Ultrasound in Med. & Biol., Vol. 42, No. 11, pp. 2650–2661, 2016Copyright � 2016 World Federation for Ultrasound in Medicine & Biology

Printed in the USA. All rights reserved0301-5629/$ - see front matter

/j.ultrasmedbio.2016.07.013

http://dx.doi.org/10.1016

d Original Contribution

EXAMINATION OF EFFECTS OF LOW-FREQUENCY ULTRASOUND ONSCLERAL PERMEABILITYAND COLLAGEN NETWORK

WAI-LEUNG LANGSTON SUEN,* JUN JIANG,y HOI SANG WONG,* JIANAN QU,y and YING CHAU*z

*Department of Chemical and Biomolecular Engineering, Hong Kong University of Science and Technology, Hong Kong,China; yDepartment of Electronic and Computer Engineering, Hong Kong University of Science and Technology, Hong Kong,China; and zDivision of Biomedical Engineering, Hong Kong University of Science and Technology, Hong Kong, China

(Received 2 September 2015; revised 23 June 2016; in final form 13 July 2016)

ASciencBay, K

Abstract—Delivery of therapeutics to the intraocular space or to targeted tissues in the posterior segment is chal-lenging because of the structural and dynamic barriers surrounding the eye. Previously, we reported the feasibilityof using ultrasound (US) irradiation to deliver macromolecules to the posterior segment of the eye via the trans-scleral route, which consists of sclera as the outermost anatomic barrier. In this study, we found that although ul-trasound increases scleral permeability for macromolecules, the scleral collagen arrangement remainsundisturbed. In an ex vivo experiment, protein permeation across the sclera was significantly enhanced by ultra-sound in the stable cavitation regime. The scleral collagen network was further examined by second harmonic gen-eration imaging. Quantitative image analysis techniques were adopted to examine the density, anisotropy andinterlacing pattern of collagen fibers before and after ultrasound irradiation. Repeated ultrasound applicationsdid not induce significant changes in the arrangement of collagen fibrils at 40 kHz with a spatial average temporalaverage intensity (ISATA) ,1.8 W/cm2. These parameters correspond to a mechanical index (MI) below 0.8 in oursetting. These data suggested that enhanced permeation of macromolecules across the sclera was achieved withoutdisturbing the collagen network of the sclera. This evidence supports that low-frequency, low-intensity ultrasoundis a tolerable approach to transscleral drug delivery. (E-mail: ying.chau@ust.hk) � 2016 World Federation forUltrasound in Medicine & Biology.

Key Words: Second harmonic generation imaging, Drug delivery, Mechanical index, Texture analysis, Collagen,Scleral permeability.

INTRODUCTION

Transscleral delivery of high-molecular-weight com-pounds is an emerging technique for the delivery of ther-apeutic molecules for treatment of eye diseases (Ambatiand Adamis 2002; Ambati et al. 2000; Geroski andEdelhauser, 2001; Thrimawithana et al. 2011). Theoutmost barrier in the transscleral route is the sclera. Ithas a large and accessible surface area and is proximalto the retina. However, the permeability of the sclera tomacromolecules is quite low because of the densenetwork arrangement of acellular collagen fibrils inter-linked with proteoglycans (Young 1985). The outwardbulk fluid flow arising from osmotic and hydrostatic pres-sure further decreases the amount of drug molecules thatcan be effectively delivered (Kim et al. 2007). Hence,

ddress correspondence to: Ying Chau, Hong Kong University ofe and Technology, Room 4353, Academic Building, Clear Waterowloon, Hong Kong, China. E-mail: ying.chau@ust.hk

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there is a need to increase the transscleral flux to over-come the aforementioned static barriers and dynamicclearance to enhance the transport of macromoleculesinto the intraocular space.

Our group recently discovered that, with the applica-tion of low-frequency, low-intensity ultrasound, trans-scleral delivery of macromolecules to the posteriorsegment of the eye was significantly enhanced in live rab-bits. In that study, 40-kHz ultrasound was applied threetimes to the sclera of a live rabbit at an intensity ISATA(spatial average temporal average intensity) of 0.12 W/cm2 with a mechanical index (MI) of 0.20. Permeabilityof macromolecules across the sclera was returned tonormal 2 wk after ultrasound irradiation. No damage tothe ocular tissues was observed, and the visual functionof the rabbits was not affected (Suen et al. 2013). In thestudy described here, our aim was to investigate the effectof ultrasound on the sclera. Answering this question leadsto an understanding of ultrasound-enhanced transscleraltransport by first focusing on the sclera. Bovine serum

Low-frequency US effects on scleral permeability d W.-L. L. SUEN et al. 2651

albumin protein was used in this study to evaluate trans-scleral transport enhancement postsonication. We hy-pothesized that ultrasound increases the permeability ofthe sclera. The effect of scleral permeability enhance-ment after ultrasound applications was evaluated at anISATA (spatial average and temporal average intensity)ranging from 0.002 to 1.8 W/cm2. The selected intensityrange covers both the stable cavitation regime and tran-sient cavitation regime, which is equivalent to the rangeof the MI of ultrasound from 0.03 to 0.8 (Holland andApfel 1989). The MI is a measure of the extent of cavita-tion and can be categorized into two regimes: stable cavi-tation (MI5 0.03–0.20), in which there is gentle periodicpulsation of cavities, and transient cavitation (MI$ 0.8),in which there occur vigorous pulsation and collapse ofthe cavities and the violent collapse of cavities that cre-ates microjets. We further hypothesized that acousticcavitation is essential for enhanced transscleral transport.Thus, protein penetration into the sclera after ultrasoundapplication was measured at different MIs to determine ifthere was any correlation. If all these hypotheses held, itwould remain to be determined whether the enhancementwas related to changes in collagen fiber arrangement.This explanation could be challenged because micron-size pores within collagen network should not restrictthe movement of macromolecules (Chopra et al. 2010).Also, concern over safety arises if the scleral collagennetwork is permanently altered as it is important formaintaining the shape of the eyeball and, therefore, visualfunction. The sclera helps to maintain the overall archi-tecture of the eyeball (Ethier et al. 2004). Changes inthe elastic properties of the sclera have been reported toimpair vision (Watson and Young 2004). Anotherpossible explanation for the improved scleral perme-ability would be changes in non-collagen constituentsof scleral tissue (Young 1985). Proteoglycans, forexample, influence hydration, solute diffusion, and fluidmovement through the sclera, both from the uvea andvia the trabecular meshwork (Watson and Young 2004).Modulation of the proteoglycan arrangement in thescleral matrix might also influence transscleral transport.Owing to the features of the imaging techniques, wefocused on the effect of ultrasound on the scleral collagenfiber network in this study.

