RESUMEN
Bell's palsy is a form of temporary facial nerve paralysis that occurs primarily in young adults. Previously, various methods were used to assess outcomes in facial nerve disease. The aim of the present study was to characterize the main branches of the normal and abnormal facial nerve using high-frequency ultrasonography (HFUS). A total of 104 healthy volunteers, 40 patients with acute onset of Bell's palsy and 30 patients who underwent 3-month routine therapy for Bell's palsy disease were included in the study. The healthy volunteers and patients were selected for HFUS examination and VII nerve conduction. The results showed significant differences in nerve diameter, echogenicity, delitescence and amplitude in different groups. Statistically significant correlations were identified for severity grading in one of the experimental groups during HFUS examinations. In conclusion, HFUS as a complementary technique paired with neural electrophysiology may establish the normal values of facial nerve. Additionally, HFUS was beneficial in the process of evaluation and prognosis of Bell's palsy disease.
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To explore the mechanism of soybean isoflavones (SI) on OVX-induced osteoporosis, we investigated the effect of SI on Wnt signaling that emerged as a novel key pathway for promoting bone formation. Results showed that SI decreased bone mineral elements loss, improved biomechanics parameters in OVX rats. Wnt3a activation can promote the dissociation of ß-catenin complexes, release of ß-catenin monomer and inhibition of ß-catenin monomer degradation. SI decreased sost mRNA and sclerosteosis protein expression in a dose-dependent manner, and increased ß-catenin proteins expression in femur of OVX rats. These data suggest that SI suppresses the canonical Wnt signal in OVX rats, partially through the enhancement of the dickkopf-1 production. OVX results in decreased estrogen level in rats. SI act as inhibitors of Wnt-mediated activation of by competitively binding to LRP5, and subsequently downregulating ß-catenin gene.
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Fémur/efectos de los fármacos , Glycine max/química , Isoflavonas/farmacología , Osteoporosis/prevención & control , Vía de Señalización Wnt/efectos de los fármacos , Proteína Wnt3A/metabolismo , Animales , Densidad Ósea/efectos de los fármacos , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Estrógenos/metabolismo , Femenino , Fémur/metabolismo , Fémur/patología , Regulación de la Expresión Génica , Marcadores Genéticos/genética , Péptidos y Proteínas de Señalización Intercelular/agonistas , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Isoflavonas/aislamiento & purificación , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Osteoporosis/genética , Osteoporosis/metabolismo , Osteoporosis/patología , Ovariectomía , Unión Proteica , Proteolisis/efectos de los fármacos , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Proteína Wnt3A/genética , beta Catenina/antagonistas & inhibidores , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND: To investigate the relationship between extracellular matrix parameters and texture of prostatic lesions evaluated by transrectal real-time tissue elastography (TRTE). METHODS: 120 patients suspicious for prostate cancer underwent TRTE. Targeted biopsies were carried out after 12-core systematic biopsy. Epithelia were stained with hematoxylin-eosin, and Victoria blue and Ponceau S were used to stain elastic-collagen fibers, and picric acid-sirius red for visualization of collagen type I (Col1) and III (Col3). Smooth muscles were visualized by immunohistochemistry. All image analyses were performed in a blind manner using Image Pro Plus 6.0, and the area ratios of epithelium, elastic fibers, collagen fibers and Col1/Col3 were determined. RESULTS: 42 patients with typical elastograms were included in the final data analysis. Significant differences were detected between the benign and malignant groups in the area ratios of epithelium (P = 0.01), smooth muscles and Col1/Col3 (P = 0.04, P = 0.02, respectively). There were no significant differences in the area ratios of epithelium, smooth muscle and elastic fibers between the stiff and soft lesion groups. The area ratio of Col1 was (0.05 ± 0.03) in the stiff group, and (0.02 ± 0.01) in the soft group (P= 0.00). However, the area ratio of Col3 was (0.03 ± 0.02) in the stiff group, and (0.05 ± 0.04) in the soft group (P = 0.16). Col1/Col3 in the stiff group (1.99 ± 1.59) was greater than in the soft group (0.71 ± 0.64) (P = 0.01). CONCLUSIONS: Tissue hardness of prostatic tumors was mainly dependent on the Col1 content, Col1/Col3 being higher in malignant than in benign lesions, so the prostate tissue texture can be used as a target for distinguishing between the two with TRTE.
