The systemic influence of chronic smoking on skin structure and mechanical function.

One of the major functions of human skin is to provide protection from the environment. Although we cannot entirely avoid, for example, sun exposure, it is likely that exposure to other environmental factors could affect cutaneous function. A number of studies have identified smoking as one such factor that leads to both facial wrinkle formation and a decline in skin function. In addition to the direct physical effects of tobacco smoke on skin, its inhalation has additional profound systemic effects for the smoker. The adverse effects on the respiratory and cardiovascular systems from smoking are well known. Central to the pathological changes associated with smoking is the elastic fibre, a key component of the extracellular matrices of lungs. In this study we examined the systemic effect of chronic smoking (>40 cigarettes/day; >5 years) on the histology of the cutaneous elastic fibre system, the nanostructure and mechanics of one of its key components, the fibrillin-rich microfibril, and the micromechanical stiffness of the dermis and epidermis. We show that photoprotected skin of chronic smokers exhibits significant remodelling of the elastic fibre network (both elastin and fibrillin-rich microfibrils) as compared to the skin of age- and sex-matched non-smokers. This remodelling is not associated with increased gelatinase activity (as identified by in situ zymography). Histological remodelling is accompanied by significant ultrastructural changes to extracted fibrillin-rich microfibrils. Finally, using scanning acoustic microscopy, we demonstrated that chronic smoking significantly increases the stiffness of both the dermis and the epidermis. Taken together, these data suggest an unappreciated systemic effect of chronic inhalation of tobacco smoke on the cutaneous elastic fibre network. Such changes may in part underlie the skin wrinkling and loss of skin elasticity associated with smoking. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Combining AFM and acoustic probes to reveal changes in the elastic stiffness tensor of living cells.

Knowledge of how the elastic stiffness of a cell affects its communication with its environment is of fundamental importance for the understanding of tissue integrity in health and disease. For stiffness measurements, it has been customary to quote a single parameter quantity, e.g., Young's modulus, rather than the minimum of two terms of the stiffness tensor required by elasticity theory. In this study, we use two independent methods (acoustic microscopy and atomic force microscopy nanoindentation) to characterize the elastic properties of a cell and thus determine two independent elastic constants. This allows us to explore in detail how the mechanical properties of cells change in response to signaling pathways that are known to regulate the cell's cytoskeleton. In particular, we demonstrate that altering the tensioning of actin filaments in NIH3T3 cells has a strong influence on the cell's shear modulus but leaves its bulk modulus unchanged. In contrast, altering the polymerization state of actin filaments influences bulk and shear modulus in a similar manner. In addition, we can use the data to directly determine the Poisson ratio of a cell and show that in all cases studied, it is less than, but very close to, 0.5 in value.

A pilot study of scanning acoustic microscopy as a tool for measuring arterial stiffness in aortic biopsies.

This study explores the use of scanning acoustic microscopy (SAM) as a potential tool for characterisation of arterial stiffness using aortic biopsies. SAM data is presented for human tissue collected during aortic bypass graft surgery for multi-vessel coronary artery disease. Acoustic wave speed as determined by SAM was compared to clinical data for the patients namely, pulse wave velocity (PWV), blood pressure, cholesterol and glucose levels. There was no obvious trend relating acoustic wave speed to PWV values, and an inverse relationship was found between systolic and diastolic blood pressure and acoustic wave speed. However, in patients with a higher cholesterol or glucose level, the acoustic wave speed increased. A more detailed investigation is needed to relate SAM data to clinical measurements.

Biomechanical Changes of Collagen Cross-Linking on Human Keratoconic Corneas Using Scanning Acoustic Microscopy.

PURPOSE: To assess the biomechanical changes of collagen cross-linking on keratoconic corneas in vitro. METHODS: Six keratoconic corneal buttons were included in this study. Each cornea was divided into two halves, where one half was cross-linked and the other half was treated with riboflavin only and served as control. The biomechanical changes of the corneal tissue were measured across the stroma using scanning acoustic microscopy (SAM). RESULTS: In the cross-linked corneas, there was a steady decrease in the magnitude of speed of sound from the anterior region through to the posterior regions of the stroma. The speed of sound was found to decrease slightly across the corneal thickness in the control corneas. The increase in speed of sound between the cross-linked and control corneas in the anterior region was by a factor of 1.039×. CONCLUSION: A higher speed of sound was detected in cross-linked keratoconic corneal tissue when compared with their controls, using SAM. This in vitro model can be used to compare to the cross-linking results obtained in vivo, as well as comparing the results obtained with different protocols.

Biomechanical changes after repeated collagen cross-linking on human corneas assessed in vitro using scanning acoustic microscopy.