Second harmonic generation (SHG) imaging wasemployed to visualize the collagen fibril network. SHGimaging is a non-linear optical imaging technique. It isexcellent for intrinsic 3-D image sectioning, producinghigh contrast superior to that of traditional linear opticalimaging methods, such as confocal microscopy (Denket al. 1990). Although SHG imaging does not exceedSEM (scanning electron microscopy) and AFM (atomicforce microscopy) in terms of image resolution, it canbe used to examine specimens non-invasively. Collagen

has a highly crystalline triple-helix structure that is notcentrosymmetric. This unique feature makes collagen avery effective ‘‘upconverter’’ of light for visualizationby SHG, with a hyperpolarizability just 10-fold lessthan that of crystalline quartz (Fine and Hansen 1971;Han et al. 2005). Image texture analysis was conductedto quantitatively evaluate changes in the collagennetwork after ultrasound irradiation, including thedensity, alignment direction and interlacing pattern ofthe collagen network. These changes were measured byentropy analysis, GLCM analysis and Fourier transformanalysis, respectively.

METHODS

MaterialsFluorescein isothiocyanate-labeled bovine serum al-

bumin (FITC-BSA) (molecular weight: 65 kDa; Sigma,St. Louis, MO, USA) was used as the ex vivo drug model.FITC-BSA was selected because its molecular weight iscomparable to the molecular weight of ranibizumab (Lu-centis, molecular weight: 48 kDa) (Schmucker et al.2010). FITC-BSA was dissolved in phosphate-bufferedsaline (PBS) solution to a final concentration of 0.1 wt% and is denoted as ‘‘BSA solution’’ hereinafter. The so-lution was light protected until the fluorescein measure-ment to reduce the photobleaching effect.

AnimalsAll experimental and animal handling procedures

were in accordance with the requirements of the Animals(Control of Experiments) Ordinance (Cap. 340) and allrelevant legislation and Codes of Practice in HongKong, All procedures were approved by the Faculty Com-mittee on the Use of Live Animals in Teaching andResearch at Hong Kong University of Science and Tech-nology (APCF 2012010).

Ultrasound device and calibrationA 40-kHz ultrasound transducer (Beijing Cheng-

Cheng Weiye Science and Technology, Beijing, China)was powered by a function generator (GFG-8216 A,GW Instek, Montclair, CA, USA), which was connectedto an amplifier (Model 7500 Amplifier; Krohn-Hite,Brockton,MA, USA) to emit ultrasoundwaves. The inputsignal was monitored by an oscilloscope (DS1002 BOscilloscope; Tektronix, Beaverton, OR, USA), whichwas connected in parallel to the amplifier (Suen et al.2013). The driving frequency (which is the same as thepulse repetition frequency of a continuous wave) wasmeasured with a membrane hydrophone (HMB-0500,Onda, Sunnyvale, CA, USA), which was connected tothe oscilloscope. The center frequency was confirmedto be 40 kHz. The ultrasound intensity was measured

2652 Ultrasound in Medicine and Biology Volume 42, Number 11, 2016

using the hydrophone. The average intensity was calcu-lated by measuring the pressure variation across the ultra-sound radiation zone. Both the ultrasound transducer andthe hydrophone were fixed on two separate metal rails.The rail on which the hydrophone was fixed was mademovable in three perpendicular axis directions. Both theultrasound transducer and the hydrophone were dippedinto a tank of degassed water. The hydrophone was thenmoved to a position at which the pressure generated bythe ultrasound wave was the greatest (denoted as thespatial peak position). The plane that is parallel to the ul-trasound transducer surface at the spatial peak positionwas scanned at a 13 1-cm grid by the hydrophone underthe ‘‘pressure-square integral’’ mode. The data obtainedwere filtered by omitting those values smaller than 0.25of the spatial peak (National Council on RadiationProtection and Measurements [NCRP] 1983), and the in-tensity can be calculated with the equation

ISATA 5prr SnðPi2.0:25 Max Pi2Þ

nrc(1)

where ISATA is the spatial-average-temporal-average in-tensity (W/cm2); prr is the pulse repetition rate (equalto the frequency of ultrasound for a continuous wave)(Hz); Max Pi2 is the maximum pressure-square integral;SnPi2.0:25 Max Pi is the sum of all data within the ma-trix with a value greater than 0.25 Max Pi2 (Pa2$s); n isthe number of data points greater than 0.25 Max Pi2 timesthe size of the grid (1 3 1 cm in the sample calculation),or the effective radiation area (cm2); r is the density of themedium (1000 kg/m3 for water in the sample calcula-tion); and c is the speed of sound in the medium(1482 m/s in the sample calculation).

The MI of ultrasound was calculated using thefollowing equation (Szabo 2004) without derating asthe hydrophone measurement and the experimental setupwere both done in the water-base environment:

MI5Pr

f1=2c

(2)

Here, Pr is the peak rarefactional pressure in mega-pascals, and fc is the center frequency in megahertz. Thepeak rarefactional pressure was measured using a hydro-phone and a conditioning amplifier (Br€uel & Kjær,Denmark; Model 2691-A-0 S2, two-channel [singleprobe] Intensity Conditioning Amplifier).

