PURPOSE. The aim of this study was to determine and characterize the effect of topical application of benzalkonium chloride (BAK) on corneal nerves in vivo and in vitro.METHODS. Thy1-YFP+ neurofluorescent mouse eyes were treated topically with vehicle or BAK (0.01% or 0.1%). Widefield stereofluorescence microscopy was performed to sequentially image the treated corneas in vivo every week for 4 weeks, and changes in stromal nerve fiber density (NFD) and aqueous tear production were determined. Whole-mount immunofluorescence staining of corneas was performed with antibodies to axonopathy marker SMI-32. Western immunoblot analyses were performed on trigeminal ganglion and corneal lysates to determine abundance of proteins associated with neurotoxicity and regeneration. Compartmental culture of trigeminal ganglion neurons was performed in Campenot devices to determine whether BAK affects neurite outgrowth.RESULTS. BAK-treated corneas exhibited significantly reduced NFD and aqueous tear production, and increased inflammatory cell infiltration and fluorescein staining at 1 week (P < 0.05). These changes were most significant after 0.1% BAK treatment. The extent of inflammatory cell infiltration in the cornea showed a significant negative correlation with NFD. Sequential in vivo imaging of corneas showed two forms of BAK-induced neurotoxicity: reversible neurotoxicity characterized by axonopathy and recovery, and irreversible neurotoxicity characterized by nerve degeneration and regeneration. Increased abundance of beta III tubulin in corneal lysates confirmed regeneration. A dose-related significant reduction in neurites occurred after BAK addition to compartmental cultures of dissociated trigeminal ganglion cells. Although both BAK doses (0.0001% and 0.001%) reduced nerve fiber length, the reduction was significantly more with the higher dose (P < 0.001).CONCLUSION. Topical application of BAK to the eye causes corneal neurotoxicity, inflammation, and reduced aqueous tear production. (Invest Ophthalmol Vis Sci.

Ornithine decarboxylase (ODC) is a key enzyme in mammalian polyamine biosynthesis that is up-regulated in various types of cancer. We previously showed that treating human neuroblastoma (NB) cells with the ODC inhibitor A-difluoromethylornithine (DFMO) depleted polyamine pools and induced G 1 cell cycle arrest without causing apoptosis. However, the precise mechanism by which DFMO provokes these changes in NB cells remained unknown. Therefore, we further examined the effects of DFMO, alone and in combination with phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or Akt/protein kinase B (PKB) inhibitor IV, on the regulation of cell survival and cell cycle-associated pathways in LAN-1 NB cells. In the present study, we found that the inhibition of ODC by DFMO promotes cell survival by inducing the phosphorylation of Akt/PKB at residue Ser 473 and glycogen synthase kinase-3B at Ser 9 . Intriguingly, DFMO also induced the phosphorylation of p27Kip1 at residues Ser 10 (nuclear export) and Thr 198 (protein stabilization) but not Thr 187 (proteasomal degradation). The combined results from this study provide evidence for a direct cross-talk between ODC-dependent metabolic processes and well-established cell signaling pathways that are activated during NB tumorigenesis. The data suggest that inhibition of ODC by DFMO induces two opposing pathways in NB: one promoting cell survival by activating Akt/PKB via the PI3K/Akt pathway and one inducing p27 Kip1 /retinoblastomacoupled G 1 cell cycle arrest via a mechanism that regulates the phosphorylation and stabilization of p27 Kip1 . This study presents new information that may explain the moderate efficacy of DFMO monotherapy in clinical trials and reveals potential new targets for DFMO-based combination therapies for NB treatment. [Cancer Res 2008;68(23):9825-31]

Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in many forms of cancer including NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested in NB. We propose that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501 will result in enhanced NB growth inhibition. Single and combination drug treatments were conducted on three NB cell lines. DFMO IC 50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC 50 values ranged from 14.13 to 17.72 mM in NB. The combination treatment resulted in hypophosphorylation of retinoblastoma protein (Rb), suggesting growth inhibition via G 1 cell cycle arrest. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hr suggested apoptosis. The combination treatment depleted intracellular polyamine pools and decreased intracellular ATP, further verifying growth inhibition. Given the current lack of effective therapies for patients with relapsed/refractory NB and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB.Neuroblastoma (NB) is the most common extracranial solid pediatric tumor, accounting 8-10% of all pediatric cancers and for 15% of cancer-related deaths in children. 1 Approximately 650 new cases of NB arise in the United States each year. Of the children diagnosed, roughly 70% have disease that has already metastasized to other parts of the body. Children with high-risk NB have a long-term survival of less than 50%, despite the use of intensive multimodality therapy. 2 Of the children with relapsed or refractory disease, there are no known curative measures and the 5-year survival is less than 10%. Biomarkers are powerful tools for determining diagnosis and prognosis for different cancers, including NB. The oncogenic transcription factor MYCN is amplified in roughly 30% of all NBs and is generally associated with high-risk disease and poor survival. 3,4 As a transcription factor, MYCN induces and represses a large number of genes involved in multiple biological processes including cell growth and differentiation. However, the genes necessary or sufficient to initiate neuroblastoma tumorigenesis downstream of MYCN remain to be established. 5 Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis in mammalian cells, is directly activated by c-MYC and MYCN 6-8 and is overexpressed in NB. 9-11 ODC decarboxylates t...

Sema7a is expressed constitutively in the cornea, and potently stimulates nerve regeneration and inflammatory cell influx. Therefore, this immune semaphorin links nerve regeneration and inflammatory processes in the cornea.

Although corneal NFD recovers to normal levels by 8 weeks after nerve transection, the arrangement of regenerated nerves is abnormal.