Second harmonic generation microscopy of electromechanical reshaping on corneal collagen.

Refractive errors remain a global health concern, as a large proportion of the world's population is myopic. Current ablative approaches are costly, not without risks, and not all patients are candidates for these procedures. Electromechanical reshaping (EMR) has been explored as a viable cost-effective modality to directly shape tissues, including cartilage. In this study, stromal collagen structure and fibril orientation was examined before and after EMR with second-harmonic generation microscopy (SHG), a nonlinear multiphoton imaging method that has previously been used to study native corneal collagen with high spatial resolution. EMR, using a milled metal contact lens and potentiostat, was performed on the corneas of five extracted rabbit globes. SHG was performed using a confocal microscopy system and all images underwent collagen fibril orientation analysis. The collagen SHG signal in controls is uniform and is similarly seen in samples treated with pulsed potential, while continuous EMR specimens have reduced, nonhomogeneous signal. Collagen fibril orientation in native tissue demonstrates a broad distribution with suggestion of another peak evolving, while with EMR treated eyes a bimodal characteristic becomes readily evident. Pulsed EMR may be a means to correct refractive errors, as when comparing its SHG signal to negative control, preservation of collagen structures with little to no damage is observed. From collagen fiber orientation analysis, it can be inferred that simple DC application alters the structure of collagen. Future studies will involve histological assessment of these layers and multi-modal imaging analysis of dosimetry.

In vivo electrochemical lipolysis of fat in a Yucatan pig model: A proof of concept study.

OBJECTIVES: Traditional fat contouring is now regularly performed using numerous office- based less invasive techniques. However, some limitations of these minimally invasive techniques include high cost or limited selectivity with performing localized contouring and reduction of fat. These shortcomings may potentially be addressed by electrochemical lipolysis (ECLL), a novel approach that involves the insertion of electrodes into tissue followed by application of a direct current (DC) electrical potential. This results in the hydrolysis of tissue water creating active species that lead to fat necrosis and apoptosis. ECLL can be accomplished using a simple voltage-driven system (V-ECLL) or a potential-driven feedback cell (P-ECLL) both leading to water electrolysis and the creation of acid and base in situ. The aim of this study is to determine the long-lasting effects of targeted ECLL in a Yucatan pig model. METHODS: A 5-year-old Yucatan pig was treated with both V-ECLL and P-ECLL in the subcutaneous fat layer using 80:20 platinum:iridium needle electrodes along an 8 cm length. Dosimetry parameters included 5 V V-ECLL for 5, 10, and 20 minutes, and -1.5 V P-ECLL, -2.5 V P-ECLL, -3.5 V P-ECLL for 5 minutes. The pig was assessed for changes in fat reduction over 3 months with digital photography and ultrasound. After euthanasia, tissue sections were harvested and gross pathology and histology were examined. RESULTS: V-ECLL and P-ECLL treatments led to visible fat reduction (12.1%-27.7% and 9.4%-40.8%, respectively) and contour changes across several parameters. An increased reduction of the superficial fat layer occurred with increased dosimetry parameters with an average charge transfer of 12.5, 24.3, and 47.5 C transferred for 5 V V-ECLL for 5, 10, and 20 minutes, respectively, and 2.0, 11.5, and 24.0 C for -1.5 V P-ECLL, -2.5 V P-ECLL, -3.5 V P-ECLL for 5 minutes, respectively. These dose-dependent changes were also evidenced by digital photography, gross pathology, ultrasound imaging, and histology. CONCLUSIONS: ECLL results in selective damage and long-lasting changes to the adipose layer in vivo. These changes are dose-dependent, thus allowing for more precise contouring of target areas. P-ECLL has greater efficiency and control of total charge transfer compared to V-ECLL, suggesting that a low-voltage potentiostat treatment can result in fat apoptosis equivalent to a high-voltage DC system.

Exploring feedback-controlled versus open-circuit electrochemical lipolysis in ex vivo and in vivo porcine fat: A feasibility study.