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Tejido Elástico/fisiología , Diagnóstico por Imagen de Elasticidad/métodos , Hiperplasia Prostática/diagnóstico , Neoplasias de la Próstata/diagnóstico , Anciano , Anciano de 80 o más Años , Biopsia , Colágeno Tipo I/análisis , Colágeno Tipo III/análisis , Diagnóstico Diferencial , Detección Precoz del Cáncer/métodos , Epitelio/fisiología , Matriz Extracelular , Humanos , Masculino , Persona de Mediana Edad , Músculo Liso/fisiología , Próstata/patología , Neoplasias de la Próstata/cirugíaRESUMEN
The principle of using ultrasonic computed tomography (UCT) clinically for mapping tissue acoustic properties was suggested almost 40 years ago. Despite strong research activity, UCT been unable to rival its x-ray counterpart in terms of the ability to distinguish tissue pathologies. Conventional piezoelectric detectors deployed in UCT are termed phase-sensitive (PS) and it is well established that this property can lead to artefacts related to refraction and phase-cancellation that mask true tissue structure, particularly for reconstructions involving attenuation. Equally, it has long been known that phase-insensitive (PI) detectors are more immune to this effect, although sufficiently sensitive devices for clinical use have not been available. This paper explores the application of novel PI detectors to UCT. Their operating principle is based on exploiting the pyroelectric properties of the piezoelectric polymer polyvinylidene difluoride. An important detector performance characteristic which makes it particularly suited to UCT, is the lack of directionality of the PI response, relative to the PS detector mode of operation. The performance of the detectors is compared to conventional PS detection methods, for quantitatively assessing the attenuation distribution within various test objects, including a two-phase polyurethane phantom. UCT images are presented for a range of single detector apertures; tomographic reconstruction images being compared with the known structure of phantoms containing inserts as small as 3 mm, which were readily imaged. For larger diameter inserts (>10 mm), the transmitter-detector combination was able to establish the attenuation coefficient of the insert to within ±10% of values determined separately from plane-wave measurements on representative material plaques. The research has demonstrated that the new PI detectors are significantly less susceptible to refraction and phase-cancellation artefacts, generating realistic images in situations where conventionally-employed through-transmission PS detection techniques were unable to do so. The implications of the study to the potential screening of breast disease are discussed.
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Conductividad Eléctrica , Tomografía/instrumentación , Ultrasonido , Procesamiento de Imagen Asistido por Computador , Fantasmas de Imagen , PoliuretanosRESUMEN
We studied molecular mechanism of Cistanches Herba aqueous extract (CHAE) in ovariectomized (OVX) rats, as an experimental model of postmenopausal osteoporosis. Female rats were either sham-operated or bilaterally OVX; and at 60 days postoperatively. The OVX group (n = 8) received an ovariectomy and treatment with normal saline for 90 days commencing from 20th post ovariectomy day. The ovariectomized +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy and were treated with Cistanches Herba aqueous extract of 100 mg/kg body weight daily for 90 days commencing from 22nd post ovariectomy day. The ovariectomy +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy, and were treated with the of 200 mg/kg body weight daily for 90 days commencing from 20th post ovariectomy day. Serum BGP and TRAP, E2, FSH and LH level, bone marrow Smad1, Smad5, TGF-ß1 and TIEG1 mRNA expression levels were examined. Results showed that serum BGP and TRAP, FSH and LH levels were significantly increased, whereas E2, Smad1, Smad5, TGF-ß1 and TIEG1 mRNA and proteins expression levels were significantly decreased in OVX rats compared to sham rats. 90 days of CHAE treatment could significantly decrease serum BGP and TRAP, FSH and LH levels, and increase E2, Smad1, Smad5, TGF-ß1 and TIEG1 mRNA and proteins expression levels in OVX rats. It can be concluded that CHAE play its protective effect against OVX-induced bone degeneration partly by regulating some bone metabolism related genes, e.g. Smad1, Smad5, TGF-ß1 and TIEG1.
