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Image-based deformation estimation is an important tool used in a variety of engineering problems, including crack propagation, fracture, and fatigue failure. These tools have been important in biomechanics research where measuring in vitro and in vivo tissue deformations are important for evaluating tissue health and disease progression. However, accurately measuring tissue deformation in vivo is particularly challenging due to limited image signal-to-noise ratio. Therefore, we created a novel deep-learning approach for measuring deformation from a sequence of images collected in vivo called StrainNet. Utilizing a training dataset that incorporates image artifacts, StrainNet was designed to maximize performance in challenging, in vivo settings. Artificially generated image sequences of human flexor tendons undergoing known deformations were used to compare benchmark StrainNet against two conventional image-based strain measurement techniques. StrainNet outperformed the traditional techniques by nearly 90%. High-frequency ultrasound imaging was then used to acquire images of the flexor tendons engaged during contraction. Only StrainNet was able to track tissue deformations under the in vivo test conditions. Findings revealed strong correlations between tendon deformation and applied forces, highlighting the potential for StrainNet to be a valuable tool for assessing rehabilitation strategies or disease progression. Additionally, by using real-world data to train our model, StrainNet was able to generalize and reveal important relationships between the effort exerted by the participant and tendon mechanics. Overall, StrainNet demonstrated the effectiveness of using deep learning for image-based strain analysis in vivo.
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Aprendizaje Profundo , Tendones , Ultrasonografía , Tendones/diagnóstico por imagen , Tendones/fisiología , Humanos , Ultrasonografía/métodos , Fenómenos Biomecánicos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Two-dimensional ultrasound (2DUS) echocardiography is the main noninvasive method used to evaluate cardiac function in animal models of myocardial infarction (MI). However, 2DUS echocardiography does not capture regional differences in cardiac contractility since it relies on planar images to estimate left ventricular (LV) geometry and global function. Thus, the current study was designed to evaluate the efficacy of a newly developed 4-dimensional ultrasound (4DUS) method in detecting cardiac functional differences between two models of MI, permanent ligation (PL), and ischemia/reperfusion (I/R) in rats. We found that only 4DUS was able to detect LV global functional differences between the two models and that 4DUS-derived surface area strain accurately detected infarcted regions within the myocardium that correlated well with histological infarct size analysis. We also found that 4DUS-derived strain, which includes circumferential, longitudinal, and surface area strain, correlated with the peak positive of the first derivative of left ventricular pressure (+dP/dtmax). In conclusion, 4DUS strain echocardiography effectively assesses myocardial mechanics following experimentally induced ischemia in rats and accurately estimates infarct size as early as 1 day after injury. 4DUS also correlates well with +dP/dtmax, a widely used marker of cardiac contractility.
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Infarto del Miocardio , Daño por Reperfusión Miocárdica , Ratas Sprague-Dawley , Animales , Masculino , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/patología , Ratas , Daño por Reperfusión Miocárdica/diagnóstico por imagen , Daño por Reperfusión Miocárdica/fisiopatología , Ecocardiografía Tetradimensional/métodos , Contracción Miocárdica , Función Ventricular Izquierda , Modelos Animales de EnfermedadRESUMEN
Cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD), however, in the mdx mouse model of DMD, the cardiac phenotype differs from that seen in DMD-associated cardiomyopathy. Although some have used pharmacologic stress to enhance the cardiac phenotype in the mdx model, many methods lead to high mortality, variable cardiac outcomes, and do not recapitulate the structural and functional cardiac changes seen in human disease. Here, we describe a simple and effective method to enhance the cardiac phenotype model in mdx mice using advanced 2D and 4D high-frequency ultrasound to monitor cardiac dysfunction progression in vivo. For our study, mdx and wild-type (WT) mice received daily low-dose (2 mg/kg/day) isoproterenol injections for 10 days. Histopathologic assessment showed that isoproterenol treatment increased myocyte injury, elevated serum cardiac troponin I levels, and enhanced fibrosis in mdx mice. Ultrasound revealed reduced ventricular function, decreased wall thickness, increased volumes, and diminished cardiac reserve in mdx mice compared to wild-type. Our findings highlight the utility of low-dose isoproterenol in mdx mice as a valuable model for exploring therapies targeting DMD-associated cardiac complications.