PURPOSE: To explore the biomechanical changes induced by repeated cross-linking using scanning acoustic microscopy (SAM). METHODS: Thirty human corneas were divided into three groups. In group A, five corneas were cross-linked once. In group B, five corneas were cross-linked twice, 24 hours apart. In group C, five corneas were cross-linked three times, 24 hours apart. The contralateral controls in all groups had similar treatment but without UV-A. The speed of sound, which is directly proportional to the square root of the tissue's elastic modulus, was assessed using SAM. RESULTS: In group A, the speed of sound of the treated corneas was 1677.38 ± 10.70 ms(-1) anteriorly and 1603.90 ± 9.82 ms(-1) posteriorly, while it was 1595.23 ± 9.66 ms(-1) anteriorly and 1577.13 ± 8.16 ms(-1) posteriorly in the controls. In group B, the speed of sound of the treated corneas was 1746.33 ± 23.37 ms(-1) anteriorly and 1631.60 ± 18.92 ms(-1) posteriorly, while it was 1637.57 ± 22.15 ms(-1) anteriorly and 1612.30 ± 22.23 ms(-1) posteriorly in the controls. In group C, the speed of sound of the treated corneas was 1717.97 ± 18.92 ms(-1) anteriorly and 1616.62 ± 17.58 ms(-1) posteriorly, while it was 1628.69 ± 9.37 ms(-1) anteriorly and 1597.68 ± 11.97 ms(-1) posteriorly in the controls. The speed of sound in the anterior (200 × 200 µm) region between the cross-linked and control corneas in groups A, B, and C was increased by a factor of 1.051 (P = 0.005), 1.066 (P = 0.010), and 1.055 (P = 0.005) respectively. However, there was no significant difference among the cross-linked corneas in all groups (P = 0.067). CONCLUSIONS: A significant increase in speed of sound was found in all treated groups compared with the control group; however, the difference among the treated groups is not significant, suggesting no further cross-links are induced when collagen cross-linking treatment is repeated.

Scanning acoustic microscopy for mapping the microelastic properties of human corneal tissue.

PURPOSE: To assess the feasibility of applying scanning acoustic microscopy (SAM) on UV cross-linked corneal tissue for mapping and analyzing its biomechanical properties. MATERIALS AND METHODS: Five corneal pairs (10 corneas) were used. In each pair, one cornea was cross-linked (epithelium removed, riboflavin application for 45 min and UVA irradiation for 30 min) and the contralateral control cornea was epithelial debrided and treated only with riboflavin for 45 min. Histological sections were prepared and their mechanical properties were examined using SAM. A line profile technique and 2D analysis was used to analyze the mechanical properties of the corneas. Then the corneal paraformaldehyde and unfixed sections were examined histologically using hematoxylin and eosin (H&E) staining. RESULTS: In the frozen fresh corneal tissue, the speed of sound of the treated corneas was 1672.5 ± 36.9 ms(-1), while it was 1584.2 ± 25.9 ms(-1) in the untreated corneas. In the paraformaldehyde fixed corneal tissue, the speed of sound of the treated corneas was 1863.0 ± 12.7 ms(-1), while it was 1739.5 ± 30.4 ms(-1) in the untreated corneas. The images obtained from the SAM technique corresponded well with the histological images obtained with H&E staining. CONCLUSION: SAM is a novel tool for examining corneal tissue with a high spatial resolution, providing both histological and mechanical data.

Biomechanical properties of human corneas following low- and high-intensity collagen cross-linking determined with scanning acoustic microscopy.

PURPOSE: To assess and compare changes in the biomechanical properties of the cornea following different corneal collagen cross-linking protocols using scanning acoustic microscopy (SAM). METHODS: Ten donor human corneal pairs were divided into two groups consisting of five corneal pairs in each group. In group A, five corneas were treated with low-fluence (370 nm, 3 mW/cm(2)) cross-linking (CXL) for 30 minutes. In group B, five corneas were treated with high-fluence (370 nm, 9 mW/cm(2)) CXL for 10 minutes. The contralateral control corneas in both groups had similar treatment but without ultraviolet A. The biomechanical properties of all corneas were tested using SAM. RESULTS: In group A, the mean speed of sound in the treated corneas was 1677.38 ± 10.70 ms(-1) anteriorly and 1603.90 ± 9.82 ms(-1) posteriorly, while it was 1595.23 ± 9.66 ms(-1) anteriorly and 1577.13 ± 8.16 ms(-1) posteriorly in the control corneas. In group B, the mean speed of sound of the treated corneas was 1665.06 ± 9.54 ms(-1) anteriorly and 1589.89 ± 9.73 ms(-1) posteriorly, while it was 1583.55 ± 8.22 ms(-1) anteriorly and 1565.46 ± 8.13 ms(-1) posteriorly in the untreated control corneas. The increase in stiffness between the cross-linked and control corneas in both groups was by a factor of 1.051×. CONCLUSIONS: SAM successfully detected changes in the corneal stiffness after application of collagen cross-linking. A higher speed-of-sound value was found in the treated corneas when compared with the controls. No significant difference was found in corneal stiffness between the corneas cross-linked with low- and high-intensity protocols.