Fig. 1. Schematic of ex vivo ultrasound experiment setup.PBS 5 phosphate-buffered saline solution.

Detection of cavitation by acoustic emissionThe method of detection was similar to that

described by Farny et al. (2009). Briefly, the ultrasoundprobe was connected to a cylindrical tube 14 mm in diam-eter and 15 mm in height made with silicone rubber. The

tube was filled with degassed phosphate-buffered saline,and a single-element ultrasonic atomizing transducer of1.70-MHz central frequency (Model S-UAT03 A, SoundComponents, Hong Kong) was placed at the other end ofthe tube. The 40-kHz ultrasound probe acted as the trans-mitter, and the single element transducer as the receiver,which was connected to an oscilloscope (ModelTDS1002 B, Tektronix, Beaverton, OR, USA) with asampling rate of 1 Giga sample/s. The signal generatedby the receiver in response to the ultrasound producedby the transmitter was recorded by the oscilloscope.The oscilloscope was set to fast Fourier transform(FFT) mode to display the frequency spectrum. The spec-trum was obtained by averaging 128 data points over timeusing the ‘‘Averaging’’ function in the oscilloscope. Theaveraged linear frequency spectrum was first normalizedto the maximum value, and the relative amplitude of thebroadband noise signal to the fundamental signal wascalculated to determine the presence of inertial cavitationactivity.

Ex vivo ultrasound experiments

FITC-BSA penetration measurement. Fresh sclerafrom the posterior segment of the rabbit eye was mountedon a Franz diffusion cell with a contact area of 0.238 mm2

at room temperature (206 2�C). The orbital side was fac-ing the donor chamber, and the uveal side was facing thereceiver chamber. An ultrasound absorber was placed atthe bottom of the receiver chamber (Fig. 1). Two millili-ters of degassed BSA solution was placed in the donorchamber, and 4 mL of degassed phosphate-buffered sa-line was placed in the receiver chamber.

Continuous ultrasound wave at 40 kHz using theaforementioned system was applied 3 cm from the scleraat ISATA 5 0.002, 0.01, 0.05, 0.38 or 1.8 W/cm2 for 30 s.The whole setup was covered with aluminum foil to

Low-frequency US effects on scleral permeability d W.-L. L. SUEN et al. 2653

minimize the photobleaching of fluorescence materials.The sclera was in contact with BSA solution after ultra-sound application for 15 min before cryosectioning.The surface temperature of the sclera was monitoredbefore and after the ultrasound treatment using a thermo-couple (206-3738, RS Components, Hong Kong). No ul-trasound was applied in the control group.

Forty-eight pieces of sclera tissue were cryosec-tioned into 10-mm-thick sections using a cryostat(CM1850, Leica, Wetzlar, Germany). The sectioned tis-sues were viewed with a fluorescence microscope todetermine the penetration distance of FITC-dextran(Cheung et al. 2010). Experiments under each conditionwere repeated at least three times (n$ 3). The penetrationdistance of FITC-BSA across sclera was measured usingImageJ software. The measurements were taken perpen-dicular to the general orientation of the sclera. The pene-tration distance is defined as the distance from the sclerasurface to the deepest depth at which the fluorescentsignal reached the statistical threshold. The statisticalthreshold (i.e., background signal of the slide) is definedas the average signal of the control sclera (not exposed tomodel drug). The same microscope setting and exposuretime were used in capturing images from different exper-iments to obtain comparable contrast in the fluorescenceimages. The permeation distance was measured in threeseparate zones (zones are separated from each other byat least 100 mm) in each cryosectioned sclera sample.The arithmetical mean penetration distance of FITC-BSA in each intensity group was obtained from 12 sclerasections with a minimum of 20 measurements. Compari-son between different conditions was performed by two-tailed t-test, and differences were considered statisticallysignificant at p , 0.05.

Examination of scleral collagen arrangement. Thesetup was similar to that described above except that2 mL of degassed phosphate-buffered saline solutionwas placed in the donor chamber. A continuous ultrasoundwave at 40 kHz with ISATA5 0.002, 0.05, 0.12 or 1.80 wasapplied 3 cm away to the sclera. The sclera was immedi-ately removed from the setup for SHG imaging to deter-mine the collagen fibril arrangement in the sclera.

Imaging experimentAn SHG microscope system was instrumented as

described in previous work (Zeng et al. 2014). The exci-tation source was a femtosecond Ti:sapphire laser tuningat 740 nm. Two single-channel photomultiplier tubesconnecting to a TCSPC board were used as the detectionsystem, which covered two separate spectral bands: 370–400 nm and 500–700 nm. An actuator was used to controlthe imaging depth. Thirty consecutive images with theinterval depth of 2 mm below the surface of sclera were

collected for analysis in each site. In total, 48 pieces ofsclera were used and were evenly divided into four groupsaccording to the ultrasound parameter, that is,ISATA 5 0.002, 0.05, 0.12 and 1.8 W/cm2. In each pieceof sclera, six different sites were examined with theSHG microscope before and after insonation.

Image texture analysis. Three texture analyses tech-niques were used to quantitatively examine the changes inthe sclera interfibrillar matrix, that is, the space betweenindividual fibrils, the relative position of individual fi-brils, the dominant orientation of the fibrils in a regionand the interlacing pattern of the collagen network. Allthese methods captured the texture information ofcollagen fibrils on the XY image plane (parallel to the sur-face of the sclera) of each layer, which was obtained usingthe procedures illustrated in Figure 2 (Ferguson 2007).Briefly, after the 3-D image stacks are obtained fromseveral different sites on the sclera, we first performtexture analysis on each layer and then average the datafrom all the layers in a stack. Finally, the data fromdifferent stacks are further averaged to obtain the final re-sults for this sclera (this step is not illustrated in Fig. 2). Inaddition to the texture analysis, we created projection im-ages for each 3-D stack using ImageJ software, whichcould provide a perspective view of the arrangement ofcollagen fibers (see Fig. 2).