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Background: Cardiomyopathy (CMP) is the leading cause of death in Duchenne muscular dystrophy (DMD). Characterization of disease trajectory can be challenging, especially in the early stage of CMP where onset and clinical progression may vary. Traditional metrics from cardiovascular magnetic resonance (CMR) imaging such as LVEF (left ventricular ejection fraction) and LGE (late gadolinium enhancement) are often insufficient for assessing disease trajectory. We hypothesized that strain patterns from a novel 4D (3D+time) CMR regional strain analysis method can be used to predict the rate of DMD CMP progression. Methods: We compiled 115 short-axis cine CMR image stacks for n=40 pediatric DMD patients (13.6±4.2 years) imaged yearly for 3 consecutive visits and computed regional strain metrics using custom-built feature tracking software. We measured regional strain parameters by determining the relative change in the localized 4D endocardial surface mesh using end diastole as the initial reference frame. Results: We first separated patients into two cohorts based on their initial CMR: LVEF≥55% (n=28, normal cohort) and LVEF<55% (n=12, abnormal cohort). Using LVEF decrease measured two years following the initial scan, we further subclassified these cohorts into slow (ΔLVEF%≤5) or fast (ΔLVEF%>5) progression groups for both the normal cohort (n=12, slow; n=15, fast) and the abnormal cohort (n=8, slow; n=4, fast). There was no statistical difference between the slow and fast progression groups in standard biomarkers such as LVEF, age, or LGE status. However, basal circumferential strain (Ecc) late diastolic strain rate and basal surface area strain (Ea) late diastolic strain rate magnitude were significantly decreased in fast progressors in both normal and abnormal cohorts (p<0.01, p=0.04 and p<0.01, p=0.02, respectively). Peak Ea and Ecc magnitudes were also decreased in fast progressors, though these only reached statistical significance in the normal cohort (p<0.01, p=0.24 and p<0.01, p=0.18, respectively). Conclusion: Regional strain metrics from 4D CMR can be used to differentiate between slow or fast CMP progression in a longitudinal DMD cohort. These results demonstrate that 4D CMR strain is useful for early identification of CMP progression in patients with DMD. Clinical Perspective: Cardiomyopathy is the number one cause of death in Duchenne muscular dystrophy, but the onset and progression of the disease are variable and heterogeneous. In this study, we used a novel 4D cardiovascular magnetic resonance regional strain analysis method to evaluate 40 pediatric Duchenne patients over three consecutive annual visits. From our analysis, we found that peak systolic strain and late diastolic strain rate were early indicators of cardiomyopathy progression. This method offers promise for early detection and monitoring, potentially improving patient outcomes through timely intervention and management.
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BACKGROUND: Cardiomyopathy (CMP) is the most common cause of mortality in Duchenne muscular dystrophy (DMD), though the age of onset and clinical progression vary. We applied a novel 4D (3D + time) strain analysis method using cine cardiovascular magnetic resonance (CMR) imaging data to determine if localized strain metrics derived from 4D image analysis would be sensitive and specific for characterizing DMD CMP. METHODS: We analyzed short-axis cine CMR image stacks from 43 DMD patients (median age: 12.23 yrs [10.6-16.5]; [interquartile range]) and 25 male healthy controls (median age: 16.2 yrs [13.3-20.7]). A subset of 25 male DMD patients age-matched to the controls (median age: 15.7 yrs [14.0-17.8]) was used for comparative metrics. CMR images were compiled into 4D sequences for feature-tracking strain analysis using custom-built software. Unpaired t-test and receiver operator characteristic area under the curve (AUC) analysis were used to determine statistical significance. Spearman's rho was used to determine correlation. RESULTS: DMD patients had a range of CMP severity: 15 (35% of total) had left ventricular ejection fraction (LVEF) > 55% with no findings of myocardial late gadolinium enhancement (LGE), 15 (35%) had findings of LGE with LVEF > 55% and 13 (30%) had LGE with LVEF < 55%. The magnitude of the peak basal circumferential strain, basal radial strain, and basal surface area strain were all significantly decreased in DMD patients relative to healthy controls (p < 0.001) with AUC values of 0.80, 0.89, and 0.84 respectively for peak strain and 0.96, 0.91, and 0.98 respectively for systolic strain rate. Peak basal radial strain, basal radial systolic strain rate, and basal circumferential systolic strain rate magnitude values were also significantly decreased in mild CMP (No LGE, LVEF > 55%) compared to a healthy control group (p < 0.001 for all). Surface area strain significantly correlated with LVEF and extracellular volume (ECV) respectively in the basal (rho = - 0.45, 0.40), mid (rho = - 0.46, 0.46), and apical (rho = - 0.42, 0.47) regions. CONCLUSION: Strain analysis of 3D cine CMR images in DMD CMP patients generates localized kinematic parameters that strongly differentiate disease from control and correlate with LVEF and ECV.
