Hypocrellin B-mediated sonodynamic action induces apoptosis of hepatocellular carcinoma cells

Objective

The present study aims to investigate apoptosis of hepatocellular carcinoma cells induced by hypocrellin B-mediated sonodynamic action.

Methods

The hypocrellin B concentration was kept constant at 2.5 μM and cells from the hepatocellular carcinoma HepG2 cell line were exposed to ultrasound with an intensity of 0.46 W/cm² for 8 s. Cell cytotoxicity was quantified using an MTT assay 24 h after sonodynamic therapy (SDT) of hypocrellin B. Apoptosis was investigated using a flow cytometry with Annexin V-FITC and propidium iodine staining. Intracellular reactive oxygen species (ROS) levels were detected using a flow cytometry with 2,7-dichlorodihydrofluorecein diacetate (DCFH-DA) staining.

Results

The cytotoxicity of hypocrellin B-mediated sonodynamic action on HepG2 cells was significantly higher than those of other treatments including ultrasound alone, hypocrellin B alone and sham treatment. Flow cytometry showed that hypocrellin B-induced sonodynamic action markedly enhanced the apoptotic rate of HepG2 cells. Increased ROS was observed in HepG2 cells after being treated with hypocrellin B-mediated sonodynamic action.

Conclusions

Our data demonstrated that hypocrellin B-mediated sonodynamic action remarkably induced apoptosis of HepG2 cells, suggesting that apoptosis is an important mechanism of cell death induced by hypocrellin B-mediated SDT.     

Protoporphyrin IX-mediated sonodynamic action induces apoptosis of K562 cells

Objectives

The present study aims to investigate apoptosis of human leukemia K562 cells induced by protoporphyrin IX (PpIX)-mediated sonodynamic therapy (PpIX-SDT).

Methods

The uptakes of intracellular PpIX in K562 cells were detected by flow cytometry. The sub-cellular localization of PpIX was imaged by confocal microscope. The cytotoxic effect of PpIX-SDT was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium) assay. Apoptosis was evaluated by chromatin condensation with DAPI (4′-6-diamidino-2-phenylindole) staining, decrease of mitochondria membrane potential (MMP), re-distribution of Bax, and the expression changes of the key apoptosis-associated protein (Caspase-3 and polypeptide poly (ADP-robose) polymerase). The possible mechanism of SDT-induced apoptosis was investigated by detecting by intracellular ROS (reactive oxygen species) generation and effect of ROS scavenger-NAC (N-acetylcysteine) on SDT induced apoptosis.

Results

The intracellular PpIX increased quickly within 2 h after PpIX administration and PpIX mainly localized in the mitochondria. Compared with PpIX alone and ultrasound alone groups, the synergistic cytotoxicity of PpIX plus ultrasound was significantly boosted. In addition, the ultrasound induced some extent of chromatin condensation and MMP loss was greatly enhanced by the presence of 2 μg/ml PpIX, where PpIX alone treatment showed no or only slight effect. Time-dependent Bax translocation, caspase-3 activation and PARP cleavage were detected in SDT treatment groups. Besides, intracellular ROS production was significantly enhanced after SDT, and the general ROS scavenger NAC could obviously alleviate the SDT-caused cell viability loss, MMP loss, Bax redistribution and nuclear changes.

Conclusions

These results indicated that PpIX-mediated sonodynamic action could induce apoptosis on K562 cells, and the intracellular ROS was involved in the PpIX-SDT induced apoptosis.     

Sonodynamically induced anti-tumor effect with protoporphyrin IX on hepatoma-22 solid tumor

Objective

The purpose of this study was to evaluate sonodynamically induced anti-tumor effect of protoporphyrin IX (PPIX) in mice bearing hepatoma-22 (H-22) solid tumors, and the possible in vivo cell damage mechanism was also investigated.