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Conservadores de la Densidad Ósea/farmacología , Médula Ósea/metabolismo , Cistanche/química , Expresión Génica/efectos de los fármacos , Osteoporosis Posmenopáusica/tratamiento farmacológico , Extractos Vegetales/farmacología , Fosfatasa Ácida/sangre , Animales , Conservadores de la Densidad Ósea/aislamiento & purificación , Médula Ósea/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Evaluación Preclínica de Medicamentos , Estradiol/sangre , Femenino , Fémur/efectos de los fármacos , Fémur/patología , Hormona Folículo Estimulante/sangre , Humanos , Isoenzimas/sangre , Hormona Luteinizante/sangre , Osteocalcina/sangre , Osteoporosis Posmenopáusica/sangre , Ovariectomía , Extractos Vegetales/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Ratas , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Solventes/química , Fosfatasa Ácida Tartratorresistente , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Agua/químicaRESUMEN
An improved method for noninvasive measurement of the local velocity of arterial pulse wave propagation by an echo-tracking-based ultrasound system is described. A data acquisition image interface was programmed in the ultrasound machine simultaneously to record M-mode ultrasound signals at two locations of a given distance apart along an artery. The selections of measurement sites, separation, and time resolution were performed on the control interface. The temporal sampling frequency could be as high as 10 kHz. The displacements of the blood vessel wall along the time axis were calculated from the M-mode signals by cross-correlation of the radio-frequency data and the distension waveforms were obtained. The temporal separation of the feet of the distension curves from the two measurement locations was derived to give the travel time of the pulse wave. Measurements were made in vivo on human carotid arteries. The pulse wave velocities measured from four volunteers were from 4.1 to 7.2 m/s with coefficients of variation from 5.9 to 29.5%. Some of the factors contributing to the variation in measured values of the velocity are discussed. The method is simple to implement and should be suitable for clinical research into local pulse wave velocity.
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Arterias Carótidas/diagnóstico por imagen , Arterias Carótidas/fisiología , Procesamiento de Señales Asistido por Computador , Ultrasonografía/métodos , Rigidez Vascular/fisiología , Adulto , Presión Sanguínea/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
Ultrasonic imaging is a mature and widely used medical diagnostic technology but it is also a field of intense research activity. Innovations are viewed with differing perspectives by the stakeholders- users, industrialists, regulators, and researchers and research funders. The more important recent developments include advances in transducers, scanning schemes, coded excitation, three-dimensional, high-resolution and high-speed imaging, contrast agents, harmonic, elasticity and strain imaging, point-of-care devices, computed tomography, thermoacoustic, photoacoustic, acousto-optic and Hall effect imaging. Viewed from diverse perspectives, the assessment of ultrasonic imaging technologies is intellectually challenging. This is a general problem, which demands a multidisciplinary approach. An emerging, integrated, context for such assessment is presented. Given the straitened economies around the world, the need to articulate value for each and all stakeholders is becoming increasingly important.
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Ultrasonografía/métodos , Humanos , Ultrasonografía/economía , Ultrasonografía/instrumentación , Ultrasonografía/tendenciasRESUMEN
After X-radiography, ultrasound is now the most common of all the medical imaging technologies. For millennia, manual palpation has been used to assist in diagnosis, but it is subjective and restricted to larger and more superficial structures. Following an introduction to the subject of elasticity, the elasticity of biological soft tissues is discussed and published data are presented. The basic physical principles of pulse-echo and Doppler ultrasonic techniques are explained. The history of ultrasonic imaging of soft tissue strain and elasticity is summarized, together with a brief critique of previously published reviews. The relevant techniques-low-frequency vibration, step, freehand and physiological displacement, and radiation force (displacement, impulse, shear wave and acoustic emission)-are described. Tissue-mimicking materials are indispensible for the assessment of these techniques and their characteristics are reported. Emerging clinical applications in breast disease, cardiology, dermatology, gastroenterology, gynaecology, minimally invasive surgery, musculoskeletal studies, radiotherapy, tissue engineering, urology and vascular disease are critically discussed. It is concluded that ultrasonic imaging of soft tissue strain and elasticity is now sufficiently well developed to have clinical utility. The potential for further research is examined and it is anticipated that the technology will become a powerful mainstream investigative tool.