Multi-layer phase analysis: quantifying the elastic properties of soft tissues and live cells with ultra-high-frequency scanning acoustic microscopy.

Scanning acoustic microscopy is potentially a powerful tool for characterizing the elastic properties of soft biological tissues and cells. In this paper, we present a method, multi-layer phase analysis (MLPA), which can be used to extract local speed of sound values, for both thin tissue sections mounted on glass slides and cultured cells grown on cell culture plastic, with a resolution close to 1 µm. The method exploits the phase information that is preserved in the interference between the acoustic wave reflected from the substrate surface and internal reflections from the acoustic lens. In practice, a stack of acoustic images are captured beginning with the acoustic focal point 4 µm above the substrate surface and moving down in 0.1-µm increments. Scanning parameters, such as acoustic wave frequency and gate position, were adjusted to obtain optimal phase and lateral resolution. The data were processed offline to extract the phase information with the contribution of any inclination in the substrate removed before the calculation of sound speed. Here, we apply this approach to both skin sections and fibroblast cells, and compare our data with the V(f) (voltage versus frequency) method that has previously been used for characterization of soft tissues and cells. Compared with the V(f) method, the MPLA method not only reduces signal noise but can be implemented without making a priori assumptions with regards to tissue or cell parameters.

Cardiotrophin 1 is involved in cardiac, vascular, and renal fibrosis and dysfunction.

Cardiotrophin 1 (CT-1), a cytokine belonging to the interleukin 6 family, is increased in hypertension and in heart failure. We aimed to study the precise role of CT-1 on cardiac, vascular, and renal function; morphology; and remodeling in early stages without hypertension. CT-1 (20 µg/kg per day) or vehicle was administrated to Wistar rats for 6 weeks. Cardiac and vascular functions were analyzed in vivo using M-mode echocardiography, Doppler, and echo tracking device and ex vivo using a scanning acoustic microscopy method. Cardiovascular and renal histomorphology were measured by immunohistochemistry, RT-PCR, and Western blot. Kidney functional properties were assessed by serum creatinine and neutrophile gelatinase-associated lipocalin and microalbuminuria/creatininuria ratio. Without alterations in blood pressure levels, CT-1 treatment increased left ventricular volumes, reduced fractional shortening and ejection fraction, and induced myocardial dilatation and myocardial fibrosis. In the carotid artery of CT-1-treated rats, the circumferential wall stress-incremental elastic modulus curve was shifted leftward, and the acoustic speed of sound in the aorta was augmented, indicating increased arterial stiffness. Vascular media thickness, collagen, and fibronectin content were increased by CT-1 treatment. CT-1-treated rats presented unaltered serum creatinine concentrations but increased urinary and serum neutrophile gelatinase-associated lipocalin and microalbuminuria/creatininuria ratio. This paralleled a glomerular and tubulointerstitial fibrosis accompanied by renal epithelial-mesenchymal transition. CT-1 is a new potent fibrotic agent in heart, vessels, and kidney able to induce cardiovascular-renal dysfunction independent from blood pressure. Thus, CT-1 could be a new target simultaneously integrating alterations of heart, vessels, and kidney in early stages of heart failure.

Quantifying Micro-mechanical Properties of Soft Biological Tissues with Scanning Acoustic Microscopy.

In this study we have established a new approach to more accurately map acoustic wave speed (which is a measure of stiffness) within soft biological tissues at micrometer length scales using scanning acoustic microscopy. By using thin (5 µm thick) histological sections of human skin and porcine cartilage, this method exploits the phase information preserved in the interference between acoustic waves reflected from the substrate surface as well as internal reflections from the acoustic lens. A stack of images were taken with the focus point of acoustic lens positioned at or above the substrate surface, and processed pixel by pixel using custom software developed with LABVIEW and IMAQ (National Instruments) to extract phase information. Scanning parameters, such as acoustic wave frequency and gate position were optimized to get reasonable phase and lateral resolution. The contribution from substrate inclination or uneven scanning surface was removed prior to further processing. The wave attenuation was also obtained from these images.