Methods

The pharmacokinetics of PPIX was analyzed in plasma, skin, muscle and tumor of H-22 bearing mice. Tumors were irradiated with ultrasound (1.43 MHz, I_SATA 3 W/cm², 3 min) for three times at 8, 12 and 24 h after 5.0 mg/kg PPIX administration, respectively. The anti-tumor effects of sonodynamic therapy (SDT) were estimated by the tumor inhibition ratio (volume and weight). The bio-effects of SDT were evaluated by hematoxylin and eosin (H&E) staining, transmission electron microscope (TEM) observation, lipid peroxidation (LPO) measurement and anti-oxidative enzymes (glutathione peroxidase (GSH-PX), catalase (CAT) and superoxide dismutase (SOD)) assay.

Results

A significant anti-tumor effect was obtained by PPIX-mediated sonodynamic therapy (PPIX-SDT). At the fifteenth day after PPIX-SDT, the tumor growth and tumor weight inhibition ratios were 53.84% and 45.86%, respectively. In addition, the structure of tumor tissues and the anti-oxidative enzymes were obviously destroyed after SDT treatment.

Conclusions

A biochemical mechanism was involved in PPIX-SDT in vivo, and the free radicals produced by the synergistic treatment destroying the anti-oxidative system of tumor cells in vivo may play an important role in this action. Also, the thermal effect could not be excluded in inducing damage of cellular structures, like membrane disruption and chromatin condensation under current evaluation in this paper.     

Characterization of ultrasound-mediated destruction of drug-loaded microbubbles using an improved in vitro model

Introduction

Ultrasound mediated destruction of microbubbles (MBs) has become a promising tool for site specific drug and gene delivery. One of the most important properties of drug-loaded MBs is their destructibility by ultrasound. Therefore, the aim of this study was to establish a new in vitro model that allows evaluation of the kinetics of ultrasound-mediated MB destruction at near physiological conditions.In this work, a newly developed drug-loaded MB formulation was compared with unloaded MBs in order to assess the influence of drug-loading on their acoustic destructibility. Furthermore, drug-loaded MBs were compared to acoustically active lipospheres (AALs), comprising an additional layer of triacetin, as well as to a marketed MB contrast agent (SonoVue^®, Bracco Diagnostics, USA), used as standard.

Methods

MBs with phospholipid monolayer shells were produced by mechanical agitation of liposomal dispersions and octafluoropropane gas. AALs were accordingly produced by agitation of phospholipid-stabilized aqueous triacetin microemulsions with gas.The in vitro experimental setup for acoustic destructibility testing comprised a membrane cell, pressurized and brought to 37 °C in order to imitate human blood pressure and body temperature. The optimized egg-like cell shape provided optimal flow conditions and a minimized dead volume.Ultrasound with frequencies of 1 and 3 MHz and intensities, varying from 1 to 4 W/cm², was applied through a silicone membrane window to the cell. MB size distribution and concentration were measured by light blockage in equal time intervals during the sonication.

Results

The optimized in vitro setup demonstrated differences in the ultrasound destructibility of the MB formulations used. The fastest decay upon ultrasound exposure was found for SonoVue^®. Unloaded and drug-loaded MBs appeared to be comparably destructible to SonoVue^®. AALs were about 4.5-fold more stable than SonoVue^®. MB destructibility was also found to depend on particle diameter, corresponding to theoretical models described in the literature.

Conclusion

The optimized in vitro setup has rendered a fast and reliable laboratory tool for characterization of MB formulations.     

Dual-high-frequency ultrasound excitation on microbubble destruction volume

Objective and motivation

The goal of this work was to test experimentally that exposing air bubbles or ultrasound contrast agents in water to amplitude modulated wave allows control of inertial cavitation affected volume and hence could limit the undesirable bioeffects.

Methods

Focused transducer operating at the center frequency of 10 MHz and having about 65% fractional bandwidth was excited by 3 μs 8.5 and 11.5 MHz tone-bursts to produce 3 MHz envelope signal. The 3 MHz frequency was selected because it corresponds to the resonance frequency of the microbubbles used in the experiment. Another 5 MHz transducer was used as a receiver to produce B-mode image. Peak negative acoustic pressure was adjusted in the range from 0.5 to 3.5 MPa. The spectrum amplitudes obtained from the imaging of SonoVue^TM contrast agent when using the envelope and a separate 3 MHz transducer were compared to determine their cross-section at the - 6 dB level.

