Development and characterization of transfontanelle photoacoustic imaging system for detection of intracranial hemorrhages and measurement of brain oxygenation: Ex-vivo.

We have developed and optimized an imaging system to study and improve the detection of brain hemorrhage and to quantify oxygenation. Since this system is intended to be used for brain imaging in neonates through the skull opening, i.e., fontanelle, we called it, Transfontanelle Photoacoustic Imaging (TFPAI) system. The system is optimized in terms of optical and acoustic designs, thermal safety, and mechanical stability. The lower limit of quantification of TFPAI to detect the location of hemorrhage and its size is evaluated using in-vitro and ex-vivo experiments. The capability of TFPAI in measuring the tissue oxygenation and detection of vasogenic edema due to brain blood barrier disruption are demonstrated. The results obtained from our experimental evaluations strongly suggest the potential utility of TFPAI, as a portable imaging modality in the neonatal intensive care unit. Confirmation of these findings in-vivo could facilitate the translation of this promising technology to the clinic.

Three-dimensional mapping of the greater palatine artery location and physiology.

OBJECTIVE: To develop a novel technique for localizing and reconstructing the greater palatine artery (GPA) using three-dimensional (3D) technology. METHODS: A miniaturized intraoral ultrasound transducer was used to imaging landmarks including the GPA, gingival margin (GM), and palatal masticatory mucosa (PMM). A 5-mm-thick solid hydrogel couplant was integrated to replace traditional ultrasound gel and avoid bubbles when moving the transducer. RESULTS: A panorama image provided the relative localization of landmarks including the GPA, PMM, and hard palate. Short- and long-axis imaging of GPA was performed in five subjects including 3D mapping of GPA branches and surrounding tissues in a volume of 10 mm × 8 mm × 10 mm. Full-mouth Doppler imaging was also demonstrated on both the dorsal and ventral tongue as well as buccal mucosa and sublingual region on two subjects. CONCLUSIONS: This study can measure the vertical distance from the GM to the GPA and depth from PMM to GPA and visualize the GPA localization in a 3D manner, which is critical to evaluate the available volume of palatal donor tissues and avoid sectioning of GPA during surgical harvesting of the tissues. Finally, the transducer's small size facilitates full-mouth Doppler imaging with the potential to improve the assessment, diagnosis, and management of oral mucosa.

A miniaturized ultrasound transducer for monitoring full-mouth oral health: a preliminary study.

OBJECTIVE: To customize a miniaturized ultrasound transducer to access full-mouth B-mode, color Doppler, and spectral Doppler imaging for monitoring oral health. METHODS: A customized periodontal ultrasound transducer SS-19-128 (19 MHz, 128 channels) 1.8-cm wide and 1-cm thick was developed and connected to a data acquisition (DAQ) system. B-mode, color Doppler, and spectral Doppler data could all be collected with SS-19-128. The imaging resolution and penetration capacity of SS-19-128 were characterized on phantoms. The gingival thickness was measured on 11 swine teeth by SS-19-128 for comparison with conventional transgingival probing via Bland-Altman analysis and Pearson correlation. Five human subjects were then recruited to demonstrate B-mode and Doppler imaging by SS-19-128. RESULTS: The axial and lateral spatial resolution at 5.5 mm depth is 102.1 µm and 142.9 µm, respectively. The penetration depth in a tissue-mimicking phantom is over 30 mm. In vivo B-mode imaging of all 28 teeth was demonstrated on one human subject, and imaging of tooth #18 was accessed on five human subjects. Gingival thickness measurement compared with transgingival probing showed a bias of -0.015 mm and SD of 0.031 mm, and a r = 0.9235 (p < 0.0001) correlation. In vivo color and spectral Doppler imaging of the supraperiosteal artery in human gingiva was performed to generate hemodynamic information. CONCLUSIONS: The small size of SS-19-128 offers important advantages over existing ultrasound technology-more specifically, whole-mouth scanning/charting reminiscent of radiography. This is nearly a two-fold increase in the number of teeth that can be assessed versus conventional transducers.

Venglustat, a Novel Glucosylceramide Synthase Inhibitor, in Patients at Risk of Rapidly Progressing ADPKD: Primary Results of a Double-Blind, Placebo-Controlled, Phase 2/3 Randomized Clinical Trial.