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Cardiomiopatías , Distrofia Muscular de Duchenne , Humanos , Masculino , Niño , Adolescente , Distrofia Muscular de Duchenne/complicaciones , Distrofia Muscular de Duchenne/diagnóstico por imagen , Volumen Sistólico , Función Ventricular Izquierda , Medios de Contraste , Fenómenos Biomecánicos , Valor Predictivo de las Pruebas , Gadolinio , Imagen por Resonancia Cinemagnética/métodos , Cardiomiopatías/diagnóstico por imagen , Cardiomiopatías/etiología , Cardiomiopatías/patología , Espectroscopía de Resonancia MagnéticaRESUMEN
Cardiomyopathy (CM) is the leading cause of death for individuals with Duchenne muscular dystrophy (DMD). While DMD CM progresses rapidly and fatally for some in teenage years, others can live relatively symptom-free into their thirties or forties. Because CM progression is variable, there is a critical need for biomarkers to detect early onset and rapid progression. Despite recent advances in imaging and analysis, there are still no reliable methods to detect the onset or progression rate of DMD CM. Cardiac strain imaging is a promising technique that has proven valuable in DMD CM assessment, though much more work has been done in adult CM patients. In this review, we address the role of strain imaging in DMD, the mechanical and functional parameters used for clinical assessment, and discuss the gaps where emerging imaging techniques could help better characterize CM progression in DMD. Prominent among these emerging techniques are strain assessment from 3D imaging and development of deep learning algorithms for automated strain assessment. Improved techniques in tracking the progression of CM may help to bridge a crucial gap in optimizing clinical treatment for this devastating disease and pave the way for future research and innovation through the definition of robust imaging biomarkers and clinical trial endpoints.
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Ischemic heart disease is the leading cause of death in the United States, Canada, and worldwide. Severe disease is characterized by coronary artery occlusion, loss of blood flow to the myocardium, and necrosis of tissue, with subsequent remodeling of the heart wall, including fibrotic scarring. The current study aims to demonstrate the efficacy of quantitating infarct size via two-dimensional (2-D) echocardiographic akinetic length and four-dimensional (4-D) echocardiographic infarct volume and surface area as in vivo analysis techniques. We further describe and evaluate a new surface area strain analysis technique for estimating myocardial infarction (MI) size after ischemic injury. Experimental MI was induced in mice via left coronary artery ligation. Ejection fraction and infarct size were measured through 2-D and 4-D echocardiography. Infarct size established via histology was compared with ultrasound-based metrics via linear regression analysis. Two-dimensional echocardiographic akinetic length (r = 0.76, P = 0.03), 4-D echocardiographic infarct volume (r = 0.85, P = 0.008), and surface area (r = 0.90, P = 0.002) correlate well with histology. Although both 2-D and 4-D echocardiography were reliable measurement techniques to assess infarct, 4-D analysis is superior in assessing asymmetry of the left ventricle and the infarct. Strain analysis performed on 4-D data also provides additional infarct sizing techniques, which correlate with histology (surface strain: r = 0.94, P < 0.001, transmural thickness: r = 0.76, P = 0.001). Two-dimensional echocardiographic akinetic length, 4-D echocardiography ultrasound, and strain provide effective in vivo methods for measuring fibrotic scarring after MI.NEW & NOTEWORTHY Our study supports that both 2-D and 4-D echocardiographic analysis techniques are reliable in quantifying infarct size though 4-D ultrasound provides a more holistic image of LV function and structure, especially after myocardial infarction. Furthermore, 4-D strain analysis correctly identifies infarct size and regional LV dysfunction after MI. Therefore, these techniques can improve functional insight into the impact of pharmacological interventions on the pathophysiology of cardiac disease.