Results

The conventional 3 MHz tone-burst excitation resulted in the region of interest (ROI) cross-section of 2.47 mm while amplitude modulated, dual-frequency excitation with difference frequency of 3 MHz produced cross-section equal to 1.2 mm.

Conclusion

These results corroborate our hypothesis that, in addition to the considerably higher penetration depth of dual-frequency excitation due to the lower attenuation at 3 MHz than that at 8.5 and 11.5 MHz, the sample volume of dual-frequency excitation is also smaller than that of linear 3-MHz method for more spatially confined destruction of microbubbles.     

The effects of Ce6-mediated sono-photodynamic therapy on cell migration,apoptosis and autophagy in mouse mammary 4T1 cell line

Purpose

Sono-Photodynamic therapy (SPDT) is an alternative therapy which claims to enhance the anti-cancer effects by combining sonodynamic therapy (SDT) with photodynamic therapy (PDT). In the present study, we investigated the effects of chlorin e6 (Ce6) mediated SPDT on migration, apoptosis and autophagy in mouse mammary 4T1 cancer cells, and its underlying mechanisms.

Materials

Cell migration was determined by wound healing assay. Apoptosis was analyzed using annexin V-PE/7-ADD staining as well as Hoechst 33342 staining. Changes of mitochondria membrane potential (MMP) was evaluated by flow cytometry. Formation of acidic vesicular organelles (AVOs) during autophagy was observed with fluorescence microscope by MDC staining. Immunofluorescence assays were performed to detect the co-localization of LC3 and Lamp2. Western blotting was employed to analyze the activity of the apoptosis related proteins Caspase-3, PARP, Bax and Bcl-2, as well as the autophagy associated processing of LC3-I to LC3-II and Beclin-1 expression.

Results

Ce6 mediated SPDT further enhanced cell migration inhibition, significantly triggered cell apoptosis, nuclear condensation and MMP drop. Cleaved Caspase-3 and PARP increased dramatically after Ce6-SPDT, accompanied by decreased Bcl-2 expression, while the expression of Bax remained stable. Additionally, AVOs formation, co-localization of LC3 and Lamp2 occurred following Ce6-SPDT and simultaneously accompanied by LC3-II processing and increased Beclin-1 expression.

Conclusions

Ce6-SPDT could enhance cell migration inhibition, and induce mitochondria-dependent apoptosis as well as autophagy in 4T1 cells.     

Ultrasound induces cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin

Objectives

Curcumin, a natural pigment from the traditional Chinese herb, has shown promise as an efficient enhancer of ultrasound. The present study aims to investigate ultrasound-induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin in vitro.

Methods

Nasopharyngeal carcinoma cell line CNE2 cells were incubated by 10 μm curcumin and then were treated by ultrasound for 8 s at the intensity of 0.46 W/cm². Cytotoxicity was evaluated using MTT assay and light microscopy. Mitochondrial damage was analyzed using a confocal laser scanning microcopy with Rhodamine 123 and ultrastructural changes were observed using a transmission electron microscopy (TEM).

Results

MTT assay showed that cytotoxicity induced by ultrasound treatment alone and curcumin treatment alone was 18.16 ± 2.37% and 24.93 ± 8.30%, respectively. The cytotoxicity induced by the combined treatment of ultrasound and curcumin significantly increased up to 86.67 ± 7.78%. TEM showed that microvillin disappearance, membrane blebbing, chromatin condensation, swollen mitochondria, and mitochondrial myelin-like body were observed in the cells treated by ultrasound and curcumin together. The significant collapse of mitochondrial membrane potential (MMP) was markedly observed in the CNE2 cells after the combined treatment of curcumin and ultrasound.