RATIONALE & OBJECTIVE: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of multiple kidney cysts that leads to growth in total kidney volume (TKV) and progression to kidney failure. Venglustat is a glucosylceramide synthase inhibitor that has been shown to inhibit cyst growth and reduce kidney failure in preclinical models of ADPKD. STUDY DESIGN: STAGED-PKD was a 2-stage, multicenter, double-blind, randomized, placebo-controlled phase 2/3 study in adults with ADPKD at risk of rapidly progressive disease, who were selected based on Mayo Clinic imaging classification of ADPKD class 1C, 1D, or 1E and an estimated glomerular filtration rate (eGFR) of 30-89.9mL/min/1.73m2. SETTING & PARTICIPANTS: Enrollment included 236 and 242 patients in stages 1 and 2, respectively. INTERVENTIONS: In trial stage 1, the patients were randomized 1:1:1 to venglustat, 8mg; venglustat, 15mg; or placebo. In stage 2, the patients were randomized 1:1 to venglustat, 15mg (highest dose identified as safe and well tolerated in stage 1), or placebo. OUTCOMES: Primary end points were rate of change in TKV over 18 months in stage 1 and eGFR slope over 24 months in stage 2. Secondary end points were eGFR slope over 18 months (stage 1), rate of change in TKV (stage 2), and safety/tolerability, pain, and fatigue (stages 1 and 2). RESULTS: A prespecified interim futility analysis showed that venglustat treatment had no effect on the annualized rate of change in TKV over 18 months (stage 1) and had a faster rate of decline in eGFR slope over 24 months (stage 2). Due to this lack of efficacy, the study was terminated early. LIMITATIONS: The short follow-up period after the end of treatment and limited generalizability of the findings. CONCLUSIONS: In patients with rapidly progressing ADPKD, treatment with venglustat at either 8mg or 15mg showed no change in the rate of change in TKV and a faster rate of eGFR decline in STAGED-PKD despite a dose-dependent decrease in plasma glucosylceramide levels. FUNDING: This study was funded by Sanofi. TRIAL REGISTRATION: Registered at ClinicalTrials.gov with study number NCT03523728.

Clinical relevance of globotriaosylceramide accumulation in Fabry disease and the effect of agalsidase beta in affected tissues.

Fabry disease (FD) is a rare lysosomal storage disorder, characterized by a reduction in α-galactosidase A enzyme activity and the progressive accumulation of globotriaosylceramide (GL3) and its metabolites in the cells of various organs. Agalsidase beta, an enzyme replacement therapy (ERT), is approved for use in patients with FD in Europe, Canada, Australia, South America, and Asia, and is the only ERT approved for use in the United States. In this review, we discuss the clinical relevance of GL3 accumulation, the effect of agalsidase beta on GL3 in target tissues, and the association between treatment-related tissue GL3 clearance and long-term structure, function, or clinical outcomes. Accumulation of GL3 in the kidney, heart, vasculature, neurons, skin, gastrointestinal tract and auditory system correlates to cellular damage and irreversible organ damage, as a result of sclerosis, fibrosis, apoptosis, inflammation, and endothelial dysfunction. Damage leads to renal dysfunction and end-stage renal disease; myocardial hypertrophy with heart failure and arrhythmias; ischemic stroke; neuropathic pain; skin lesions; intestinal ischemia and dysmotility; and hearing loss. Treatment with agalsidase beta is effective in substantially clearing GL3 in a range of cells from the tissues affected by FD. Agalsidase beta has also been shown to slow renal decline and lower the overall risk of clinical progression, demonstrating an indirect link between treatment-related GL3 clearance and stabilization of FD.

Posterior photoacoustic/ultrasound imaging of the periodontal pocket with a compact intraoral transducer.