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Efecto Doppler , Elasticidad/fisiología , Traumatismos de los Tejidos Blandos/diagnóstico por imagen , Ultrasonografía/historia , Ultrasonografía/métodos , Ultrasonografía/tendencias , Historia del Siglo XX , Historia del Siglo XXI , Palpación/historiaRESUMEN
Ultrasonic imaging based on the pulse-echo principle is widely used throughout the world, particularly in medical applications. However, its spatial resolution is poor (around 2 times the wavelength, or 200 µm at 15 MHz), limiting its ability to detect small but clinically important lesions (such as microcalcifications in breast cancer). The work presented here is different from the traditional approach. Continuous-wave ultrasound is transmitted to insonate a rotating object, then the amplitude and phase of the returned signals are coherently processed to reconstruct a Doppler tomographic image of the object's backscatter field. It is demonstrated numerically that the spatial resolution is up to 0.19 wavelengths and the sampling requirement and image formation method are given. To show the performance of the method, we present the results obtained by applying the new technique in simulation and experiment. A string phantom consisting of very thin copper wires and two cylindrical phantoms constructed by tissue-mimicking-material were scanned. It is demonstrated that the copper wires were located very accurately with very high spatial resolution, and good shape approximation for the cylindrical phantoms was achieved.
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Algoritmos , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Tomografía de Coherencia Óptica/métodos , Ultrasonografía Doppler/métodos , Humanos , Fantasmas de Imagen , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tomografía de Coherencia Óptica/instrumentación , Ultrasonografía Doppler/instrumentaciónRESUMEN
The aim of the study was to assess if low-frequency ultrasound (US), in the range of 30-35 kHz, increases non-viral gene transfer to the mouse lung. US is greatly attenuated in the lung due to large energy losses at the air/tissue interfaces. The advantages of low-frequency US, compared with high-frequency US are: (i) increased cavitation (responsible for the formation of transient pores in the cell membrane) and (ii) reduced energy losses during lung penetration. Cationic lipid GL67/plasmid DNA (pDNA), polyethylenimine (PEI)/pDNA and naked pDNA were delivered via intranasal instillation and the animals were then exposed to US (sonoporation) at 0.07 or 0.1 MPa for 10 min. Under these conditions, US did not enhance GL67 or PEI-mediated transfection. It did, however, increase naked pDNA gene transfer by approximately 4 folds. Importantly, this was achieved in the absence of microbubbles, which are crucial for the commonly used high-frequency (1 MHz) sonoporation but may not be able to withstand nebulization in a clinically relevant setup. Lung hemorrhage was also assessed and shown to increase with US pressure in a dose-dependent manner. We have thus, established that low-frequency US can enhance lung gene transfer with naked pDNA and this enhancement is more effective than the previously reported 1 MHz US.