Entropy analysis. Entropy was a first-order statisti-cal description of texture. Entropy analysis was used tomeasure the texture randomness and determine the pres-ence of collagen fibrils in an image; these were treated asrandom variables (Ihara 1993; Kuczynski andMikolajczak 2003). Images with low entropy had littlegray-level contrast. (An image that was perfectly flathas zero entropy.) On the other hand, images with highentropy had higher levels of contrast from one pixel tothe next and were more ‘‘chaotic.’’ In this study, the en-tropy of the 2-D image I was calculated using MATLABsoftware’s built-in function; this was followed by takingthe reciprocal of the value (1/entropy).

Fast Fourier transform (FFTAR) analysis. Fourieranalysis examines the texture in the frequency domain,and was regarded as a higher-order statistical analysismethod. After the transformed image was obtained,texture information could be extracted to describe themorphology of the original image. Here, we focused onthe orderliness of texture by calculating the asymmetryratio: for a well-aligned pattern, its FFT image in the fre-quency domain would have higher values along the direc-tion orthogonal to alignment. This would result in ellipticbehavior in the respective intensity plot, indicating thatthe examined image has a high asymmetry ratio. On thecontrary, the FFT image of an image with a random

Fig. 2. Schematic of the image processing procedure. Texture analysis was performed on each layer, and then the valueswere averaged. The projection image here was actually obtained using ImageJ software and is equal to the sum all the

images in a stack. SHG 5 second harmonic generation.

2654 Ultrasound in Medicine and Biology Volume 42, Number 11, 2016

pattern would exhibit a circular plot, which indicates alow asymmetry ratio. The values of the asymmetric ratioof FFT (denoted as FFTAR) images were obtained as thedifference between the maximum and minimum of theline integral value (Zheng et al. 2011).

Gray-level co-occurrence matrix analysis. Gray-level co-occurrence matrix (GLCM) texture analysiswas based on the second-order statistics (i.e., statisticsgiven by pairs of pixels) of gray-level pixel distribution(Haralick 1979; Zheng et al. 2011). The jointprobability distribution of pairs of pixels (i.e., howoften a pixel with the gray-level value i occurs in a spe-cific spatial relationship to a pixel with the value j) wasdefined as the GLCM, and different texture features couldbe deduced from the GLCM. In this study, we usedGLCM correlation to evaluate the linear dependence be-tween each pixel pair in the image. The higher value indi-cated that there were more similar structures in the image.Therefore, it could provide an estimate of the organiza-tion and structure of the collagen fibrils. Because

GLCM was sensitive to direction and pixel distances,GLCM correlation was averaged from 0 to 180� and pixeldistances from 1 to 48 in our analysis. The GLCM corre-lation was abbreviated GLCMCORR and was obtainedfrom MATLAB codes (Haralick 1979).

Intraocular pressure measurementThe intraocular pressure of the eye of New Zealand

white rabbits was recorded with Tonopen XL (Ametek,Berwyn, PA, USA) before and after application of 40-kHz ultrasound at the indicated intensities. Five sampleswere tested for each ultrasound intensity. Measurementswith the Tonopen XL were repeated until the coefficientof variation was less than 5% (Lim et al. 2005).

RESULTS

Enhanced transscleral transport of macromolecules byultrasound ex vivo

Penetration distance of FITC-BSA across ex vivosclera was measured after application of 40-kHz

Fig. 3. Penetration of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) after application of 40-kHzultrasound for 30 s and 15 min of immersion: (a) control; (b) ISATA 5 0.002 W/cm2; (c) ISATA 5 0.01 W/cm2; (d)ISATA 5 0.05 W/cm2; (e) ISATA 5 0.38 W/cm2; (f) ISATA 5 1.8 W/cm2; A representative cryosection viewed by fluores-

cence microscope is shown. U 5 uveal side; S 5 sclera; O 5 orbital side.

Low-frequency US effects on scleral permeability d W.-L. L. SUEN et al. 2655

ultrasound at different intensities (Fig. 3). The bright-field and fluorescence images of cryosectioned sclerawere combined before measurement of protein penetra-tion distance using ImageJ software. The representativeimages of cryosectioned sclera and the measurement ofpenetration distance with ImageJ software are illustrated

Fig. 4. Penetration distance of 0.1 wt% isothiocyanate-labeled40 kHz at different intensity values, followed by 15 min of i

compared with control group.

in Figure 3. The arithmetical mean thickness of the sclerais �300 6 32 mm; this value was obtained by averagingthe thickness of 48 pieces of sclera tissue. The tempera-ture on the surface of the sclera was measured beforeand after sonication. Negligible temperate changes(,0.1�C) were detected after the sclera was sonicated

bovine serum albumin after 30 s of ultrasound radiation atmmersion (n 5 3). p , 0.05, ultrasound-treated groupsMI 5 mechanical index.

Fig. 5. Projection images: (a) ISATA 5 0.002 W/cm2; (b) ISATA 5 0.05 W/cm2; (c) ISATA 5 0.12 W/cm2; (d)ISATA 5 1.8 W/cm2. This imaging area measured 200 3 200 mm, 256 3 256 pixels.

2656 Ultrasound in Medicine and Biology Volume 42, Number 11, 2016

at ISATA5 0.0002, 0.05 and 0.12W/cm2. The temperatureon the scleral surface increased 1.02 6 0.8�C after soni-cation at an intensity of 1.8 W/cm2. The changes in thescleral surface temperature were comparable to the calcu-lation results based on ultrasound energy input (seeSupplementary Material, online only, available at http://dx.doi.org/10.1016/j.ultrasmedbio.2016.07.013).

