Quantification of microvascular change of retinal degeneration in Royal College of Surgeons rats using high-resolution spectral domain optical coherence tomography angiography.

Significance: For research on retinitis pigmentosa in humans, the Royal College of Surgeons (RCS) rat is commonly used as the primary animal model since the disease process is similar. Therefore, it is necessary to understand how the disease develops and determine whether the treatment is effective. Aim: In this study, structural and microvascular change of retinal degeneration in RCS rats was assessed non-invasively on specific dates over 3.5 months. Approach: Using a high-resolution spectral domain (SD) optical coherence tomography angiography (OCTA), the retinal degeneration in RCS rats, from day 14 until day 126, was qualitatively and quantitatively analyzed. Results: Aside from the thinning of the retina thickness starting from 2 weeks of age, blood vessels in the deep layer of the retina also began to degenerate at about 4 weeks of age. Hole structures appeared at the inner nuclear layer and the inner plexiform layer by the age of 10 weeks. Observations of abnormal angiogenesis in the choroid began by 12 weeks of age. Conclusions: We conducted a longitudinal study of retina degeneration structure and vascular changes in an RCS rat model using a supercontinuum laser based high-resolution SD-OCTA. Combined with OCTA, OCT leads to a better understanding of photoreceptor pathology as retinal degeneration by identifying tissue and vessel loss.

Handheld common-path swept-source optical coherence tomography angiography.

This study develops a handheld optical coherence tomography angiography (OCTA) system that uses a high-speed (200 kHz) swept laser with a dual-reference common-path configuration for stable and fast imaging. The common-path design automatically avoids polarization and dispersion mismatches by using one circulator as the primary system element, ensuring a cost-effective and compact design for handheld probe use. With its stable envelope (i.e., sub-µm shifts) and phase variation (corresponding to nm changes in axial displacement), the minimum detectable flow velocity is ∼ 0.08 mm/s in our experiment, which gives the common-path setup a high potential for application in a handheld OCTA system for clinical skin screening. In vivo skin structures and microvasculature networks on the dorsum of the hand and cheek of a healthy human are imaged successfully.

Inhibition of oxidative stress-induced epithelial-mesenchymal transition in retinal pigment epithelial cells of age-related macular degeneration model by suppressing ERK activation.

INTRODUCTION: Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is related to the pathogenesis of various retinopathies including age-related macular degeneration (AMD). Oxidative stress is the major factor that induces degeneration of RPE cells associated with the etiology of AMD. OBJECTIVES: Sodium iodate (NaIO3) generates intracellular reactive oxygen species (ROS) and is widely used to establish a model of AMD due to the selective induction of retinal degeneration. This study was performed to clarify the effects of multiple NaIO3-stimulated signaling pathways on EMT in RPE cells. METHODS: The EMT characteristics in NaIO3-treated human ARPE-19 cells and RPE cells of the mouse eyes were analyzed. Multiple oxidative stress-induced modulators were investigated and the effects of pre-treatment with Ca2+ chelator, extracellular signal-related kinase (ERK) inhibitor, or epidermal growth factor receptor (EGFR) inhibitor on NaIO3-induced EMT were determined. The efficacy of post-treatment with ERK inhibitor on the regulation of NaIO3-induced signaling pathways was dissected and its role in retinal thickness and morphology was evaluated by using histological cross-sections and spectral domain optical coherence tomography. RESULTS: We found that NaIO3 induced EMT in ARPE-19 cells and in RPE cells of the mouse eyes. The intracellular ROS, Ca2+, endoplasmic reticulum (ER) stress marker, phospho-ERK, and phospho-EGFR were increased in NaIO3-stimulated cells. Our results showed that pre-treatment with Ca2+ chelator, ERK inhibitor, or EGFR inhibitor decreased NaIO3-induced EMT, interestingly, the inhibition of ERK displayed the most prominent effect. Furthermore, post-treatment with FR180204, a specific ERK inhibitor, reduced intracellular ROS and Ca2+ levels, downregulated phospho-EGFR and ER stress marker, attenuated EMT of RPE cells, and prevented structural disorder of the retina induced by NaIO3. CONCLUSIONS: ERK is a crucial regulator of multiple NaIO3-induced signaling pathways that coordinate EMT program in RPE cells. Inhibition of ERK may be a potential therapeutic strategy for the treatment of AMD.