OBJECTIVES: Minimally invasive fat sculpting techniques are becoming more widespread with the development of office-based devices and therapies. Electrochemical lipolysis (ECLL) is a needle-based technology that uses direct current (DC) to electrolyze tissue water creating acid and base in situ. In turn, fat is saponified and adipocyte cell membrane lysis occurs. The electrolysis of water can be accomplished using a simple open-loop circuit (V-ECLL) or by incorporating a feedback control circuit using a potentiostat (P-ECLL). A potentiostat utilizes an operational amplifier with negative feedback to allow users to precisely control voltage at specific electrodes. To date, the variation between the two approaches has not been studied. The aim of this study was to assess current and charge transfer variation and lipolytic effect created by the two approaches in an in vivo porcine model. METHODS: Charge transfer measurements from ex vivo V-ECLL and P-ECLL treated porcine skin and fat were recorded at -1 V P-ECLL, -2 V P-ECLL, -3 V P-ECLL, and -5 V V-ECLL each for 5 min to guide dosimetry parameters for in vivo studies. In follow-up in vivo studies, a sedated female Yorkshire pig was treated with both V-ECLL and P-ECLL across the dorsal surface over a range of dosimetry parameters, including -1.5 V P-ECLL, -2.5 V P-ECLL, -3.5 V P-ECLL, and 5 V V-ECLL each treated for 5 min. Serial biopsies were performed at baseline before treatment, 1, 2, 7, 14, and 28 days after treatment. Tissue was examined using fluorescence microscopy and histology to compare the effects of the two ECLL approaches. RESULTS: Both V-ECLL and P-ECLL treatments induced in-vivo fat necrosis evident by adipocyte membrane lysis, adipocyte denuclearization, and an acute inflammatory response across a 28-day longitudinal study. However, -1.5 V P-ECLL produced a smaller spatial necrotic effect compared to 5 V V-ECLL. In addition, 5 V V-ECLL produced a comparable necrotic effect to that of -2.5 V and -3.5 V P-ECLL. CONCLUSIONS: V-ECLL and P-ECLL at the aforementioned dosimetry parameters both achieved fat necrosis by adipocyte membrane lysis and denuclearization. The -2.5 V and -3.5 V P-ECLL treatments created spatially similar fat necrotic effects when compared to the 5 V V-ECLL treatment. Quantitatively, total charge transfer between dosimetry parameters suggests that -2.5 V P-ECLL and 5 V V-ECLL produce comparable electrochemical reactions. Such findings suggest that a low-voltage closed-loop potentiostat-based system is capable of inducing fat necrosis to a similar extent compared to that of a higher voltage direct current system.

Costal Cartilage Considerations: Novel Use of Handheld Ultrasound Device in Rhinoplasty.

Handheld ultrasound devices can be used in revision rhinoplasty to evaluate the calcification of costal rib cartilage that is to be harvested for grafting. This article provides instructions on how to perform this technique. Laryngoscope, 134:651-653, 2024.

Boomerang Modification of the Septal Extension Graft: Graft Design and Functional Outcomes.

Background: A "boomerang" graft is an end-to-end caudal septal extension graft (SEG) that conforms to the geometry of the anterior septal angle, and avoids septal overlap, unlike a side-to-side SEG. Objective: To compare breathing improvements in rhinoplasty patients receiving boomerang SEGs and patients receiving side-to-side SEGs. Methods: Retrospective cohort analysis of patients undergoing rhinoplasty with either end-to-end boomerang SEG or a side-to-side SEG. Functional outcomes were assessed through the Nasal Obstruction Symptom Evaluation (NOSE) survey. Results: The boomerang SEG cohort had a mean age of 34 years and were 68% female compared with 38 years and 67% female in the side-to-side SEG cohort (p > 0.05). The cohorts did not differ in the proportion of the lateral crural tensioning, spreader graft placement, or history of rhinoplasty. The boomerang cohort demonstrated a 67% reduction in NOSE scores compared with a 70% reduction among the side-to-side SEG cohort (p = 0.14). Men undergoing boomerang graft placement reported significantly less postoperative functional improvement than men undergoing placement of a side-to-side SEG (62% vs. 77%, p = 0.01). Conclusion: Use of a boomerang graft is not likely to negatively affect rhinoplasty functional outcomes when compared with a side-to-side SEG.

Impact of Tissue Handling and Size Modification on Septal Chondrocyte Viability.