From Figure 4, the enhancement of transscleralpenetration of FITC-BSA ex vivowas significantly higherin the low-intensity regime (ISATA 5 0.002, 0.01 and0.05 W/cm2) than in the high-intensity regime(ISATA 5 0.38 and 1.8 W/cm2). Compared with control,40-kHz ultrasound irradiation, regardless of the level ofintensity, could significantly enhance the permeation ofFITC-BSA along the transscleral route, with an increasefrom 48% to 63%. It is interesting to note that the increasein ultrasound intensity did not lead to a further increase inpenetration distance of BSA along the transscleral route.Rather, the ultrasound intensity that leads to maximumpenetration distance of BSA was observed only in thelower-intensity regime (ISATA between 0.002 and0.05 W/cm2), which corresponds to the stable cavitationregime with the MI between 0.003 and 0.136.

Second harmonic generation imagingSecond harmonic generation images of the ex vivo

sclera tissue were acquired by scanning the tissue fromthe outmost surface to the inner part, layer by layer. Afterscanning, the images obtained were stacked to generateZ-stack images before image projection to generate a 2-D image plane (Fig. 2). Figure 5 illustrates the typical

projection images of ex vivo sclera tissue after ultrasoundirradiation at ISATA 5 0.002, 0.05, 0.12 and 1.8 W/cm2,respectively. Note that the same area of the sclera wasimaged before and after ultrasound applications.

Image texture analysisThe final results of texture analysis are illustrated in

Figure 6. The t-test reveals that the difference between the‘‘control group’’ and all ‘‘treated groups’’ is not statisti-cally significant (p . 0.01). Here, 1/entropy provides in-formation on the density of collagen fibrils at a scale of200 3 200 mm. Ex vivo sclera treated with 40 kHz atthe ultrasound intensities tested did not exhibit significantchanges in the 1/entropy value. Therefore, we canconclude that the overall interlacing pattern of collagenfibrils was not affected by 40-kHz ultrasound in boththe low-intensity and high-intensity regimes. Becausecollagen fibril is reported to have a high level of anisot-ropy, fast Fourier transform analysis was conducted tocapture the changes in anisotropy, that is, the dominantalignment direction of collagen fibrils in sclera after ultra-sound irradiation. Again, ex vivo sclera tissue did notexhibit significant changes in collagen anisotropy on ul-trasound irradiation at all tested intensities. Both entropyanalysis and the asymmetric ratio of fast Fourier trans-form analysis were conducted on the entire image planeto provide information on the collagen interlacing patternand collagen anisotropy at a macroscale (2003 200 mm).On the contrary, GLCM correlation analysis focuses onthe local correlation and variation of the collagen fibrilpattern, which grasps the texture on a smaller scale

Fig. 6. Results of the texture analysis of the control (untreated sclera) and treated (ultrasound-exposed) groups. Results ofthe t-test indicate that the differences between the control and treated groups were not statistically significant.FFTAR5 asymmetric ratio of fast Fourier transform; GLCM5 gray-level co-occurrence matrix; MI5mechanical index.

Low-frequency US effects on scleral permeability d W.-L. L. SUEN et al. 2657

(20 3 20 mm). In this context, we are interested in eval-uating if ultrasound irradiation would induce changes inthe spacing, relative position and alignment direction ofscleral collagen at a local level. From the mathematicsperspective, collagen interlacing pattern and morphology(spacing, relative positions and alignment direction) wereevaluated by first-order, second-order and higher-orderstatistical analyses, corresponding to entropy analysis,GLCM analysis and FFTAR, respectively. Figure 6 illus-trates that there were no observable changes in thecollagen arrangement in both the low- and high-intensity regimes (ISATA 5 0.005, 0.05, 0.12 and 1.8 W/cm2) from FFTAR and GLCM analysis.

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Intraocular pressure measurement

The typical intraocular pressure (IOP) e of untreatedrabbit eye is 9.4 6 0.4 mm Hg (Fig. 7). After 40-kHz ul-trasound applications at the indicated intensities, the IOPvalue recorded after sonication did not change signifi-cantly (p . 0.05) compared with the IOP value recordedbefore sonication (Fig. 7).

00.03 0.06 0.133 0.20 Before

sonica on

IOP

of u

Mechanical Index

Fig. 7. Intraocular pressure (IOP) measurement of rabbit eyebefore and after 40-kHz ultrasound application at MI 5 0.03–0.20. N 5 5. There were no statistically significant differences(p . 0.05) between the ultrasound-exposed groups (MI 5

0.03–0.20) and the control group.

Passive acoustic detection of cavitationThe presence of cavitation was verified by passive

acoustic detection of bubble activity. Figure 8 illustratesthe frequency spectra at 40 kHz at ISATA 5 0.05, 0.38and 1.8 W/cm2. Peaks at subharmonic (f/2) and funda-mental (f) frequency, which denote the stable oscillationof bubbles, are present in the low-intensity regime

(Fig. 8a). In the low-intensity regime (ISATA 5 0.05 W/cm2), the distance between the fundamental and the noisefloor was 54.44 dB (Fig. 8a). The distance between thefundamental and the noise floor decreased from 62.69to 59.5 dB after ultrasound intensity increased from0.38 to 1.8 W/cm2 (Fig. 8b, c). This indicated the pres-ence of inertial cavitation in the higher-intensity regime(Leighton 1994, 1995, 1997).

DISCUSSION

Herein, we have reported that low-frequency ultra-sound did not induce changes in the collagen network

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Fig. 8. Frequency spectrum for 40-kHz ultrasound at (a)ISATA 5 0.05 W/cm2, (b) ISATA 5 0.38 W/cm2, and (c)ISATA 5 1.8 W/cm2. The ‘‘f’’ and ‘‘f/2’’ marks indicate thepeak of the driving frequency and the subharmonic frequency.The presence of inertial cavitation at higher intensity regimewas indicated by the reduction of the distance between thefundamental peak and the broadband noise. FFT5 fast Fourier

transform.