En-face polarization-sensitive optical coherence tomography to characterize early-stage esophageal cancer and determine tumor margin.

Current imaging tools are insufficiently sensitive to the early diagnosis of esophageal squamous cell carcinoma (ESCC). The application of polarization-sensitive optical coherence tomography (PS-OCT) to detect tumor-stroma interaction is an interesting issue in cancer diagnosis. In this translational study, we found that en-face PS-OCT effectively characterizes protruding, flat, and depressive type ESCC regardless of animal or human specimens. In addition, the tumor contour and margin could also be drawn and determined on a broad en-face view. The determined tumor margin could be in the proximity of 2 mm to the actual tumor margin, which was proved directly using histology.

Human Umbilical Mesenchymal Stem Cell Xenografts Repair UV-Induced Photokeratitis in a Rat Model.

Most patients with a corneal injury are administered anti-inflammatory medications and antibiotics, but no other treatments are currently available. Thus, the corneal injury healing is unsatisfactory, affects the vision, and has a risk of blindness in severe cases. Human umbilical mesenchymal stem cells exhibit pluripotent and anti-inflammatory properties and do not cause immunological rejection in the host. Rats were irradiated with type B ultraviolet (UVB) light to generate a stable animal model of photokeratitis. After irradiation-induced photokeratitis, human umbilical mesenchymal stem cells were implanted into the subconjunctival space of the lateral sclera, and the changes in the corneal pathology were evaluated. Three weeks after implantation, many mesenchymal stem cells were visible in the subconjunctival space. These mesenchymal stem cells effectively reduced the extent of injury to the adjacent corneal tissue. They accelerated the epithelial layer repair, reduced the inflammatory response and neovascularization, and improved the disorganization of collagen and fibronectin in the corneal stroma caused by the injury. In conclusion, xenografted human umbilical mesenchymal stem cells can survive in rat eye tissues for a long time, effectively support the structural integrity of injured corneal tissues, restore corneal permeability, and reduce abnormal neovascularization. This study provides a new approach to the treatment of photokeratitis.

Needle-Probe Optical Coherence Tomography for Real-Time Visualization of Veress Peritoneal Needle Placement in a Porcine Model: A New Safety Concept for Pneumoperitoneum Establishment in Laparoscopic Surgery.

The safe establishment of pneumoperitoneum is a critical step in all laparoscopic surgeries. A closed pneumoperitoneum is usually obtained by inserting a Veress needle into the peritoneal cavity. However, there is no definite measure to visually confirm the position of the Veress needle tip inside the peritoneal cavity. This study aimed to describe a method of real-time visual detection of peritoneal placement of the Veress needle using an incorporated optical coherence tomography (OCT) probe in a porcine model. A 14-gauge Veress needle was incorporated with a miniature fiber probe to puncture the piglet's abdominal wall into the peritoneal cavity. A total of 80 peritoneal punctures were attempted in four piglets. For each puncture, continuous two-dimensional OCT images of the abdominal wall were acquired for real-time visual detection of the needle placement into the peritoneal cavity. Characteristic OCT image patterns could be observed during the puncturing process, especially a deep V-shaped concave pattern before the peritoneum puncture, which was a crucial feature. A statistical difference in the OCT signal standard deviation value also indicated the differentiability of images between the peritoneum and extra-peritoneal tissue layers. A success rate of 97.5% could be achieved with the guidance of the OCT images. OCT images translate the blind closed technique of peritoneal access into a visualized procedure, thus improving peritoneal access safety.

Inhibition of DUSP6 Activates Autophagy and Rescues the Retinal Pigment Epithelium in Sodium Iodate-Induced Retinal Degeneration Models In Vivo and In Vitro.