Periodontitis is a public issue and imaging periodontal pocket is important to evaluate periodontitis. Regular linear transducers have limitations in imaging the posterior teeth due to their geometry restrictions. Here we characterized a transducer that can image the posterior teeth including assessment of periodontal pockets via a combination of photoacoustic and ultrasound imaging. Unlike conventional transducer design, this device has a toothbrush-shaped form factor with a side-view transducer to image molars (total size: 1 ×1.9 cm). A laser diode was integrated as the light source to reduce the cost and size and facilitates clinical transition. The in vivo imaging of a molar of a periodontal patient demonstrated that the transducer could image in the posterior area of gum in vivo; the value determined by imaging was within 7 % of the value measured clinically.

The STAGED-PKD 2-Stage Adaptive Study With a Patient Enrichment Strategy and Treatment Effect Modeling for Improved Study Design Efficiency in Patients With ADPKD.

Rationale & Objective: Venglustat, a glucosylceramide synthase inhibitor, inhibits cyst growth and reduces kidney failure in mouse models of autosomal dominant polycystic kidney disease (ADPKD). STAGED-PKD aims to determine the safety and efficacy of venglustat and was designed using patient enrichment for progression to end-stage kidney disease and modeling from prior ADPKD trials. Study Design: STAGED-PKD is a 2-stage, international, double-blind, randomized, placebo-controlled trial in adults with ADPKD (Mayo Class 1C-1E) and estimated glomerular filtration rate (eGFR) 45-<90 mL/min/1.73 m2 at risk of rapidly progressive disease. Enrichment for rapidly progressing patients was identified based on retrospective analysis of total kidney volume (TKV) and eGFR slope from the combined Consortium for Radiologic Imaging Studies of Polycystic Kidney Disease and HALT Progression of Polycystic Kidney Disease A studies. Setting & Participants: Target enrollment in stages 1 and 2 was 240 and 320 patients, respectively. Interventions: Stage 1 randomizes patients 1:1:1 to venglustat 8 mg or 15 mg once daily or placebo. Stage 2 randomizes patients 1:1 to placebo or venglustat, with the preferred dose based on stage 1 safety data. Outcomes: Primary endpoints are TKV growth rate over 18 months in stage 1 and eGFR slope over 24 months in stage 2. Secondary endpoints include: annualized rate of change in eGFR from baseline to 18 months (stage 1); annualized rate of change in TKV based on magnetic resonance imaging from baseline to 18 months (stage 2); and safety, tolerability, pain, and fatigue (stages 1 and 2). Limitations: If stage 1 is unsuccessful, patients enrolled in the trial may develop drug-related adverse events that can have long-lasting effects. Conclusions: Modeling allows the design and powering of a 2-stage combined study to assess venglustat's impact on TKV growth and eGFR slope. Stage 1 TKV assessment via a nested approach allows early evaluation of efficacy and increased efficiency of the trial design by reducing patient numbers and trial duration. Funding: This study was funded by Sanofi. Trial registration: STAGED-PKD has been registered at ClinicalTrials.gov with study number NCT03523728.

Anti-microRNA-21 Therapy on Top of ACE Inhibition Delays Renal Failure in Alport Syndrome Mouse Models.

Col4a3-/- Alport mice serve as an animal model for renal fibrosis. MicroRNA-21 (miR-21) expression has been shown to be increased in the kidneys of Alport syndrome patients. Here, we investigated the nephroprotective effects of Lademirsen anti-miR-21 therapy. We used a fast-progressing Col4a3-/- mouse model with a 129/SvJ background and an intermediate-progressing F1 hybrid mouse model with a mixed genetic background, with angiotensin-converting enzyme inhibitor (ACEi) monotherapy in combination with anti-miR-21 therapy. In the fast-progressing model, the anti miR-21 and ACEi therapies showed an additive effect in the reduction in fibrosis, the decline of proteinuria, the preservation of kidney function and increased survival. In the intermediate-progressing F1 model, the anti-miR-21 and ACEi therapies individually improved kidney pathology. Both also improved kidney function and survival; however, the combination showed a significant additive effect, particularly for survival. RNA sequencing (RNA-seq) gene expression profiling revealed that the anti-miR-21 and ACEi therapies modulate several common pathways. However, anti-miR-21 was particularly effective at normalizing the expression profiles of the genes involved in renal tubulointerstitial injury pathways. In conclusion, significant additive effects were detected for the combination of anti-miR-21 and ACEi therapies on kidney function, pathology and survival in Alport mouse models, as well as a strong differential effect of anti-miR-21 on the renal expression of fibrotic factors. These results support the addition of anti-miR-21 to the current standard of care (ACEi) in ongoing clinical trials in patients with Alport syndrome.