2658 Ultrasound in Medicine and Biology Volume 42, Number 11, 2016

at all intensities tested, whereas transport of macromole-cules along the transscleral route was significantlyenhanced (Suen et al. 2013).

Rabbit sclera was chosen in this study to investigatethe effect of ultrasound on scleral collagen arrangement.It has been reported that there is similar proteoglycan–collagen organization in human and rabbit sclera(Young 1985). It is interesting that the scleral collagenarrangement remained undisturbed after sonication atISATA values ranging from 0.002 to 1.8 W/cm2. The pres-ence of cavitation in the low-intensity regime appears tohave had no effect on the scleral collagen matrix (Cheunget al. 2010). It is known that ultrasound can induce acous-tic cavitation in which cavities alternately shrink andgrow in size, oscillating in response to the subsequent

high- and low-pressure portions of the ultrasound waveduring stable cavitation (Merino et al. 2003). Stable cavi-tation occurs at low MIs (MI 5 0.03–0.133, which isequivalent to ISATA 5 0.002–0.05 W/cm2 in our setting),which is manifested by the periodic pulsation of bubblesor cavities in the medium. These pulsations create micro-streaming, that is, fluid flow around the cavities(Hitchcock and Holland 2010; Soltani et al. 2008).Transient cavitation, on the other hand, is a vigorousform of cavity oscillations that results in violentcollapse of the cavities. When this occurs, the bubblewalls attain supersonic speeds on the collapse. Whencavitations occur near or within the sclera, thesemicrostreams could exert force on the collagen fibers.At the highest intensity tested (1.8 W/cm2) in thisstudy, however, the energy did not appear to induce anypermanent changes in the collagen network.

The enhancement of the transscleral penetration ofFITC-BSA ex vivo, interestingly, was higher in the stablecavitation regime (MI between 0.03 and 0.133, which isequivalent to ISATA 5 0.002–0.05 W/cm2 in our setting).The enhancement of transscleral transport can be due tothe microstreaming effects created by pulsating cavitiesin the medium during ultrasound application. The chaoticmixing induced by pulsating bubbles could enhance thepermeation of macromolecules along the transscleralroute. Though sufficient to induce microstreams thatassist the permeation of FITC-BSA across the scleral tis-sue, the energy from stable cavitation did not producechanges in the collagen network. Because there are noguidelines on the use of ultrasound for delivery of oculardrugs, we benchmarked the ultrasound setting in ourstudy to the U.S. Food and Drug Administration(USFDA) regulation for diagnostic imaging (USFDA2008) that the MI of ultrasound should not exceed 0.23for ophthalmic use (USFDA 2008). With respect to theultrasound-induced heating effect, USFDA regulationsrequire that the ultrasound system for ophthalmic useshould have a thermal index,1. We observed negligibletemperature changes on the scleral surface (,1�C) at allacoustic outputs tested. The negligible changes in tem-perature on the scleral surface were supported by thelow energy input by the ultrasound probe. Negligibletemperate changes (,0.1�C) were detected after thesclera was sonicated at ISATA 5 0.0002, 0.05 and0.12 W/cm2. The temperature on the scleral surfaceincreased 1.02 6 0.8�C after sonication at an intensityof 1.8 W/cm2. The changes in scleral surface temperaturewere comparable to the results calculated on the basis ofultrasound energy input (see Supplementary Material).As we have shown that the enhancement by ultrasoundin transscleral delivery is optimal under stable cavitation,maximum enhancement of delivery occurs withoutviolating the current regulation.

Low-frequency US effects on scleral permeability d W.-L. L. SUEN et al. 2659

The collagen fibril is a triple-helix molecular assem-bly that enables SHG imaging (Han et al. 2005). Weadopted the two-photon excited fluorescence (TPEF)-SHG imaging technique to visually analyze the arrange-ment of collagen fibrils in the sclera before and afterultrasound applications. By taking advantage of theintrinsic properties of the collagen triple-helix arrange-ment, we could examine the collagen structure whileminimizing artifacts caused by sample processing and la-beling (Han et al. 2005). In our study, the aforementionedTPEF-SHG system can generate images with a resolutionlevel that can distinguish between individual collagen fi-brils and yield images for quantifying packing density inthe formation of collagen bundles or lamellae (Denk et al.1990).

Quantitative SHG image analysis techniques wereemployed to extract information on the collagen arrange-ment along the XY plane. Komai and Ushiki (1991) re-ported that collagen bundles or lamellae mostly runparallel to the XY plane, and therefore, it contains mostof the features of the collagen network structure.Collagen fibrils in the sclera interweave with each otherto form a network structure: the outer region consists ofparallel-aligned collagen fibrils, which are packed intoslender lamellae; increased interweaving of collagen fi-ber bundles replaces the lamellar arrangement in thedeep sclera, with scattered elastic fibers throughout thestroma (Komai and Ushiki 1991). The overall texturerandomness of collagen fibrils after ultrasound irradiationdid not reveal significant changes as indicated by datafrom SHG image entropy analysis, which implies thereis no thinning of collagen fibers. FFT analysis has beenused to evaluate the orientation index (or anisotropy) ofSHG images of skin and corneal collagen fibers, whichis effective for discrimination of collagen fibers withand without a particular orientation (Hamanaka 1989;Matteini et al. 2009; Rayleigh 1879). From our results,collagen fibrils did not exhibit significant changes inalignment orientation after ultrasound irradiation atvarious MIs. To further differentiate morphologicpatterns of collagen fibrils (linear, curved ordisordered), we adopted orientation-dependent GLCManalysis. GLCM analysis revealed that there was insignif-icant change in the regularity and continuity of collagenbundles after ultrasound irradiation at both low intensity(ISATA between 0.002 and 0.05W/cm2) and high intensity(ISATA 5 0.38 and 1.8 W/cm2). These findings clearlyindicate that ultrasound application in the low-intensityregime (with an equivalent MI from 0.03 to 0.20) andhigh-intensity regime (with an equivalent MI from 0.38and 0.8) does not raise safety concerns, as scleral collagenremained unchanged. The elaborate intertwining natureof the collagen bundles is functionally important becausethis structure is considered to be effective in giving the

