effect of 29D7 on RGC neurite growth. Main RGC cultures.