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Pulmón/virología , Polietileneimina/química , Transfección/métodos , Animales , Técnicas de Transferencia de Gen , Pulmón/química , Ratones , Transfección/estadística & datos numéricos , UltrasonidoRESUMEN
Over the years, there has been a substantial increase in acoustic exposure in diagnostic ultrasound as new imaging modalities with higher intensities and frame rates have been introduced; and more electronic components have been packed into the probe head, so that there is a tendency for it to become hotter. With respect to potential thermal effects, including those which may be hazardous occurring during ultrasound scanning, there is a correspondingly growing need for in vivo techniques to guide the operator as to the actual temperature rise occurring in the examined tissues. Therefore, an in vivo temperature estimator would be of considerable practical value. The commonly-used method of tissue thermal index (TI) measurement with a hydrophone in water could underestimate the actual value of TI (in one report by as much as 2.9 times). To obtain meaningful results, it is necessary to map the temperature elevation in 2-D (or 3-D) space. We present methodology, results and validation of a 2-D spatial and temporal thermal strain ultrasound temperature estimation technique in phantoms, and its apparently novel application in tracking the evolution of heat deposition at diagnostic exposure levels. The same ultrasound probe is used for both transmission and reception. The displacement and thermal strain estimation methods are similar to those used in high-intensity focused ultrasound thermal monitoring. The use of radiofrequency signals permits the application of cross correlation as a similarity measurement for tracking feature displacement. The displacement is used to calculate the thermal strain directly related to the temperature rise. Good agreement was observed between the temperature rise and the ultrasound power and scan duration. Thermal strain up to 1.4% was observed during 4000-s scan. Based on the results obtained for the temperature range studied in this work, the technique demonstrates potential for applicability in phantom (and possibly in vivo tissue) temperature measurement for the determination of TI.
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Ultrasonografía/métodos , Humanos , Fantasmas de Imagen , Temperatura , TransductoresRESUMEN
OBJECTIVE: To investigate phospholipids-based microbubbles induced sonoporation and cell membrane reseal in vitro under various conditions. METHODS: A breast cancer cell line SK-BR-3 was used to investigate ultrasonic sonoporation under various conditions. Atomic force microscopy (AFM) scanning techniques were employed to observe the change of membrane pores. RESULTS: Normal SK-BR-3 cells membrane pores were evenly distributed and less than 1 microm. After ultrasound exposure, membrane pores were enlarged at different degree depending on ultrasound exposure durations, filling gas species and microbubble suspension concentration. With microbubble suspension concentration being increased to 5% or ultrasound exposure reached 30 s, membrane pores in fluorocarbon (C(3)F(8) or SF(6))-filled microbubble groups exceeded 1 microm, which were significantly larger than that of air-filled microbubble group. Membrane pores were about 2-3 microm under ultrasound 60 s with 5% fluorocarbon-filled microbubble suspension. After 24 h of incubation, most of the enlarged membrane pores could reseal to normal size, which corresponded to cell viability. CONCLUSIONS: Membrane pores can be obviously enlarged by ultrasonic sonoporation of fluorocarbon-filled microbubbles, whose reseal time depended on ultrasound exposure duration and microbubble suspension concentration.
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Membrana Celular/química , Membrana Celular/ultraestructura , Fosfolípidos/química , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/fisiología , Femenino , Gases , Humanos , Microscopía de Fuerza Atómica , Microesferas , Porosidad , Embarazo , UltrasonidoRESUMEN
OBJECTIVE: To compare transfection efficiency and safety for antisense oligodeoxynucleotides (AS-ODNs) between two type of phospholipids-based vectors. METHODS: An AS-ODNs sequence HA824 combined with luciferase reporter plasmid was used. Under low intensity ultrasound (US), a breast cancer cell line SK-BR-3 was exposed to different concentration of microbubbles and liposomes. Transfection efficiency was detected by fluorescence microscopy. Cell viability was verified by propidium iodide assay. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the inhibitory effect of HA824 on HER-2 expression at mRNA level. Atomic force microscopy (AFM) scanning techniques was employed to observe the change of membrane pore size. RESULTS: AS-ODNs transfection efficiency showed an increasing tend with microbubble concentration, but not with liposome concentration. Maximum transfection efficiency with minimum cell viability was achieved under 2% microbubble concentration. Too strong sonoporation activity would enlarge membrane pores significantly and cause low cell viability. CONCLUSION: US-mediated AS-ODNs transfection enhanced by phospholipids-based microbubbles represents an effective and safe avenue.