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Infarto del Miocardio/diagnóstico por imagen , Ultrasonografía/métodos , Algoritmos , Animales , Gasto Cardíaco , Femenino , Ventrículos Cardíacos/diagnóstico por imagen , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Imagenología Tridimensional/métodos , Imagenología Tridimensional/normas , Masculino , Ratones , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Sensibilidad y Especificidad , Ultrasonografía/normasRESUMEN
Right ventricular (RV) strain measurements from ultrasound via speckle-tracking techniques are being used more frequently as a non-invasive diagnostic tool for a variety of cardiopulmonary pathologies. However, despite the clinical utility of ultrasound RV strain measurements, quantification of RV strain in rodents remains difficult owing to unique image artifacts and non-standardized methodologies. We demonstrate here a simple approach for measuring RV strain in both mice and rats using high-frequency ultrasound and automated speckle tracking. Our results show estimated peak RV free-wall longitudinal strain values (mean ± standard error of the mean) in mice (n = 15) and rats (n = 5) of, respectively, -10.38% ± 0.4% and -4.85% ± 0.42%. We further estimated the 2-D Green-Lagrange strain within the RV free wall, with longitudinal components estimated at -5.7% ± 0.48% in mice and -2.1% ± 0.28% in rats. These methods and data may provide a foundation for future work aimed at evaluating murine RV strain levels in different disease models.
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Ventrículos Cardíacos , Disfunción Ventricular Derecha , Animales , Ecocardiografía , Corazón , Ventrículos Cardíacos/diagnóstico por imagen , Ratones , Ratas , Ultrasonografía , Disfunción Ventricular Derecha/diagnóstico por imagenRESUMEN
Tissue engineered vascular grafts (TEVGs) using scaffolds fabricated from braided poly(glycolic acid) (PGA) fibers coated with poly(glycerol sebacate) (PGS) are developed. The approach relies on in vivo tissue engineering by which neotissue forms solely within the body after a scaffold has been implanted. Herein, the impact of altering scaffold braid design and scaffold coating on neotissue formation is investigated. Several combinations of braiding parameters are manufactured and evaluated in a Beige mouse model in the infrarenal abdominal aorta. Animals are followed with 4D ultrasound analysis, and 12 week explanted vessels are evaluated for biaxial mechanical properties as well as histological composition. Results show that scaffold parameters (i.e., braiding angle, braiding density, and presence of a PGS coating) have interdependent effects on the resulting graft performance, namely, alteration of these parameters influences levels of inflammation, extracellular matrix production, graft dilation, neovessel distensibility, and overall survival. Coupling carefully designed in vivo experimentation with regression analysis, critical relationships between the scaffold design and the resulting neotissue that enable induction of favorable cellular and extracellular composition in a controlled manner are uncovered. Such an approach provides a potential for fabricating scaffolds with a broad range of features and the potential to manufacture optimized TEVGs.
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Prótesis Vascular , Ingeniería de Tejidos , Animales , Matriz Extracelular , Ratones , Andamios del TejidoRESUMEN
Cell-free protein synthesis has emerged as a promising platform for the production of therapeutic proteins due to its inherently open reaction environment, flexible reaction conditions and rapid protein synthesis capabilities. In recent years, lyophilized cell-free systems have widened the application space of cell-free technology by improving reagent stability outside of cold-chain storage. Current embodiments of the system, however, demonstrate poor stability at elevated temperatures. Lyoprotectants have long been recognized for the ability to preserve the activity of biological molecules during drying processes, but the application of this technology to lyophilized cell-free systems has been limited and has failed to address the negative effects that such lyoprotectants may have on cell-free systems. Here, several lyoprotected, lyophilized cell-free protein synthesis systems are demonstrated using antiplasticized sugar glasses as lyoprotectants, showing significant improvement over standard lyophilized systems or trehalose-preserved systems. Furthermore, we demonstrate for the first time, preservation and therapeutic expression, specifically of FDA-approved crisantaspase, from a truly single-pot lyophilized, endotoxin-free, cell-free protein synthesis system, exemplifying the potential for on-site therapeutic synthesis.