Conclusions

Our findings demonstrated that ultrasound sonication in the presence of curcumin significantly killed the CNE2 cells and induced ultrastructural damage and the dysfunction of mitochondria, suggesting that ultrasound treatment remarkably induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin.     

Surface modification with fibronectin or collagen to improve the cell adhesion

Objective

The surface of biomaterials plays a critical role in determining bioactivity. The aim of this study was to evaluate the cell adhesion and proliferation of ADSCs on the surface of biomaterial which is modified with fibronectin or collagen.

Materials and methods

Adipose-derived stromal cells (ADSCs) were obtained from SD rats, expanded in culture, and seeded onto scaffold surface-modified with fibronectin or collagen. To characterize cellular attachment, cells were incubated on scaffold for 1 and 2 h and then counted the cells attached onto the scaffold. The MTT assay was chosen to evaluate the proliferation at days 1, 4, 7 and 14. After 7 d of culture, scanning electron microscope was chosen to observe cell morphology and attachment of ADSCs on the scaffolds.

Results

Attachment at 1 and 2 h of cells on scaffold modified with fibronectin was significantly greater than in control, but not with collagen. The MTT assay revealed that ADSCs proliferation tendency was nearly parallel to that in control. The scanning electron microscope (SEM) showed that ADSCs in experiment expanded thoroughly and excreted much extracellular materials.

Conclusions

Surface modification with fibronectin or collagen can enhance the attachment of cultured ADSCs on the scaffold, but it had not evident effect to proliferation.     

Sonodynamic action of pyropheophorbide-a methyl ester induces mitochondrial damage in liver cancer cells

Sonodynamic therapy with pyropheophorbide-a methyl ester (MPPa) presents a promising aspect in treating liver cancer. The present study aims to investigate the mitochondrial damage of liver cancer cells induced by MPPa-mediated sonodynamic action. Mouse hepatoma cell line H₂₂ cells were incubated with MPPa (2 μM) for 20 h and then exposed to ultrasound with an intensity of 0.97 W/cm² for 8 s. Cytotoxicity was investigated 24 h after sonodynamic action using MTT assay and light microscopy. Mitochondrial membrane potential (ΔΨm) was analyzed using flow cytometry with rhodamine 123 staining and ultrastructural changes were observed using transmission electron microscopy (TEM).The cytotoxicity of MPPa-mediated SDT on H₂₂ cell line was 73.00 ± 3.42%, greater than ultrasound treatment alone (28.12 ± 5.19%) significantly while MPPa treatment alone had no significant effect on H₂₂ cells. Moreover, after MPPa-mediated SDT cancer cells showed swollen mitochondria under TEM and a significant collapse of mitochondrial membrane potential. Our findings demonstrated that MPPa-mediated SDT could remarkably induce cell death of H₂₂ cells, and highlighted that mitochondrial damage might be an important cause of cell death induced by MPPa-mediated SDT.     

Effects of therapeutic pulsed ultrasound and dimethylsulfoxide phonophoresis on oxidative stress parameters after injury induced by eccentric exercise

Introduction

The aim of the study was to evaluate the effects of TPU together with DMSO on oxidative stress parameters after eccentric exercise.

Methods

Thirty and six animals were divided in control; eccentric exercise (EE); EE + saline gel 0.9%; EE + TPU 0.8 W/cm²; EE + DMSO gel; EE + TPU + DMSO gel and submitted to one 90-min downhill run (1.0 km h⁻¹). TPU was used 2, 12, 24, 46 h after exercise session and 48 h after the animals were killed and the gastrocnemius muscles were surgically removed. Production of superoxide anion, creatine kinase (CK) levels, lipoperoxidation, carbonylation, and antioxidants enzymes were analyzed.

Results

Showed that TPU and gel-DMSO improved muscle healing. Moreover, superoxide anion production, TBARS level and protein carbonyls levels, superoxide dismutase and catalase activity were all decreased in the group TPU plus gel-DMSO.

Discussion

Our results show that DMSO is effective in the reduction of the muscular lesion and in the oxidative stress after eccentric exercise only when used with TPU.     