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Oligodesoxirribonucleótidos Antisentido/efectos adversos , Oligodesoxirribonucleótidos Antisentido/farmacología , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos , Excipientes , Femenino , Técnicas de Transferencia de Gen , Genes Reporteros/genética , Genes erbB-2/genética , Humanos , Liposomas , Luciferasas/genética , Microesferas , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Fosfolípidos , Porosidad , Propidio , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrofotometría Atómica , TransfecciónRESUMEN
Pluronics have been investigated as vectors for drug and gene delivery in vitro and in vivo and were demonstrated to have high efficiency for gene transfer in vivo. However, they alone do not enhance gene transfer in vitro. We examined three pluronics, F127, L61 and P85, for their effects on ultrasound (US)-mediated gene transfer in three cell lines, 3T3-MDEI, C2C12 and CHO. The polymers showed differential effects on cell viability and transfection efficiency in a dose-dependent manner. All the polymers were unable to facilitate gene transfer when used alone, but enhanced US-mediated gene transfer significantly at concentrations around the critical micelle concentration in the three cell lines. F127 showed no significant toxicity at any concentration and protected the cells against US-mediated damage at a high concentration. L61 decreased cell viability significantly in a dose-dependent manner, whereas P85 showed mild toxicity when its concentration was at or above 0.05%.
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Técnicas de Transferencia de Gen , Poloxaleno/farmacología , Poloxámero/farmacología , Polietilenos/farmacología , Polipropilenos/farmacología , Tensoactivos/farmacología , Ultrasonido , Células 3T3 , Animales , Células CHO , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Relación Dosis-Respuesta a Droga , Ratones , TransfecciónRESUMEN
The objective of this work was to prepare echogenic phospholipid-based gas-filled microbubbles (PGM) and investigate their physical characteristics, echogenicity and loading ability of hirudin under various NaCl concentrations. PGM were prepared by a sonication-lyophilization method. Hirudin was used as a model drug to evaluate the drug encapsulation efficiency of the PGM. PGM loaded with hirudin were prepared by dissolving lyophilized powder with hirudin solution. The morphology, particle size and microbubble concentration of PGM were measured. The hirudin encapsulation efficiency as a function of NaCl concentration was determined. The mean particle size and microbubble concentration of PGM were unchanged by the presence of hirudin for at least 60 min after preparation. Hirudin encapsulation quantity was proportional to the hirudin concentration until saturation occurred at high concentration, and the encapsulation efficiency had an inverse relationship. Hirudin encapsulation efficiency was affected by NaCl concentration. When NaCl concentration was increased from 10 mg mL(-1) to 20 mg mL(-1) in PGM solution, hirudin encapsulation efficiency decreased from 35.8 to 26.7%, and microbubble concentration decreased from 2.7 x 10(8) to 1.7 x 10(8) microbubbles per mL. The PGM were shown easily to be visible in in vivo rabbit liver. There was no difference in echogenicity between the loaded and unloaded bubbles. PGM prepared by the sonication-lyophilization method exhibited satisfactory physical characteristics and loading ability and are suitable for use in imaging and ultrasound-triggered delivery.
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Sistemas de Liberación de Medicamentos , Hirudinas/administración & dosificación , Microburbujas , Fosfolípidos , Animales , Medios de Contraste , Composición de Medicamentos , Liofilización , Hirudinas/análisis , Hígado/diagnóstico por imagen , Masculino , Tamaño de la Partícula , Conejos , Cloruro de Sodio , Ultrasonido , UltrasonografíaRESUMEN
PURPOSE: To compare three commercial microbubble contrast agents (Optison, SonoVue, and Levovist) for their effect on gene delivery in skeletal muscle in conjunction with the use of therapeutic ultrasound. MATERIALS AND METHODS: The study was approved by the Animal Care and Use Committee. Plasmid DNA (10 microg) encoding green fluorescent protein (GFP) was mixed with microbubbles (or saline control) and injected into the tibialis anterior muscle of mice with and without adjunct ultrasound (1 MHz, 2 W/cm2, 30 seconds, 20% duty cycle). The efficiencies of GFP transgene expression were determined with four experimental conditions: (a) plasmid and saline as control (six mice), (b) plasmid and Optison (six mice), (c) plasmid and SonoVue (four mice), and (d) plasmid and Levovist (air based, four mice). The right legs were exposed to ultrasound, while the left legs were unexposed. Transfection efficiency was assessed by counting the number of GFP-positive fibers. Tissue damage was assessed by measuring the maximal-damage area on serial sections. RESULTS: When ultrasound was applied, both SonoVue and Optison significantly improved (P < .05) gene transfection efficiency. Optison was also effective (P < .05) even when no ultrasound was applied, which is consistent with previous studies. Levovist without ultrasound decreased the level of transfection (P < .05), with increased tissue damage. CONCLUSION: Both non-air-based agents show promise in gene delivery in skeletal muscle with undetectable tissue damage. Enhanced gene transfer with additional ultrasound was achieved only with SonoVue.