eyeball its rigidity and flexibility against changes in intra-ocular pressure. Previous studies have reported that thisscleral thinning is associated with a narrowing and disso-ciation of the collagen fiber bundles and a reduction incollagen fibril diameter (Curtin et al. 1979). Changes inthe biochemical structure of the sclera, such as alteredglycosaminoglycan (GAG) and collagen content(Avetisov et al. 1983), have also been reported in highlymyopic postmortem human eyes and are further evidenceof scleral pathology in high myopia. In our study, we didnot observe changes from the scleral collagen arrange-ment; thus, ultrasound application for enhanced trans-scleral transport is a safe approach for delivery ofocular drugs.

In addition to the texture analysis of SHG images,the results of IOP measurements further supported thenotion that ultrasound application at the intensities testeddid not alter scleral structure. It is known that changes inIOP indicate that the mechanical property of the sclera isaltered (Palko et al. 2011). No significant changes inintraocular pressure were observed, and thus, weconcluded that ultrasound did not induce changes in themechanical properties of the sclera before and aftertreatment.

This finding further indicates that cavitationactivity-induced sonication in the low-MI regime(MI 5 0.03–0.20, stable cavitation) and high-MI regime(MI 5 0.38–0.8, transient cavitation) would not affectscleral collagen structure. Cavitation is the formation orinteraction of cavities in the liquid body caused by rapidchanges in pressure; often the cavities formed in thelower-pressure regions of the liquid. Effects of cavitationcan be measured using the MI (Holland and Apfel 1989).The MI is the ratio between peak negative pressuresgenerated by the ultrasound and the frequency. Whenlow-frequency, low-intensity ultrasound radiated on thesclera in the presence of a liquid medium, cavitationoccurred. In the stable cavitation regime, cavities in themedium started to pulsate periodically without violentcollapse. From the results of passive cavitation detection,our ultrasound system could trigger stable cavitationwhen ultrasound was generated at ISATA between 0.002and 0.12 W/cm2. When ultrasound intensity increasedto 0.38 W/cm2, cavities in the medium started to pulsatevigorously and eventually burst; the bubble walls attainedsupersonic speeds and created microjets (Apfel andHolland 1991). This phenomenon was observed in our ul-trasound system at ISATA5 0.38 and 1.8W/cm2, validatedby the reduction in distance between the broadband noiseand the fundamental signal. (Leighton 1997).

In combining the results of protein transscleral pene-tration with SHG imaging analysis, it is interesting tonote that enhanced permeation of macromolecules acrossthe sclera could be achieved by cavitation, at low MI,

2660 Ultrasound in Medicine and Biology Volume 42, Number 11, 2016

without upsetting the scleral collagen arrangement. Thestudy confirms that changes in the scleral collagennetwork are not necessary for transscleral penetrationenhancement. This is desirable for future ultrasound-mediated transscleral delivery of therapeutics to the eyebecause of the strategic location and function of the scleraas the outermost barrier and mechanical support of theeyeball.

As mentioned in the previous section, the sclera iscomposed of collagen fibrils interlinked with proteogly-can molecules. A thorough analysis of the effect of ultra-sound on the scleral collagen network was carried out andreported in previous sections. However, little is knownabout the effect of ultrasound on non-collagen constitu-ents. Further studies on non-collagen constituents arerequired to have a better understanding of the enhancedtransscleral penetration of macromolecules observed inprevious study.

CONCLUSIONS

Penetration of macromolecules to the sclera isenhanced by ultrasound, and no permanent changes inthe scleral collagen network are observed. Optimal trans-scleral transport enhancement lies within the low-intensity regime. We conclude that low-frequency,low-intensity ultrasound does not affect the scleralcollagen fibrillar interlacing pattern and anisotropy. Asystematic study was conducted to evaluate the effect ofultrasound on collagen fibril arrangement. The resultsfrom quantitative image analysis revealed that the differ-ences in the collagen fibril arrangement after applicationof 40-kHz continuous ultrasound to ex vivo sclera tissuein both the low-intensity and high-intensity regimeswere not significant.

Acknowledgments—The research work was supported by Hong KongUniversity Grant Committee—Research Infrastructure Grant FSGRF14EG09.

SUPPLEMENTARY DATA

Supplementary data related to this article can befound at http://dx.doi.org/10.1016/j.ultrasmedbio.2016.07.013.

REFERENCES

Ambati J, Adamis AP. Transscleral drug delivery to the retina andchoroid. Prog Retinal Eye Res 2002;21:145–151.

Ambati J, Gragoudas ES, Miller JW, You TT, Miyamoto K, Delori FC,Adamis AP. Transscleral delivery of bioactive protein to the choroidand retina. Invest Ophthalmol Visual Sci 2000;41:1186–1191.

Apfel RE, Holland CK. Gauging the likelihood of cavitation from short-pulse, low-duty cycle diagnostic ultrasound. Ultrasound Med Biol1991;17:179–185.

Avetisov ES, Savitskaya NF, Vinetskaya MI, Iomdina EN. A study ofbiochemical and biomechanical qualities of normal and myopic

eye sclera in humans of different age groups. Metab Pediatr SystOphthalmol 1983;7:183–188.

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

Chopra P, Hao J, Li SK. Iontophoretic transport of charged macromol-ecules across human sclera. Int J Pharm 2010;388:107–113.

Curtin BJ, Iwamoto T, Renaldo DP. Normal and staphylomatous scleraof high myopia: An electron microscopic study. Arch Ophthalmol1979;97:912–915.