Autophagy plays a protective role in the retinal pigment epithelium (RPE) by eliminating damaged organelles in response to reactive oxygen species (ROS). Dual-specificity protein phosphatase 6 (DUSP6), which belongs to the DUSP subfamily, works as a negative-feedback regulator of the extracellular signal-regulated kinase (ERK) pathway. However, the complex interplay between DUSP6 and autophagy induced by ROS in RPE is yet to be investigated. To investigate the relationship between DUSP6 and autophagy, we exposed the ARPE-19 cell line and C57BL/6N mice to sodium iodate (NaIO3) as an oxidative stress inducer. Our data showed that the inhibition of DUSP6 activity promotes autophagy flux through the ERK pathway via the upregulation of immunoblotting expression in ARPE-19 cells. Live imaging showed a significant increase in autophagic flux activities, which suggested the restoration autophagy after treatment with the DUSP6 inhibitor. Furthermore, the mouse RPE layer exhibited an irregular structure and abnormal deposits following NaIO3 injection. The retina layer was recovered after being treated with DUSP6 inhibitor; this suggests that DUSP6 inhibitor can rescue retinal damage by restoring the mouse retina's autophagy flux. This study suggests that the upregulation of DUSP6 can cause autophagy flux malfunctions in the RPE. The DUSP6 inhibitor can restore autophagy induction, which may serve as a potential therapeutic approach for retinal degeneration disease.

In Vivo Longitudinal Tracking of Lymphangiogenesis and Angiogenesis in Cutaneous Melanoma Mouse Model Using Multifunctional Optical Coherence Tomography.

Melanoma is a high-risk skin cancer because it tends to metastasize early and ultimately leads to death. In this study, we introduced a noninvasive multifunctional optical coherence tomography (MFOCT) for the early detection of premetastatic pathogenesis in cutaneous melanoma by label-free imaging of microstructures (i.e., providing the thickness and the scattering information) and microcirculation (i.e., providing depth-resolved angiography and lymphangiography). Using MFOCT-based approaches, we presented an in vivo longitudinal observation of the tumor microenvironment in Braf V600E/V600E ;Pten -/- mice with inducible melanoma monitored for 42 days. Quantitative analysis of MFOCT images identified an increased number of lymphatic and vascular vessels during tumor progression and faster lymphangiogenesis (beginning on day 21) than angiogenesis (beginning on day 28) in the melanoma microenvironment. We further observed lymphatic vessel enlargement from the first week of melanoma development, implying tumor cells interacting with the vessels and increased likelihood of metastasis. MFOCT identified cutaneous melanoma‒associated angiogenesis and lymphangiogenesis before the possible visual perception of the tumor (≥42 days) and before metastasis could be diagnosed using micropositron emission tomography (35 days). Thus, the proposed quantitative analysis using MFOCT has the potential for early detection of cutaneous melanoma progression or prediction of metastatic melanoma in a mouse model. However, retrospective and extensive experiments still need to be performed in the future to confirm the value of MFOCT in clinical application.

Fiber-Needle Swept-Source Optical Coherence Tomography for the Real-Time Visualization of the Transversus Abdominis Plane Block Procedure in a Swine Model.

BACKGROUND: Fascia blocks (eg, the transversus abdominis plane [TAP] block) target the intermuscular fascia layers. Ultrasound techniques have allowed peripheral blocks to be performed with accuracy and safety, however, with limitations. Optical coherence tomography (OCT) is based on low-coherence interferometry. In this study, we examined the ability of OCT to identify the TAP. METHODS: A swept-source OCT probe was placed in a 17-gauge needle to obtain imaging. The needle was inserted within 2 different angle ranges (0°-30° and 30°-60°) on a slice of pork belly to assess imaging characteristics. A series of real-time OCT imaging of the muscle, fascia, and interfascial space was obtained. The tissue location of the needle tip was identified using near-infrared (NIR) imaging. In vivo OCT imaging was further done on 3 female 6-month-old native Chinese Landrance Duroc pigs. Real-time images of tissue layers were obtained with needle insertion. Ultrasound imaging of the OCT needle probe was also performed at the same time for needle trajectory guidance. After imaging, the OCT probe was removed, and 5 mL of normal saline was injected via the needle to confirm correct fascia plane identification. RESULTS: In and ex vivo studies showed clear visual distinction of muscle, fascia, and interfascial layer with OCT, with limitations. Independent validation of OCT criteria for the muscle/fascia differentiation by 20 OCT readers for the in vivo data demonstrated the sensitivity = 0.91, specificity = 0.90, and accuracy = 0.89. Although the angle of needle entry affected the depth of OCT penetration in the muscle, the attenuation coefficient values of the fascia and muscle tissue were statistically different (P < .001) and with high area under the receiver operating characteristics (ROC) curve (AUC) (AUC = 0.93 in 0°-30° and AUC = 1 in 30°-60°) for fascia identification. CONCLUSIONS: This study introduced a novel needle imaging probe method to identify the transversus abdominis fascia plane in real-time. Quantitative calculation of the attenuation coefficients can further aid objective identification by providing direct confirmation of the tip position, increasing the first-pass success rate, and decreasing the need for needle repositioning. Combining OCT and ultrasound may improve the accuracy of anesthetics placement.