Parametric harmonic-to-fundamental ratio contrast echocardiography: a novel approach to identification and accurate measurement of left ventricular area under variable levels of ultrasound signal attenuation

Objectives

We introduced a harmonic-to-fundamental ratio (HFR) of the radiofrequency (RF) signals that reduces confounding effects of attenuation. We studied whether HFR analysis of RF signals received from contrast microbubbles allows accurate measurement of the left ventricular (LV) cavity area under varying levels of attenuation.

Background

Attenuation is a fundamental problem in ultrasound imaging and limits the use of clinical echocardiography.

Methods

RF data from short axis systolic and diastolic scans were obtained from 14 open-chest dogs following left-atrial bolus of Optison. Attenuation was induced by interposed silicone pads calibrated to induce 7 dB or 14 dB reductions of the backscattered RF signal. RF images were reconstructed from the RF signals, HFR values calculated for each image pixel for 0 dB, 7 dB and 14 dB attenuation conditions, and LV area obtained by summation of “LV cavity pixels”. A reference LV cavity area was obtained from endocardial border tracings in enhanced scans by experts.

Results

Correlation of the HFR-defined and reference areas at systole was R = 0.95, R = 0.94, and R = 0.91 for 0 dB, 7 dB and 14 dB levels of attenuation, respectively, and at diastole was R = 0.95 for 0 dB, 7 dB and 14 dB levels of attenuation. The mean difference from both systolic and diastolic values was <1.45 cm² (i.e. negligible) in all attenuation settings.

Conclusion

Our novel HFR method supports precise measurement of the LV cavity area in contrast images with simulated high attenuation of ultrasound signals.     

Phantom investigation of phase-inversion-based dual-frequency excitation imaging for improved contrast display

Objective

The goal of this work is to examine the effects of pulse-inversion (PI) technique in combination with dual-frequency (DF) excitation method to separate the high-order nonlinear responses from microbubble contrast agents for improvement of image contrast. DF excitation method has been previously developed to induce the low-frequency ultrasound nonlinear responses from bubbles by using the composition of two high-frequency sinusoids (f₁ and f₂).

Motivation

Although the simple filtering was conventionally utilized to provide signal separation, the PI approach is better in the sense that it minimizes the mutual interferences among these high-order nonlinear responses in the presence of spectral overlap. The novelty of the work is that, in addition to the common PI summation, the PI subtraction was also applied in DF excitation method.

Methods

DF excitation pulses having an envelope frequency of 3 MHz (i.e., f₁ = 8.5 MHz and f₂ = 11.5 MHz) with pulse lengths of 3-10 μs and the pressure amplitudes from 0.5 to 1.5 MPa were used to interrogate the nonlinear responses of SonoVue™ microbubbles in the phantom experiments. The high-order nonlinear responses in the DF excitation were extracted for contrast imaging using PI summation for even-order nonlinear components or PI subtraction for odd-order nonlinear ones.

Results

Our results indicated that, as compared to the conventional filtering technique, the PI processing effectively increases the contrast-to-tissue ratio (CTR) of the third-order nonlinear response at 5.5 MHz and the fourth-order nonlinear response at 6 MHz by 2-5 dB. For these high-order nonlinear components, the CTR increase varies with the transmission pressures from 0.5 to 1.5 MPa due to the microbubbles’ displacement induced by the radiation force of DF excitation.

Conclusions

For DF excitation technique, the PI processing can help to extract either the odd-order or the even-order nonlinear components for higher CTR estimates.     

Continuous-wave ultrasound reflectometry for surface roughness imaging applications

Background

Measurement of surface roughness irregularities that result from various sources such as manufacturing processes, surface damage, and corrosion, is an important indicator of product quality for many nondestructive testing (NDT) industries. Many techniques exist, however because of their qualitative, time-consuming and direct-contact modes, it is of some importance to work out new experimental methods and efficient tools for quantitative estimation of surface roughness.

