x is the mean value. Abbreviations: NP, neuronal progenitor cells; TD, neuronal progenitor cells expressing TD; IGF-1, neuronal progenitor cells expressing IGF-TD fusion protein; Scale bar: 200 m and 50 m. doi:10.1371/journal.pone.0125695.g002 co-cultured with hNPIGF-TD cells also produced more neurites per cell body, averaging 2 0 neurites/cell, while those co-cultured with hNPTD or hNP cells displayed an average of 1 neurite/cell each. These observations confirm that IGF-TD secreted by hNPIGF-TD cells was able to enhance the survival and neurite outgrowth of cultured primary RGCs. Analysis was conducted by fitting a two way linear model for treatments and time points on the inverse of the data. Multiple comparisons were conducted on the transformed scale by the Tukey test. Asterisks indicate statistically significant differences versus all Salvianic acid A chemical information groups at the same time point. IGF-1 Inhibitors Abrogate RGC Survival and Neurite Outgrowth To confirm the direct effects of IGF-TD on RGC survival and neurite outgrowth in hNPIGF-TD co-cultures, we applied various antagonists: H-1356 is an IGF-1 analog that competitively binds and blocks IGF-1R signaling; NBI-31772, a tyrosine kinase receptor disrupts the binding of IGF-1 to IGF-1 binding proteins; and a neutralizing antibody to IGF-1R. Our observations indicate that application of H-1356 completely eliminated the effects of hNPIGF-TD on RGC survival and neurite outgrowth. Similarly, NBI-31772 and a neutralizing antibody to IGF-1R also independently and completely blocked these effects. The rates of cell survival and average neurite lengths of RGCs co-cultured with hNPIGF-TD in presence 
of these inhibitors were not significantly different from the observed rates for RGCs co-cultured with hNPTD or hNP cells. Rescue of RGCs via IGF-1 Signaling in the Retina We used an established model of murine glaucoma in which injections of microbeads into the anterior chamber of the mouse eye causes blockage of aqueous humor outflow and reproducible elevation of IOP. Prior to microbead injection, baseline IOPs were measured and averaged 8.17 0.88 mmHg. Mice that received control saline injections into their anterior chambers exhibited a steady IOP level of 8.64 1.04 mmHg throughout the study period. No significant differences were found among IOPs of saline-injected mice and baseline IOP values of other groups. Significant elevation of IOPs in the microbead-injected mice was observed within 4 days after the injection, reaching peak levels around day 10. Elevated IOPs gradually dropped off around 2 weeks after injection and returned to the baseline by week 4. There were no significant PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19783802 differences in IOP levels among the four microbead-injected groups. Expectedly, these groups exhibited higher IOPs compared with the saline-injected group. Our observations showed that injection of microbeads into anterior chamber effectively induced reproducibly transient elevation of IOP. Mouse group 25 received intravitreal injections of one of the following components, in the following order, saline, hNPs, hNPTD and hNPIGF-TD. Mice were sacrificed on day 30 and retinal flatmounts were prepared and subjected to immunostaining. Transfected cells were confirmed to be of hNP origin using an antibody to human HLA Class I antigen. Interestingly, some transplanted 11 / 24 Progenitor Cells Expressing IGF-1 on Retinal Ganglion Cell Survival Fig 3. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786154 Microbead injection into the anterior chamber of C57BL/6 mice to induce elevation of IOP and