Therapeutic applications of ultrasound in ophthalmology

Figures & data

Figure 1. Cross-section (a) and two-point (b) perspective of a human eye.

Figure 2. Geometry of the six transducers: 4.5-mm width and 7-mm length segment of a 10.2 mm radius cylinder. The dotted lines represent the focal volume, similar to an elliptic cylinder.

Figure 3. Positioning of the device including circular piezoelectric transducers, inserted into a spacing cone in contact with the eye (A). Sequential activation of the transducers, producing an ultrasonic beam which selectively destroys a well-defined portion of the ciliary body (B). CC: coupling cone. CB: ciliary body. UB: ultrasound beam.

Figure 4. Histological sections at low (top) and high (low) magnification showing the difference between the intact (A and C) and treated (B and D) ciliary body: loss of the bilayered epithelium responsible for the production of aqueous humor (fluid filling the eye).

Table 1.  Summary of the experimental studies evaluating the potential of ultrasound for ocular drug, protein or gene delivery.

Reference	Molecule/protein/gene	Target	US parameters	Results
Zderic et al. [28]	Sodium fluorescein	Ex vivo rabbit cornea	880 kHz, 0.19, 0.34 or 0.56 W/cm^2 for 5 min in continuous mode.	Permeability increased by 2.1, 2.5, and 4.2 times when ultrasound was applied at 0.19, 0.34, and 0.56 W/cm^2. The surface cells of corneal epithelium exposed to ultrasound appeared swollen and lighter in colour (indications of membrane rupture). Some of the surface epithelial cells were absent. Holes 3–10 µm in diameter were observed on the epithelial surface. No structural changes were observed in the stroma
Zderic et al. [29]	Sodium fluorescein	In vivo rabbit cornea	880 kHz and intensities of 0.19 to 0.56 W/cm^2 (continuous mode) with an exposure duration of 5 min	Increase in dye concentration in the aqueous humour (relative to sham treatment) was 2.4 times at 0.19 W/cm^2, 3.8 times at 0.34 W/cm^2, and 10.6 times at 0.56 W/cm^2. Disruption of the superficial epithelial layer was observed after US, but disappeared in 90 min
Sonoda et al. [30]	Gene (plasmid DNA with GFP) ± MBs	Cultured rabbit corneal epithelial RC-1 cells (in vitro) and rabbit keratocytes (in vivo)	6 mm US probe; 1 MHz; 0.5–2 W/cm^2; 15–120 s; duty cycle 20%–100%	In vitro: DNA exposure alone, no transfer; US, enhanced gene transfer slightly (about 5% to 10% of fluorescent cells); co-exposure with MBs significantly increased gene transfer efficiency (about 7% to 35% of fluorescent cells) In vivo: DNA injection alone could transfer the gene to a limited degree, but DNA + US + MBs strongly increased gene transfer efficiency (about 1.5 to 2 times higher) without apparent tissue damage
Yamashita et al. [31]	Gene (plasmid DNA with GFP) ± MBs or PEG BL	Cultured rabbit corneal epithelial RC-1 cells (in vitro) and rat conjunctiva (in vivo)	6 mm US probe; 1 MHz; 20 or 60 s; duty cycle 50% and 0.8, 1.0 and 1.2 W/cm^2	In vitro: BL + US gene transfer efficacy 27%, US-only 1%; MB + US 11%. No apparent cell damage was found in the BL + US group In vivo: conjunctiva GFP staining score 3.6 (BL + US), 2.3 (injection alone), 2.1 (US) and 2.0 (MB + US). No apparent tissue damage
Li et al. [32]	Gene (rAAV2-CMV-EGFP) ± MBs	Cultured human retinal pigment epithelium cells (in vitro) and rat retina (in vivo)	In vitro: 1 MHz; 1, 2 and 3 W/cm^2; duration 60 and 120 s; 10, 20 and 50% duty cycle and continuous wave; pulse recurrent frequency 100 Hz In vivo: 1 MHz, 2 W/cm^2; pulse repetition frequency 100 Hz, 50% duty cycle	In vitro study: about 15–20% fluorescent cells (control, US alone, MBs alone) versus 30–35% fluorescent cells (US + MBs). No major cytotoxicity. In vivo study: fluorescence expression mainly located in the retinal layer, and significantly higher with US + MBs than US alone, MBs alone or controls (up to 35 days after transfection).
Cheung et al. [33]	FITC-BSA	Ex vivo rabbit sclera	1 cm^2 US probe, 30 s continuous wave, 1 and 3 MHz, 0.05 W/cm^2	Without US: FITC–BSA penetrated to 56.3 ± 26.1 µm by passive diffusion, which was 19 ± 9% of the thickness of the sclera With a brief application (30 s) of low-power ultrasound the penetration distance was 88 ± 22 µm, which was 29 ± 7% of the thickness of the sclera Penetration enhancing was higher at 1 MHz than 3 MHz, and likely mainly due to cavitation phenomena
Zderic et al. [27]	Atenolol, carteolol, timolol or betaxolol (antiglaucoma medications: beta-blockers)	Ex vivo rabbit cornea	1-s bursts of 20-kHz ultrasound, at SAPA of 14 W/cm^2 (SATA of 2 W/cm^2), duration of 10, 30 and 60 min	Permeability increased by 2.6 times for atenolol, 2.8 for carteolol, 1.9 for timolol and 4.4 times for betaxolol after 60 min of US US application appeared to produce epithelial disorganisation and structural changes in the corneal stroma (bubble-like structures were present in the epithelium and occasionally in the stroma)

BL, bubble liposome; FITC-BSA, Fluorescein isothiocyanate conjugated bovine serum albumin; GFP, green fluorescent protein; MB, microbubble; SAPA, spatial average pulse average; SATA, spatial average temporal average; US, ultrasound.

Zderic V, Clark JI, Martin RW, Vaezy S. Ultrasound-enhanced transcorneal drug delivery. Cornea 2004; 23: 804–811

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Zderic V, Clark JI, Vaezy S. Drug delivery into the eye with the use of ultrasound. J Ultrasound Med 2004; 23: 1349–1359

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Sonoda S, Tachibana K, Uchino E, Okubo A, Yamamoto M, Sakoda K, et al. Gene transfer to corneal epithelium and keratocytes mediated by ultrasound with microbubbles. Invest Ophthalmol Vis Sci 2006; 47: 558–564

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Yamashita T, Sonoda S, Suzuki R, Arimura N, Tachibana K, Maruyama K, et al. A novel bubble liposome and ultrasound-mediated gene transfer to ocular surface: RC-1 cells in vitro and conjunctiva in vivo. Exp Eye Res 2007; 85: 741–748

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Li HL, Zheng XZ, Wang HP, Li F, Wu Y, Du LF. Ultrasound-targeted microbubble destruction enhances AAV-mediated gene transfection in human RPE cells in vitro and rat retina in vivo. Gene Ther 2009; 16: 1146–1153

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Cheung AC, Yu Y, Tay D, Wong HS, Ellis-Behnke R, Chau Y. Ultrasound-enhanced intrascleral delivery of protein. Int J Pharm 2010; 401: 16–24

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Zderic V, Vaezy S, Martin RW, Clark JI. Ocular drug delivery using 20-kHz ultrasound. Ultrasound Med Biol 2002; 28: 823–829

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