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Albúminas , Medios de Contraste , ADN/genética , Fluorocarburos , Técnicas de Transferencia de Gen , Músculo Esquelético/diagnóstico por imagen , Fosfolípidos , Plásmidos , Polisacáridos , Hexafluoruro de Azufre , Animales , Proteínas Fluorescentes Verdes , Masculino , Ratones , Ratones Endogámicos C57BL , Microburbujas , Transfección , UltrasonografíaRESUMEN
Ultrasound (US) is a promising tool for facilitating direct gene transfer to skeletal muscle, but no systematic optimisation study has been performed. We exposed H2K myoblast cells to US with varying intensity of exposure and duration to evaluate its effect on cell viability and transfection efficiency using as endpoints transfection rate, average fluorescence intensity (fluorescence normalised by the number of transfected cells) and overall expression (the product of transfection rate and average fluorescence intensity) as indices. Cell viability decreased with exposure time and intensity, consistent with previous findings. Optimal setting of US was observed at the range of 0.5 to 1 W cm(-2) with duration of 20 s, producing maximum efficiency (transfection = 4.5%) in gene transfection with minimum cell toxicity (cell viability = 83%). Higher intensity alone or in combination with low intensity and long duration did not improve cell viability and transfection. The increase of eGFP (enhanced green fluorescence protein) plasmid concentration up to 200 microg per mL was related to an increase in average fluorescence intensity and overall expression. However, transfection rate saturated when DNA concentration reached 50 microg per mL despite initial increase with DNA concentration. The average fluorescence intensity was linearly proportional to the logarithm of DNA concentration, suggesting a diffusion-based model for DNA uptake under sonoporation. We conclude that low-intensity US irradiation provides a safe and effective alternative for gene delivery.
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Técnicas de Transferencia de Gen , Músculo Esquelético/fisiología , Ultrasonido , Animales , Supervivencia Celular/genética , Células Cultivadas , ADN/análisis , Citometría de Flujo/métodos , Proteínas Fluorescentes Verdes/análisis , Ratones , Músculo Esquelético/diagnóstico por imagen , Plásmidos/genética , Factores de Tiempo , Transfección/métodos , UltrasonografíaRESUMEN
As an object rotates with respect to a stationary planar ultrasonic beam, the scattering centres within the object return echoes that are Doppler-shifted in frequency by amounts depending on the velocities of the individual scatterers. The backscattered echo amplitude at any particular frequency is the line integral of the scattered radiation at the cross-range corresponding to that frequency. The amplitude as a function of frequency can be interpreted as a tomographic projection. A tomographic reconstruction algorithm can produce an image of the distribution of scattering centres in the insonified object from these projections. This paper describes the development and characterisation of a microscanner to investigate the approach of using continuous wave ultrasound for three-dimensional cross-sectional imaging. The results of preliminary tissue investigation, conducted using bovine coccygeal intervertebral discs, are described. The radial imaging resolution improves as the Doppler frequency resolution improves but the circumferential resolution degrades proportionally. As the number of projections increases, there is a finite increase in image quality. Two- and three-dimensional images of the intervertebral disc reveal an alternate light and dark banding pattern that is characteristic of the laminar structure of the annulus fibrosus.