@article{10.1167/iovs.10-5257,
    author = {Liu, Cailing and Cheng, Qiang and Nguyen, Tracy and Bonanno, Joseph A.},
    title = "{Knockdown of NBCe1 In Vivo Compromises the Corneal Endothelial Pump}",
    journal = {Investigative Ophthalmology & Visual Science},
    volume = {51},
    number = {10},
    pages = {5190-5197},
    year = {2010},
    month = {10},
    abstract = "{   To evaluate the role of the sodium bicarbonate cotransporter (NBCe1) as a component of the corneal endothelial pump in the in vivo rabbit eye.    Lentiviruses with NBCe1 shRNA and GFP expression cassettes were injected intracamerally. Knockdown efficacy was determined 1 week to 4 weeks later by immunofluorescence, Western blot analysis, and PCR. Functional effects were monitored by corneal thickness (CT) and brinzolamide sensitivity.    Within 24 hours there was a modest anterior chamber inflammation that resolved within 48 hours. At 4 × 106 IFU, more than 95\\% of the corneal endothelial surface showed GFP fluorescence above background within 7 days. At 14 to 21 days, signs of anterior chamber inflammation reemerged, and endothelial cell GFP fluorescence disappeared within 40 days after injection. The second phase of inflammation could be avoided by using GFP-less viruses. There was no significant difference in CT between scrambled sequence and NBCe1 shRNA–injected eyes over 3 weeks. Two drops of 1\\% brinzolamide produced 7.85\\% ± 3.3\\% corneal swelling within 5 hours of topical instillation. However, in corneas showing more than 25\\% NBCe1 knockdown (30 of 42 rabbits; 59\\% ± 15\\% knockdown), corneal swelling was significantly higher (10.1\\% ± 2.9\\%) relative to control eyes.    FIV-based lentiviral vectors can transfect CE with shRNA in rabbits. The response to GFP is consistent, with previous studies showing the production of anti-GFP antibodies. Partial knockdown of NBCe1 did not affect baseline CT, which is consistent with the corneal endothelium having a substantial functional reserve. Provocative testing using, brinzolamide, however, revealed an underlying deficiency, confirming the importance of NBCe1 bicarbonate transport and demonstrating the concerted action between NBCe1 and carbonic anhydrases.  }",
    issn = {1552-5783},
    doi = {10.1167/iovs.10-5257},
    url = {https://doi.org/10.1167/iovs.10-5257},
    eprint = {https://arvojournals.org/arvo/content\_public/journal/iovs/932962/z7g01010005190.pdf},
}