Objective and method

Here we present continuous-wave ultrasound reflectometry (CWUR) as a novel nondestructive modality for imaging and measuring surface roughness in a non-contact mode. In CWUR, voltage variations due to phase shifts in the reflected ultrasound waves are recorded and processed to form an image of surface roughness.

Results

An acrylic test block with surface irregularities ranging from 4.22 μm to 19.05 μm as measured by a coordinate measuring machine (CMM), is scanned by an ultrasound transducer having a diameter of 45 mm, a focal distance of 70 mm, and a central frequency of 3 MHz. It is shown that CWUR technique gives very good agreement with the results obtained through CMM inasmuch as the maximum average percent error is around 11.5%.

Conclusion

Images obtained here demonstrate that CWUR may be used as a powerful non-contact and quantitative tool for nondestructive inspection and imaging of surface irregularities at the micron-size level with an average error of less than 11.5%.     

Noninvasive detection of intimal xanthoma using combined ultrasound, strain rate and photoacoustic imaging

Background and motivation

The structure, composition and mechanics of carotid artery are good indicators of early progressive atherosclerotic lesions. The combination of three imaging modalities (ultrasound, strain rate and photoacoustic imaging) which could provide corroborative information about the named arterial properties could enhance the characterization of intimal xanthoma.

Methods

The experiments were performed using a New Zealand white rabbit model of atherosclerosis. The aorta excised from an atherosclerotic rabbit was scanned ex vivo using the three imaging techniques: (1) ultrasound imaging of the longitudinal section: standard ultrasound B-mode (74 Hz frame rate); (2) strain rate imaging: the artery was flushed with blood and a 1.5 Hz physiologic pulsation was induced, while the ultrasound data were recorded at higher frame rate (296 Hz); (3) photoacoustic imaging: the artery was irradiated with nanosecond pulsed laser light of low fluence in the 1210-1230 nm wavelength range and the photoacoustic data was recorded at 10 Hz frame rate. Post processing algorithms based on cross-correlation and optical absorption variation were implemented to derive strain rate and spectroscopic photoacoustic images, respectively.

Results

Based on the spatio-temporal variation in displacement of different regions within the arterial wall, strain rate imaging reveals differences in tissue mechanical properties. Additionally, spectroscopic photoacoustic imaging can spatially resolve the optical absorption properties of arterial tissue and identify the location of lipid pools.

Conclusions

The study demonstrates that ultrasound, strain rate and photoacoustic imaging can be used to simultaneously evaluate the structure, the mechanics and the composition of atherosclerotic lesions to improve the assessment of plaque vulnerability.     

In vitro comparative study of vibro-acoustography versus pulse-echo ultrasound in imaging permanent prostate brachytherapy seeds

Background

Permanent prostate brachytherapy (PPB) is a common treatment for early stage prostate cancer. While the modern approach using trans-rectal ultrasound guidance has demonstrated excellent outcome, the efficacy of PPB depends on achieving complete radiation dose coverage of the prostate by obtaining a proper radiation source (seed) distribution. Currently, brachytherapy seed placement is guided by trans-rectal ultrasound imaging and fluoroscopy. A significant percentage of seeds are not detected by trans-rectal ultrasound because certain seed orientations are invisible making accurate intra-operative feedback of radiation dosimetry very difficult, if not impossible. Therefore, intra-operative correction of suboptimal seed distributions cannot easily be done with current methods. Vibro-acoustography (VA) is an imaging modality that is capable of imaging solids at any orientation, and the resulting images are speckle free.

Objective and methods

The purpose of this study is to compare the capabilities of VA and pulse-echo ultrasound in imaging PPB seeds at various angles and show the sensitivity of detection to seed orientation. In the VA experiment, two intersecting ultrasound beams driven at f₁ = 3.00 MHz and f₂ = 3.020 MHz respectively were focused on the seeds attached to a latex membrane while the amplitude of the acoustic emission produced at the difference frequency 20 kHz was detected by a low frequency hydrophone.