Detection of Oral Dysplastic and Early Cancerous Lesions by Polarization-Sensitive Optical Coherence Tomography.

Detection of oral dysplastic and early-stage cancerous lesions is difficult with the current tools. Half of oral cancers are diagnosed in a late stage. Detection of early stromal change to predict malignant transformation is a new direction in the diagnosis of early-stage oral cancer. The application of new optical tools to image stroma in vivo is under investigation, and polarization-sensitive optical coherence tomography (PS-OCT) is potentially one of those tools. This is a preliminary study to sequentially image oral stromal changes from normal, hyperplasia, and dysplasia to early-stage cancer by PS-OCT in vivo. We used 4-Nitroquinoline-1-oxide drinking water to induce dysplasia and early-stage oral cancer in 19 K14-EGFP-miR-211-GFP transgenic mice. A total of 8 normal, 12 hyperplastic, 11 dysplastic, and 4 early-stage cancerous lesions were enrolled. A new analytic process of PS-OCT imaging was proposed, called an en-face birefringence map. From the birefringence map, the sensitivity, specificity, positive predictive value, and negative predictive values to detect dysplasia and early-stage cancer were 100.00%, 95.00%, 93.75%, and 100.00%, respectively, and the kappa value of these images between two investigators was 0.942. The mean size of malignant lesions detected in this study is 1.66 ± 0.93 mm. This pilot animal study validates the use of PS-OCT to detect small and early-stage oral malignancy with high accuracy and consistency.

Lymphatic vessel segmentation in optical coherence tomography by adding U-Net-based CNN for artifact minimization.

The lymphatic system branches throughout the body to transport bodily fluid and plays a key immune-response role. Optical coherence tomography (OCT) is an emerging technique for the noninvasive and label-free imaging of lymphatic capillaries utilizing low scattering features of the lymph fluid. Here, the proposed lymphatic segmentation method combines U-Net-based CNN, a Hessian vesselness filter, and a modified intensity-thresholding to search the nearby pixels based on the binarized Hessian mask. Compared to previous approaches, the method can extract shapes more precisely, and the segmented result contains minimal artifacts, achieves the dice coefficient of 0.83, precision of 0.859, and recall of 0.803.

Quantification of structural and microvascular changes for diagnosing early-stage oral cancer.

Changes in mucosal microvascular networks, called intraepithelial papillary capillary loops (IPCL), are an important key factor for diagnosing early-stage oral cancer in vivo. Nevertheless, there are a lack of tools to quantify these changes objectively. This is the first study to quantify the IPCL changes in vivo to differentiate benign or malignant oral lesions by the optical coherence tomography (OCT) technique. K14-EGFP-miR-211-GFP transgenic mice were inducted by 4-Nitroquinoline-1-oxide to produce oral carcinogenesis in different stages, including normal, premalignancy and cancer. The results showed significant differentiation between benign or malignant lesions by OCT quantitative parameters, including epithelial thickness, IPCL density, radius and tortuosity.

Elucidation of microstructural changes in leaves during senescence using spectral domain optical coherence tomography.

Leaf senescence provides a unique window to explore the age-dependent programmed degradation at organ label in plants. Here, spectral domain optical coherence tomography (SD-OCT) has been used to study in vivo senescing leaf microstructural changes in the deciduous plant Acer serrulatum Hayata. Hayata leaves show autumn phenology and change color from green to yellow and finally red. SD-OCT image analysis shows distinctive features among different layers of the leaves; merging of upper epidermis and palisade layers form thicker layers in red leaves compared to green leaves. Moreover, A-scan analysis showed a significant (p < 0.001) decrease in the attenuation coefficient (for wavelength range: 1100-1550 nm) from green to red leaves. In addition, the B-scan analysis also showed significant changes in 14 texture parameters extracted from second-order spatial gray level dependence matrix (SGLDM). Among these parameters, a set of three features (energy, skewness, and sum variance), capable of quantitatively distinguishing difference in the microstructures of three different colored leaves, has been identified. Furthermore, classification based on k-nearest neighbors algorithm (k-NN) was found to yield 98% sensitivity, 99% specificity, and 95.5% accuracy. Following the proposed technique, a portable noninvasive tool for quality control in crop management can be anticipated.