INTRODUCTION: The physical modification of cartilage grafts during rhinoplasty risks chondrocyte death at the margins where the tissue is cut. This study compares chondrocyte viability between diced, scaled, and pate samples in human models, and further computes percent chondrocyte viability as a function of sequential dicing size in a computational model. METHODS: Septal cartilage from 11 individuals was prepared as follows: diced (1 mm cubic), scaled (shaved to <1 mm thickness ~ translucent), pate (0.02 g of scraped cartilage surface), positive control (2 × 2 mm diced), and negative control (2 × 2 mm diced soaked in 70% EtOH). Viability analysis was performed using Live/Dead assay™ and confocal microscopy. Numerical simulation of cartilage dicing in 0.05 mm increments was performed using MATLAB assuming 250 chondrocytes/mm3 with each average chondrocyte size of 65 µm2. RESULTS: Chondrocyte viability was similar between 1 mm diced cartilage, scaled cartilage, and positive control samples (p > 0.05). Conversely, pate samples had significantly less viability compared to positive controls, diced samples, and scaled samples (all p < 0.01 after Bonferroni correction). Pate samples had similar chondrocyte viability compared to negative controls (p = 0.36). On computational modeling, cartilage viability decreased to 50% as the diced sample was cut from 1 mm edge length to 0.7-0.8 mm. Similarly, cartilage viability decreased to 26% at 0.55-0.65 mm, 11% at 0.4-0.5 mm, and <5% at <0.4 mm edge length. CONCLUSION: Modifying septal cartilage grafts into 1 mm diced or scaled samples maintains ideal chondrocyte viability whereas pate preparations result in significant chondrocyte death. According to computational analysis, chondrocyte viability sharply decreases as the cartilage is diced below 0.7-0.8 mm. LEVEL OF EVIDENCE: N/A Laryngoscope, 2024.

Long-range optical coherence tomography of pediatric airway during drug induced sleep endoscopy: A preliminary report.

OBJECTIVE: Drug induced sleep endoscopy (DISE) is often performed for pediatric obstructive sleep apnea (OSA) when initial diagnostic studies do not provide adequate information for therapy. However, DISE scoring is subjective and with limitations. This proof-of-concept study demonstrates the use of a novel long-range optical coherence tomography (LR-OCT) system during DISE of two pediatric patients. METHODS: LR-OCT was used to visualize the airway of pediatric patients during DISE. At the conclusion of DISE, the OCT probe was guided in the airway under endoscopic visual guidance, and cross-sectional images were acquired at the four VOTE locations. Data processing involved image resizing and alignment, followed by rendering of three-dimensional (3D) volumetric models of the airways. RESULTS: Two patients were included in this study. Patient one had 18.4%, 20.9%, 72.3%, and 97.3% maximal obstruction at velum, oropharynx, tongue base, and epiglottis, while patient two had 40.2%, 41.4%, 8.0%, and 17.5% maximal obstruction at these regions, respectively. Three-dimensional reconstructions of patients' airways were also constructed from the OCT images. CONCLUSION: This proof-of-concept study demonstrates the successful evaluation of pediatric airway during DISE using LR-OCT, which accurately identified sites and degrees of obstruction with respective 3D airway reconstruction.

Temperature Profile Measurement From Radiofrequency Nasal Airway Reshaping Device.

OBJECTIVE: Nasal airway obstruction (NAO) is caused by various disorders including nasal valve collapse (NVC). A bipolar radiofrequency (RF) device (VivAer®, Aerin Medical, Sunnyvale, CA) has been used to treat NAO through RF heat generation to the upper lateral cartilage (ULC). The purpose of this study is to measure temperature elevations in nasal tissue, using infrared (IR) radiometry to map the spatial and temporal evolution of temperature. STUDY DESIGN: Experimental and computational. METHODS: Composite porcine nasal septum was harvested and sectioned (1 mm and 2 mm). The device was used to heat the cartilage in composite porcine septum. An IR camera (FLIR® ExaminIR, Teledyne, Wilsonville, OR) was used to image temperature on the back surface of the specimen. These data were incorporated into a heat transfer finite element model that also calculated tissue damage using Arrhenius rate process. RESULTS: IR temperature imaging showed peak back surface temperatures of 49.57°C and 42.21°C in 1 and 2 mm thick septums respectively. Temperature maps were generated demonstrating the temporal and spatial evolution of temperature. A finite element model generated temperature profiles with respect to time and depth. Rate process models using Arrhenius coefficients showed 30% chondrocyte death at 1 mm depth after 18 s of RF treatment. CONCLUSION: The use of this device creates a thermal profile that may result in thermal injury to cartilage. Computational modeling suggests chondrocyte death extending as deep as 1.4 mm below the treatment surface. Further studies should be performed to improve dosimetry and optimize the heating process to reduce potential injury. Laryngoscope, 134:1063-1070, 2024.