Results

Finite element simulations and results of experiments conducted under well-controlled conditions in a water tank on a series of seeds indicate that the seeds can be detected at any orientation with VA, whereas pulse-echo ultrasound is very sensitive to the seed orientation.

Conclusion

It is concluded that vibro-acoustography is superior to pulse-echo ultrasound for detection of PPB seeds.     

Fractal dimension of 40 MHz intravascular ultrasound radio frequency signals

Objective

Fully automatic tissue characterization in intravascular ultrasound systems is still a challenge for the researchers. The present work aims to evaluate the feasibility of using the Higuchi fractal dimension of intravascular ultrasound radio frequency signals as a feature for tissue characterization.

Methods

Fractal dimension images are generated based on the radio frequency signals obtained using mechanically rotating 40 MHz intravascular ultrasound catheter (Atlantis SR Plus, Boston Scientific, USA) and compared with the corresponding correlation images.

Conclusion

An inverse relation between the fractal dimension images and the correlation images was revealed indicating that the hard or slow moving tissues in the correlation image usually have low fractal dimension and vice-versa. Thus, the present study suggests that fractal dimension images may be used as a feature for intravascular ultrasound tissue characterization and present better resolution then the correlation images.     

Intravascular ultrasound allows a hyperplasia diagnosis in vivo in rat as accurate as histomorphometry

Objective and motivation

Intravascular ultrasound (IVUS) allows in vivo invasive intra-luminal real-time examination of the arterial wall structure. In this study, we aimed to validate for the first time the in vivo IVUS performing as a diagnostic tool by comparison to the well-established histomorphometry approach, in the largely used rat model of carotid angioplasty model that mimics the angioplasty procedure in humans.

Methods

Atherosclerotic lesions were allowed to develop during four weeks after balloon catheter inflation of the left carotid artery, whereas the intact right carotid artery was used as control. Four weeks after injury, a Boston Scientific 40 MHz device to perform IVUS exams in vivo on both carotid arteries. Then, both carotid arteries were examined in vitro by histomorphometry and correlation between IVUS and histomorphometric parameters (plaque plus media cross-sectional areas [CSA] and eccentricity index) were researched.

Results

After ANOVA analysis, comparative statistical analysis showed significant correlations between IVUS and histomorphometry when examining the intact right carotid artery (r = 0.662 with p < 0.003 for plaque plus media CSA; r = 0.774 with p < 0.002 for eccentricity index), but also when exploring the injured left carotid artery (r = 0.805 with p < 0.0001 for plaque plus media CSA; r = 0.775 with p < 0.002 for eccentricity index).

Conclusions and outcome

We report here for the first time the ability of IVUS to study therapeutic vascular effects in vivo in alive rats. This result is of major importance since it will allow this device to be used for restenosis drug testing in rat model of carotid angioplasty.     

Acoustic Radiation Force Impulse-Imaging for the evaluation of the thyroid gland: a limited patient feasibility study

Purpose

Real-time tissue elastography, a qualitative elastography method, has shown promising results in the diagnostic work up of thyroid nodules. However, to our knowledge no study has evaluated a quantitative elastography method in the thyroid gland. The present study is a feasibility study evaluating Acoustic Radiation Force Impulse-Imaging, a novel quantitative elastography method in the thyroid gland.

Methods

ARFI-imaging involves the mechanical excitation of tissue using short-duration acoustic pulses to generate localized displacements in tissue. The displacements induce a lateral shear-wave propagation which is tracked using multiple laterally positioned ultrasound “tracking“ beams. Inclusion criteria were: thyroid nodules ?1 cm, non-functioning or hypo-functioning on radionuclide scanning, and cytological/histological assessment of thyroid nodule as reference method. All patients received conventional ultrasound, and examination of the thyroid gland including Power Doppler Ultrasound using a 9 MHz linear transducer, in addition real-time elastography (RTE) was performed at 9 MHz frequency and ARFI-imaging was performed at 4 MHz using Siemens (ACUSON S2000) B-mode-ARFI combination transducer.