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Asparaginasa/biosíntesis , Temperatura , Asparaginasa/aislamiento & purificación , Asparaginasa/metabolismo , LiofilizaciónRESUMEN
The goal of the present study was to evaluate the effects of SGLT2i on cardiac contractile function, substrate utilization, and efficiency before and during regional myocardial ischemia/reperfusion injury in normal, metabolically healthy swine. Lean swine received placebo or canagliflozin (300 mg PO) 24 h prior to and the morning of an invasive physiologic study protocol. Hemodynamic and cardiac function measurements were obtained at baseline, during a 30-min complete occlusion of the circumflex coronary artery, and during a 2-h reperfusion period. Blood pressure, heart rate, coronary flow, and myocardial oxygen consumption were unaffected by canagliflozin treatment. Ventricular volumes remained unchanged in controls throughout the protocol. At the onset of ischemia, canagliflozin produced acute large increases in left ventricular end-diastolic and systolic volumes which returned to baseline with reperfusion. Canagliflozin-mediated increases in end-diastolic volume were directly associated with increases in stroke volume and stroke work relative to controls during ischemia. Canagliflozin also increased cardiac work efficiency during ischemia relative to control swine. No differences in myocardial uptake of glucose, lactate, free fatty acids or ketones, were noted between treatment groups at any time. In separate experiments using a longer 60 min coronary occlusion followed by 2 h of reperfusion, canagliflozin increased end-diastolic volume and stroke volume and significantly diminished myocardial infarct size relative to control swine. These data demonstrate that SGLT2i with canagliflozin preserves cardiac contractile function and efficiency during regional myocardial ischemia and provides ischemia protection independent of alterations in myocardial substrate utilization.
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Canagliflozina/farmacología , Metabolismo Energético/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/tratamiento farmacológico , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocardio/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Función Ventricular Izquierda/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Masculino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Sus scrofaRESUMEN
Many diseases and disorders are linked to exposure to endocrine disrupting chemicals (EDCs) that mimic the function of natural estrogen hormones. Here we present a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting chemicals that interact with the human estrogen receptor ß (hERß). This biosensor consists of an allosteric fusion protein, which is expressed using cell-free protein synthesis technology and is directly assayed by a colorimetric response. The resultant biosensor successfully detected known EDCs of hERß (BPA, E2, and DPN) at similar or better detection range than an analogous cell-based biosensor, but in a fraction of time. We also engineered cell-free protein synthesis reactions with RNAse inhibitors to increase production yields in the presence of human blood and urine. The RAPID biosensor successfully detects EDCs in these human samples in the presence of RNAse inhibitors. Engineered cell-free protein synthesis facilitates the use of protein biosensors in complex sample matrices without cumbersome protein purification.
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Técnicas Biosensibles/métodos , Sistema Libre de Células/metabolismo , Disruptores Endocrinos/sangre , Disruptores Endocrinos/orina , Biosíntesis de Proteínas/fisiología , Sistema Libre de Células/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Disruptores Endocrinos/farmacología , Receptor beta de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/metabolismo , Humanos , Biosíntesis de Proteínas/efectos de los fármacosRESUMEN
The effectiveness and economics of polyvinyl sulfonic acid (PVSA) as a ribonuclease inhibitor for in vitro systems is reported. PVSA was shown to inhibit RNA cleavage in the presence of RNase A as well as in the presence of Escherichia coli lysate, suggesting that PVSA can act as a broader ribonuclease inhibitor. In addition, PVSA was shown to improve the integrity of mRNA transcripts by up to 5-fold in vitro as measured by their translational viability. Improved preservation of mRNA transcripts in the presence of PVSA under common RNA storage conditions is also reported. A cost comparison with commercially available RNAse inhibitors indicates the economic practicality of PVSA which is approximately 1,700 times less expensive than commonly used ribonuclease inhibitors. PVSA can also be separated from RNA by alcohol precipitation for applications that may be sensitive to the presence of PVSA.
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Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Polivinilos/farmacología , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Ribonucleasas/antagonistas & inhibidores , Ácidos Sulfónicos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/economía , Escherichia coli/enzimología , Escherichia coli/genética , Cinética , Polivinilos/química , Polivinilos/economía , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/genética , Ribonucleasas/genética , Ribonucleasas/metabolismo , Fracciones Subcelulares/metabolismo , Ácidos Sulfónicos/química , Ácidos Sulfónicos/economía , Transcripción GenéticaRESUMEN
Cell-free protein synthesis has been around for decades but it has never been close to becoming a robust tool for the production of biotherapeutic agents. In this review, we focus on how Escherichia coli-based cell-free protein synthesis can be modified in various ways to produce challenging, complex anticancer biotherapeutics. Here we report progress in extract preparation and its relation to cell-free cancer research. The future prospects of cell-free technology and its potential in various areas of cancer therapeutics production are also highlighted.