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. 2010 Oct;51(10):5190-7.
doi: 10.1167/iovs.10-5257. Epub 2010 May 5.

Knockdown of NBCe1 in Vivo Compromises the Corneal Endothelial Pump

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Free PMC article

Knockdown of NBCe1 in Vivo Compromises the Corneal Endothelial Pump

Cailing Liu et al. Invest Ophthalmol Vis Sci. .
Free PMC article

Abstract

Purpose: To evaluate the role of the sodium bicarbonate cotransporter (NBCe1) as a component of the corneal endothelial pump in the in vivo rabbit eye.

Methods: 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.

Results: Within 24 hours there was a modest anterior chamber inflammation that resolved within 48 hours. At 4 × 10(6) 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.

Conclusions: 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.

Figures

Figure 1.
Figure 1.
Dose-response of lentivirus-GFP anterior chamber fluorescence in New Zealand White rabbits. (A–E) Intracameral injection of 4 × 106 IFU. (A) En face corneal endothelial GFP fluorescence image using a confocal microscope 7 days after injection. Scale bar, 4 mm. (B) Fourteen days after injection. (C) Slit-lamp image of endothelial fluorescence 14 days after injection. (D) GFP fluorescence from endothelial peeling 21 days after injection. Scale bar, 60 μm. (E) Iris fluorescence 14 days after injection. (F) Average (SD) fluorescence intensity over time of various viral doses (n = 3 for each dose). Average fluorescence intensity was calculated by integrating the fluorescence intensity over the area of measurement (9-mm diameter) after background subtraction. (G) Average fluorescence intensity and the percentage area of the measured corneal area that had fluorescence values above threshold for each viral dose.
Figure 2.
Figure 2.
Loss of GFP fluorescence. Representative successive corneal endothelial fluorescence images from the same eye. Bottom: slit-lamp image showing keratic precipitates (arrow) in a circumferential pattern from an eye injected with 4 × 106 IFU Lenti-GFP 25 days after injection.
Figure 3.
Figure 3.
Knockdown of NBCe1 expression. (A) Representative immunofluorescence images (blue, nuclear DAPI; green, NBCe1) of corneal endothelial peelings from the right (OD, Lenti-scrambled) and left (OS, Lenti-NBCe1-shRNA) corneas 4 weeks after injection. Scale bar, 100 μm. (B, left) Representative real-time PCR products. Right: summary results from eight rabbits and only those showing >25% knockdown. (C) Representative Western blot analysis 14 and 28 days after injection. (D) Summary of Western blot analysis (n = 34) and quantitative PCR (n = 8) data from all rabbits analyzed (n = 42) and subdivided into those coexpressing GFP (n = 24) and without GFP (n = 18). (E) Rabbits that had >25% knockdown of NBCe1 (n = 30) and subdivided at 2 weeks (n = 19) and 4 weeks (n = 11) after injection.
Figure 4.
Figure 4.
Corneal thickness and IOP after injection of Lenti-scrambled (OD) and Lenti-NBCel-shRNA (OS). (A) Average (SD) central corneal thickness of all rabbits. (B) Average of paired differences (OS-OD) in corneal thickness for only rabbits with >25% NBCe1 knockdown or >50% knockdown (P > 0.05). Number of rabbits in each category is shown in parentheses. (C) Average (SD) of IOP values from all rabbits showing >25% knockdown of NBCe1. *Significantly different from preinjection (P < 0.005).
Figure 5.
Figure 5.
Brinzolamide provocative test results in rabbits with NBCe1knockdown. (A) Average (SD) of peak changes in corneal thickness 3 to 5 hours after topical application of two drops of 1% brinzolamide to both eyes. OD, control (injected with scrambled sequence). OS, >25% knockdown of NBCe1-shRNA. (B) Average (SD) of paired differences (OS-OD) in peak brinzolamide-induced corneal swelling measured at 2 and 3 weeks after injection in rabbits demonstrating <25% and >25% knockdown of NBCe1. *Significantly different from 0 (P < 0.005). Number of rabbits in each category is shown in parentheses.

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