Direct mineralogical imaging of economic ore and rock samples with multi-modal nonlinear optical microscopy.

Multi-modal nonlinear optical (NLO) microscopy, including stimulated Raman scattering (SRS) and second harmonic generation (SHG), was used to directly image mineralogical features of economic ore and rock samples. In SRS/SHG imaging, ore samples generally require minimal preparation and may be rapidly imaged, even in their wet state. 3D structural details, at submicron resolution, are revealed tens of microns deep within samples. Standard mineral imaging based on scanning electron microscopy (SEM), with elemental analysis via energy dispersive X-Ray spectroscopy, was used to independently validate the mineral composition of the samples. Spatially-resolved SRS from dominant Raman-resonant bands precisely maps the locations of specific minerals contained within the samples. SHG imaging reveals locally non-centrosymmetric structures, such as quartz grains. Competing absorption and nonlinear scattering processes, however, can reduce contrast in SRS imaging. Importantly, the correlation between standard electron microscopy and multi-modal NLO optical microscopy shows that the latter offers rapid image contrast based on the mineral content of the sample.

Intelligent epidural needle placement using fiber-probe optical coherence tomography in a piglet model.

Incorrect needle placement during an epidural block causes medical complications such as dural puncture or spinal cord injury. We propose a system combining an optical coherence tomography imaging probe with an automatic identification algorithm to objectively identify the epidural needle-tip position and thus reduce complications during epidural needle insertion. Eight quantitative features were extracted from each two-dimensional optical coherence tomography image during insertion of the needle tip from the skin surface to the epidural space. 847 in vivo optical coherence tomography images were obtained from three anesthetized piglets. The area under the receiver operating characteristic curve was used to quantify the discriminative ability of each feature. We found a combination of six image features-mean value of intensity, mean value with depth, entropy, mean absolute deviation, root mean square, and standard deviation-showed the highest differentiating performance with the shortest processing time. Finally, differentiation of the needle tip inside or outside the epidural space was automatically evaluated using five classifiers: k-nearest neighbor, linear discriminant analysis, quadratic discriminant analysis, linear support vector machines, and quadratic support vector machine. We adopted an 8-fold cross-validation strategy with five classifications. Quadratic support vector machine classification showed the highest sensitivity (97.5%), specificity (95%), and accuracy (96.2%) among the five classifiers. This study provides an intelligent method for objective identification of the epidural space that can increase the success rate of epidural needle insertion.

Combining Microbubble Contrast Agent with Pulsed-Laser Irradiation for Transdermal Drug Delivery.

The optodynamic process of laser-induced microbubble (MB) cavitation in liquids is utilized in various medical applications. However, how incident laser radiation interacts with MBs as an ultrasound contrast agent is rarely estimated when the liquid already contains stable MBs. The present study investigated the efficacy of the laser-mediated cavitation of albumin-shelled MBs in enhancing transdermal drug delivery. Different types and conditions of laser-mediated inertial cavitation of MBs were first evaluated. A CO2 fractional pulsed laser was selected for combining with MBs in the in vitro and in vivo experiments. The in vitro skin penetration by ß-arbutin after 2 h was 2 times greater in the group combining a laser with MBs than in the control group. In small-animal experiments, the whitening effect on the skin of C57BL/6J mice in the group combining a laser with MBs on the skin plus penetrating ß-arbutin increased (significantly) by 48.0% at day 11 and 50.0% at day 14, and then tended to stabilize for the remainder of the 20-day experimental period. The present results indicate that combining a CO2 laser with albumin-shelled MBs can increase skin permeability so as to enhance the delivery of ß-arbutin to inhibit melanogenesis in mice without damaging the skin.

Combination of structural and vascular optical coherence tomography for differentiating oral lesions of mice in different carcinogenesis stages.