Electrochemical Lipolysis Induces Adipocyte Death and Fat Necrosis: In Vivo Pilot Study in Pigs.

BACKGROUND: Current minimally invasive fat reduction modalities use equipment that can cost thousands of U.S. dollars. Electrochemical lipolysis (ECLL), using low-cost battery and electrodes (approximately $10), creates acid/base within fat (width, approximately 3 mm), damaging adipocytes. Longitudinal effects of ECLL have not been studied. In this pilot study, the authors hypothesize that in vivo ECLL induces fat necrosis, decreases adipocyte number/viability, and forms lipid droplets. METHODS: Two female Yorkshire pigs (50 to 60 kg) received ECLL. In pig 1, 10 sites received ECLL, and 10 sites were untreated. In pig 2, 12 sites received ECLL and 12 sites were untreated. For ECLL, two electrodes were inserted into dorsal subcutaneous fat and direct current was applied for 5 minutes. Adverse effects of excessive pain, bleeding, infection, and agitation were monitored. Histology, live-dead (calcein, Hoechst, ethidium homodimer-1), and morphology (Bodipy and Hoechst) assays were performed on day 0 and postprocedure days 1, 2, 7, 14 (pig 1 and pig 2), and 28 (pig 2). Average particle area, fluorescence signal areas, and adipocytes and lipid droplet numbers were compared. RESULTS: No adverse effects occurred. Live-dead assays showed adipocyte death on the anode on days 0 to 7 and the cathode on days 1 to 2 (not significant). Bodipy showed significant adipocyte loss at all sites ( P < 0.001) and lipid droplet formation at the cathode site on day 2 ( P = 0.0046). Histology revealed fat necrosis with significant increases in average particle area at the anode and cathode sites by day 14 (+277.3% change compared with untreated, P < 0.0001; +143.4%, P < 0.0001) and day 28 (+498.6%, P < 0.0001; +354.5%, P < 0.0001). CONCLUSIONS: In vivo ECLL induces fat necrosis in pigs. Further studies are needed to evaluate volumetric fat reduction. CLINICAL RELEVANCE STATEMENT: In vivo ECLL induces adipocyte death and fat necrosis. ECLL has the potential to be utilized in body fat contouring.

Second Harmonic Imaging of Nasal, Auricular, and Costal Cartilage.

OBJECTIVE: There is little knowledge about the histological organization of facial and costal cartilages in terms of matrix structure and cell morphology. Second harmonic generation (SHG) imaging is a nonlinear imaging technique that capitalizes on signal generation from highly ordered macromolecules such as collagen fibers. The purpose of this study was to use SHG microscopy to image collagen extracellular matrix (ECM) structure, chondrocyte size, and density of these cartilages. STUDY DESIGN: Experimental. METHODS: Surgical remnants of septal, lower lateral, rib, and auricular cartilages were collected following surgery, sectioned into 0.5-1 mm thick samples and fixed to facilitate batch process imaging. A Leica TCS SP8 MP Microscope and multiphoton laser were used to image the specimens. Images were analyzed for cell size, cell density, and collagen fiber directionality patterns using ImageJ. RESULTS: SHG images of septal specimens show mesh-like structure of the ECM. There appears to be a superficial layer, characterized by flattened lacunae and middle zone, marked by circular lacunae clusters, similar to what is observed in articular cartilage. The structure of the ECM depicts a visible orientation perpendicular to the surface of the perichondrium. Cell size and density analysis through ImageJ suggests variety across cartilage types. Directionality analysis indicates that the collagen in the ECM displays preferred direction. CONCLUSION: This study establishes clear extracellular models of facial and costal cartilages. Limitations include heterogeneous cartilage thickness due to processing difficulties. Further studies include automating the cutting process to increase uniformity of tissue thickness and increasing sample size to further validate results. LEVEL OF EVIDENCE: 2 Laryngoscope, 133:3370-3377, 2023.

Racial and ethnic disparities in the presentation size and timing of pituitary adenomas resected via endoscopic endonasal approach.