Denk W, Strickler JH, Webb WW. Two-photon laser scanning fluores-cence microscopy. Science 1990;248:73–76.

Ethier CR, JohnsonM, Ruberti J. Ocular biomechanics and biotransport.Ann Rev Biomed Eng 2004;6:249–273.

Farny CH, Holt RG, Roy RA. Temporal and spatial detection of HIFU-induced inertial and hot-vapor cavitation with a diagnostic ultra-sound system. Ultrasound Med Biol 2009;35:603–615.

Ferguson JR. Using the grey-level co-occurrence matrix to segmentand classify radar imagery. Reno, Ann Arbor, MI: ProQuest;2007. p. 174.

Fine S, Hansen WP. Optical second harmonic generation in biologicalsystems. Appl Opt 1971;10:2350–2353.

Geroski DH, Edelhauser HF. Transscleral drug delivery for posteriorsegment disease. Adv Drug Deliv Rev 2001;52:37–48.

Hamanaka T. Scleral spur and ciliary muscle in man and monkey. Jpn JPphthalmol 1989;33:221–236.

Han M, Giese G, Bille J. Second harmonic generation imaging ofcollagen fibrils in cornea and sclera. Opt Express 2005;13:5791–5797.

Haralick RM. Statistical and structural approaches to textures. ProcIEEE 1979;67:786–804.

HitchcockKE, Holland CK. Ultrasound-assisted thrombolysis for stroketherapy: Better thrombus break-up with bubbles. Stroke 2010;41:S50–S53.

Holland CK, Apfel RE. An improved theory for the prediction of micro-cavitation thresholds. IEEE Trans Ultrason Ferroelectr Freq Control1989;36:204–208.

Ihara S. Information theory for continuous system. Singapore: WorldScientific; 1993.

Kim SH, Lutz RJ, Wang NS, Robinson MR. Transport barriers in trans-scleral drug delivery for retinal diseases. Ophthalmic Res 2007;39:244–254.

Komai Y, Ushiki T. The three-dimensional organization of collagen fi-brils in the human cornea and sclera. Invest Ophthalmol VisualSci 1991;32:2244–2258.

Kuczynski K, Mikolajczak P. Information theory based medical imagesprocessing. Opto-Electron Rev 2003;11:253–259.

Leighton TG. Acoustic bubble detection: 1. The detection of stable gasbodies. Environ Eng 1994;9–16.

Leighton TG. Acoustic bubble detection: 2. The detection of transientcavitation. Environ Eng 1995;17–25.

Leighton TG. The acoustic bubble. New York/London: Academic Press;1997.

Lim KS, Wickremasinghe SS, Cordeiro MF, Bunce C, Khaw PT.Accuracy of intraocular pressure measurements in new zealandwhite rabbits. Invest Ophthalmol Visual Sci 2005;46:2419–2423.

Matteini P, Ratto F, Rossi F, Cicchi R, Stringari C, Kapsokalyvas D,Pavone FS, Pini R. Photothermally-induced disordered patterns ofcorneal collagen revealed by SHG imaging. Opt Express 2009;17:4868–4878.

Merino G, Kalia YN, Guy RH. Ultrasound-enhanced transdermal trans-port. J Pharm Sci 2003;92:1125–1137.

National Council on Radiation Protection and Measurements (NCRP).Biological effects of ultrasound: Mechanisms and clinical implica-tions. Bethesda, MD: Author; 1983.

Palko JR, Pan X, Liu J. Dynamic testing of regional viscoelasticbehavior of canine sclera. Exp Eye Res 2011;93:825–832.

Rayleigh FRS. Investigations in optics, with special reference to thespectroscope. Philos Mag 1879;58:261–274.

Schmucker C, Ehlken C, Hansen LL, Antes G, Agostini HT,Lelgemann M. Intravitreal bevacizumab (Avastin) vs. ranibizumab

Low-frequency US effects on scleral permeability d W.-L. L. SUEN et al. 2661

(Lucentis) for the treatment of age-related macular degeneration: Asystematic review. Curr Opin Ophthalmol 2010;21:218–226.

Soltani A, Volz KR, Hansmann DR. Effect of modulated ultrasound pa-rameters on ultrasound-induced thrombolysis. Phys Med Biol 2008;53:6837–6847.

Suen WL, Wong HS, Yu Y, Lau LC, Lo AC, Chau Y. Ultrasound-medi-ated transscleral delivery of macromolecules to the posteriorsegment of rabbit eye in vivo. Invest Ophthalmol Visual Sci 2013;54:4358–4365.

Szabo T. Diagnostic ultrasound imaging: Inside out. Orlando, FL: Aca-demic Press; 2004.

Thrimawithana TR, Young SA, Bunt CR, Green CR, Alany RG. In-vitroand in-vivo evaluation of carrageenan/methylcellulose polymericsystems for transscleral delivery of macromolecules. Eur J PharmSci 2011;44:399–409.

U.S. Food and Drug Administration (USFDA). Guidance for industryand FDA staff: Information for manufacturers seeking marketingclearance of diagnostic ultrasound systems and transducers. SilverSpring, MD: Author; 2008.

Watson PG, Young RD. Scleral structure, organisation and disease: A re-view. Exp Eye Res 2004;78:609–623.

Young RD. Ultrastructural organization of proteoglycans and collagensin human and rabbit scleral matrix. J Cell Sci 1985;74:95–104.

Zeng Y, Yan B, Sun Q, He S, Jiang J, Wen Z, Qu JY. In vivo micro-vascular imaging and flow cytometry in zebrafish using two-photon excited endogenous fluorescence. Biomed Express 2014;5:653–663.

Zheng W, Li D, Li S, Zeng Y, Yang Y, Qu JY. Diagnostic value ofnonlinear optical signals from collagen matrix in the detection ofepithelial precancer. Opt Lett 2011;36:3620–3622.