Differentiating between early malignancy and benign lesions in oral cavities is difficult using current optical tools. As has been shown in previous studies, microvascular changes in squamous epithelium can be regarded as a key marker for diagnosis. We propose the combination of structural and vascular optical coherence tomography (OCT) imaging for the investigation of disease related changes. Progressive thickness changes of epithelium and the destruction of underlying lamina propria was observed during cancer development in a 4- nitroquinoline-1-oxide (4NQO) mouse model. At the same time, microvascular changes in hyperplasia, dysplasia, carcinoma in situ and advanced cancer were observed. Findings from OCT imaging were compared with histology.

The use of intensity-based Doppler variance method for single vessel response to functional neurovascular activation.

This study presents 1 use of optical coherence tomography (OCT) angiography technique to examine neurovascular coupling effect. Repeated B-scans OCT recording is performed on the rat somatosensory cortex with cranial window preparation while its contralateral forepaw is electrically stimulated to activate the neurons in rest. We use an intensity-based Doppler variance (IBDV) algorithm mapped cerebral blood vessels in the cortex, and the temporal alteration in blood perfusion during neurovascular activation is analyzed using the proposed IBDV quantitative parameters. By using principal component analysis-based Fuzzy C Means clustering method, the stimulus-evoked vasomotion patterns were classified into 3 categories. We found that the response time of small vessels (resting diameter 14.9 ±6.6 µm), middle vessels (resting diameter 21.1 ±7.9 µm) and large vessels (resting diameter 50.7 ±6.5 µm) to achieve 5% change of vascular dilation after stimulation was 1.5, 2 and 5.5 seconds, respectively. Approximately 5% peak change of relative blood flow (RBF) in both small and middle vessels was observed. The large vessels react slowly and their responses nearly 4 seconds delayed, but no significant change in RBF of the large vessels was seen.

Supercontinuum source-based multi-contrast optical coherence tomography for rat retina imaging.

This study proposed an ultrahigh-resolution multi-contrast optical coherence tomography system integrated with fundus photography for in vivo retinal imaging of rodents. A supercontinuum light source was used in the system, providing an axial resolution of less than 3 µm within 1.8 mm (in the tissue). Three types of tissue contrast based on backscattered intensity, phase retardation, and microvasculature at a capillary level can be simultaneously obtained using the proposed system. Pigmented Long-Evans, non-pigmented (albino) Sprague Dawley, and Royal College of Surgeons rats were imaged and compared. In vivo imaging results were validated with histology.

In vivo images of the epidural space with two- and three-dimensional optical coherence tomography in a porcine model.

BACKGROUND: No reports exist concerning in vivo optical coherence tomography visualization of the epidural space and the blood patch process in the epidural space. In this study, we produced real-time two-dimensional and reconstructed three-dimensional images of the epidural space by using optical coherence tomography in a porcine model. We also aimed to produce three-dimensional optical coherence tomography images of the dura puncture and blood patch process. METHODS: Two-dimensional and three-dimensional optical coherence tomography images were obtained using a swept source optical coherence tomography (SSOCT) system. Four laboratory pigs were intubated and ventilated after the induction of general anesthesia. An 18-gauge Tuohy needle was used as a tunnel for the optical coherence tomography probe to the epidural space. Two-dimensional and three-dimensional reconstruction optical coherence tomography images of the epidural space were acquired in four stages. RESULTS: In stage 1, real-time two-dimensional and reconstructed three-dimensional optical coherence tomography of the lumbar and thoracic epidural space were successfully acquired. In stage 2, the epidural catheter in the epidural space was successfully traced in the 3D optical coherence tomography images. In stage 3, water injection and lumbar puncture were successfully monitored in all study animals. In stage 4, 10 mL of fresh blood was injected into the epidural space and two-dimensional and three-dimensional optical coherence tomography images were successfully acquired. CONCLUSIONS: These animal experiments suggest the potential capability of using an optical coherence tomography-based imaging needle in the directed two-dimensional and three-dimensional visualization of the epidural space. More investigations involving humans are required before optical coherence tomography can be recommended for routine use. However, three-dimensional optical coherence tomography may provide a novel, minimally invasive, and safe way to observe the spinal epidural space, epidural catheter, lumbar puncture hole, and blood patch.