KEY POINTS: In a single-center cohort of pituitary adenoma patients, non-White race independently predicted larger tumor size at initial presentation. Uninsured patients suffered a significantly higher rate of pituitary apoplexy at initial presentation. Geographically distant care appeared to present a greater barrier for non-White and Hispanic patients relative to their White and non-Hispanic counterparts.

Electromechanical Cornea Reshaping for Refractive Vision Therapy.

The corneal stroma consists of orthogonally stacked collagen-fibril lamellae that determine the shape of the cornea and provide most of the refractive power of the eye. We have applied electromechanical reshaping (EMR), an electrochemical platform for remodeling cartilage and other semirigid tissues, to change the curvature of the cornea as a potential procedure for nonsurgical vision correction. EMR relies on short electrochemical pulses to electrolyze water, with subsequent diffusion of protons into the extracellular matrix of collagenous tissues; protonation of immobilized anions within this matrix disrupts the ionic-bonding network, leaving the tissue transiently responsive to mechanical remodeling. Re-equilibration to physiological pH restores the ionic matrix, resulting in persistent shape change of the tissue. Using ex vivo rabbit eyes, we demonstrate here the controlled change of corneal curvature over a wide range of refractive powers with no loss of optical transparency. Optical coherence tomography (OCT), combined with second-harmonic generation (SHG) and confocal microscopy, establish that EMR enables extremely fine control of corneal contouring while maintaining the underlying macromolecular collagen structure and stromal cellular viability, positioning electrochemical vision therapy as a potentially simple and ultralow-cost modality for correcting routine refractive errors.

Developing the Optimal Osteotome Hand-Sharpening Method.

Background: Rhinoplasty osteotomes can be sharpened in various ways: professional sharpening or hand sharpening using whetstones or rotary powered devices. Objective: To compare the effectiveness of sharpening osteotomes using various sharpening methods with that of professional sharpening as measured by a custom edge tester. Materials and Methods: We performed repeated serial osteotome impacts on bovine femoral cortical bone. These dull osteotomes were sharpened using preidentified sharpening techniques. Edge morphology was evaluated. Sharpness was tested using a custom mechanical testing platform. Optimized sharpness was achieved with a whetstone sharpening method wherein the osteotome is flipped after every stroke. Results: Seven distinct sharpening methods were tested for sharpness five times each to determine the optimal sharpening method versus professional sharpening (control). The two sharpening methods, 5 (5.51 ± 0.32) and 6 (5.55 ± 0.32), that used this flipping technique were significantly sharper than other methods. Methods 5 (p = 1.0) and 6 (p = 1.0) were the only methods that were not significantly different from control. Conclusion: Single stroke with successively alternating surfaces created the sharpest blades that achieved results similar to professional sharpening.

Potential-Driven Electrochemical Clearing of Ex Vivo Alkaline Corneal Injuries.

Purpose: Corneal chemical injuries (CCI) obscure vision by opacifying the cornea; however, current treatments may not fully restore clarity. Here, we investigated potential-driven electrochemical treatment (P-ECT) to restore clarity after alkaline-based CCI in ex vivo rabbit corneas and examined collagen fiber orientation changes using second harmonic generation (SHG). Methods: NaOH was applied to the corneas of intact New Zealand white rabbit globes. P-ECT was performed on the opacified cornea while optical coherence tomography (OCT) imaging (∼35 frames per second) was simultaneously performed. SHG imaging evaluated collagen fiber structure before NaOH application and after P-ECT. Irrigation with water served as a control. Results: P-ECT restored local optical clarity after NaOH exposure. OCT imaging shows both progression of NaOH injury and the restoration of clarity in real time. Analysis of SHG z-stack images show that collagen fibril orientation is similar between control, NaOH-damaged, and post-P-ECT corneas. NaOH-injured corneas flushed with water (15 minutes) show no restoration of clarity. Conclusions: P-ECT may be a means to correct alkaline CCI. Collagen fibril orientation does not change after NaOH exposure or P-ECT, suggesting that no irreversible matrix level fiber changes occur. Further studies are required to determine the mechanism for corneal clearing and to ascertain the optimal electrical dosimetry parameters and electrode designs. Translational Relevance: Our findings suggest that P-ECT is a potentially effective, low-cost treatment for alkaline CCI.