Results

Sixty nodules in 55 patients were analyzed. Three nodules were papillary carcinoma. The stiffer the tissue the faster the shear wave propagates. The results obtained indicated that the shear wave velocity in thyroid lobes ranged between 0.5 and 4.9 m/s. The median velocity of ARFI-imaging in the healthy nodule-free thyroid gland, as well as in benign and malignant thyroid nodules was 1.98 m/s (range: 1.20-3.63 m/s), 2.02 m/s (range: 0.92-3.97 m/s), and 4.30 m/s (range: 2.40-4.50 m/s), respectively. While no significant difference in median velocity was found between healthy thyroid tissue and benign thyroid nodules, a significant difference was found between malignant thyroid nodules on the one hand and healthy thyroid tissue (p = 0.018) or benign thyroid nodules (p = 0.014) on the other hand. Specificity of ARFI-imaging for the differentiation of benign and malignant thyroid nodules was comparable with RTE (91-95%).

Conclusions

ARFI can be performed in the thyroid tissue with reliable results.     

Noninvasive estimation of the ocular elastic modulus for age-related macular degeneration in the human eye using sequential ultrasound imaging

Introduction

Elastic modulus estimation may be an important clinical criterion, as it seems to affect such eye parameters as intraocular pressure, ocular pulsation, blood flow, effect of topical medications, and post-refractive surgery complications. The purpose of this study was to examine the differences in elasticity in the ocular axial length, posterior wall thickness (posterior pole), and retina-choroid thickness under normal and aged-related macular degeneration (AMD) conditions in the human eye by directly estimating the elastic modulus with sequential and noninvasive ultrasound image processing.

Materials and Methods

In this study, 25 healthy subjects and 20 patients with non-neovascular AMD participated in the experiment. The deformation of the ocular axial length, posterior wall thickness and retina-choroid complex thickness was captured using high-resolution ultrasonography before and after loading. The B-mode (20 MHz) and A-mode (8 MHz) frames were obtained and processed with an echo tracking technique. The elastic modulus was estimated using changes in ocular axial length, posterior wall thickness and retina-choroid complex thickness and with applied stress measurements.

Results

There was a significant difference (p < 0.05) in the ocular axial length elastic modulus between the AMD and healthy subjects (AMD patients: 95.165 ± 26.431 kPa, vs. healthy subjects: 49.539 ± 25.867 kPa). Moreover, there was a statistically significant difference (p < 0.05) in the posterior wall thickness elastic modulus between AMD patients and healthy subjects (AMD patients: 50.519 ± 12.295 kPa, vs. healthy subjects: 20.519 ± 11.827 kPa). However, no statistically significant difference (p-value > 0.05) was found in the retina-choroid complex elastic modulus between the two groups (AMD patients: 20.134 ± 3.898 kPa, vs. healthy subjects: 15.630 ± 4.250 kPa).

Conclusion

Although the results were obtained examining a relatively low number of patients, it would appear that noninvasive ultrasound estimation of the local elastic moduli of ocular axial length and posterior wall thickness is suited to aid in detection of the non-exudative AMD thus manifesting its potential as a screening tool in symptom-free individuals.     

Direct laser light enhancement of susceptibility of bacteria to gentamicin antibiotic

Objectives

To test the effect of pulsed (Q-switched) and continuous wave (CW) laser light at wavelength of 532 nm on the viability of free-living stationary phase bacteria with and without gentamicin (an antibiotic) treatment.

Methods

Free living stationary phase gram negative bacteria (Pseudomonas aeruginosa strain PAO1) was immersed in Luria Broth (LB) solution and exposed to Q-switched and CW lasers with and without the addition of the antibiotic gentamicin. Cell viability was determined at different time points.

Results

Laser treatment alone did not reduce cell viability compared to untreated control and the gentamicin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamicin treatment, however, resulted in a synergistic effect and viability was reduced by 8 logs for P. aeruginosa PAO1.

Conclusions

Combination of laser light with gentamicin shows an improved efficacy against P. aeruginosa.