Aggregation-Enhanced Photoluminescence and Photoacoustics of Atomically Precise Gold Nanoclusters in Lipid Nanodiscs (NANO2).

The authors designed a structurally stable nano-in-nano (NANO2) system highly capable of bioimaging via an aggregation-enhanced NIR excited emission and photoacoustic response achieved based on atomically precise gold nanoclusters protected by linear thiolated ligands [Au25(SC n H2n+1)18, n = 4-16] encapsulated in discoidal phospholipid bicelles through a one-pot synthesis. The detailed morphological characterization of NANO2 is conducted using cryogenic transmission electron microscopy, small/wide angle X-ray scattering with the support of molecular dynamics simulations, providing information on the location of Au nanoclusters in NANO2. The photoluminescence observed for NANO2 is 20-60 times more intense than that of the free Au nanoclusters, with both excitation and emission wavelengths in the near-infrared range, and the photoacoustic signal is more than tripled. The authors attribute this newly discovered aggregation-enhanced photoluminescence and photoacoustic signals to the restriction of intramolecular motion of the clusters' ligands. With the advantages of biocompatibility and high cellular uptake, NANO2 is potentially applicable for both in vitro and in vivo imaging, as the authors demonstrate with NIR excited emission from in vitro A549 human lung and the KB human cervical cancer cells.

Motion-compensated noninvasive periodontal health monitoring using handheld and motor-based photoacoustic-ultrasound imaging systems.

Simultaneous visualization of the teeth and periodontium is of significant clinical interest for image-based monitoring of periodontal health. We recently reported the application of a dual-modality photoacoustic-ultrasound (PA-US) imaging system for resolving periodontal anatomy and periodontal pocket depths in humans. This work utilized a linear array transducer attached to a stepper motor to generate 3D images via maximum intensity projection. This prior work also used a medical head immobilizer to reduce artifacts during volume rendering caused by motion from the subject (e.g., breathing, minor head movements). However, this solution does not completely eliminate motion artifacts while also complicating the imaging procedure and causing patient discomfort. To address this issue, we report the implementation of an image registration technique to correctly align B-mode PA-US images and generate artifact-free 2D cross-sections. Application of the deshaking technique to PA phantoms revealed 80% similarity to the ground truth when shaking was intentionally applied during stepper motor scans. Images from handheld sweeps could also be deshaken using an LED PA-US scanner. In ex vivo porcine mandibles, pigmentation of the enamel was well-estimated within 0.1 mm error. The pocket depth measured in a healthy human subject was also in good agreement with our prior study. This report demonstrates that a modality-independent registration technique can be applied to clinically relevant PA-US scans of the periodontium to reduce operator burden of skill and subject discomfort while showing potential for handheld clinical periodontal imaging.

Polyacrylamide hydrogel phantoms for performance evaluation of multispectral photoacoustic imaging systems.

As photoacoustic imaging (PAI) begins to mature and undergo clinical translation, there is a need for well-validated, standardized performance test methods to support device development, quality control, and regulatory evaluation. Despite recent progress, current PAI phantoms may not adequately replicate tissue light and sound transport over the full range of optical wavelengths and acoustic frequencies employed by reported PAI devices. Here we introduce polyacrylamide (PAA) hydrogel as a candidate material for fabricating stable phantoms with well-characterized optical and acoustic properties that are biologically relevant over a broad range of system design parameters. We evaluated suitability of PAA phantoms for conducting image quality assessment of three PAI systems with substantially different operating parameters including two commercial systems and a custom system. Imaging results indicated that appropriately tuned PAA phantoms are useful tools for assessing and comparing PAI system image quality. These phantoms may also facilitate future standardization of performance test methodology.

Mitochondrial DNA mutations in renal disease: an overview.

Kidneys have a high energy demand to facilitate the reabsorption of the glomerular filtrate. For this reason, renal cells have a high density of mitochondria. Mitochondrial cytopathies can be the result of a mutation in both mitochondrial and nuclear DNA. Mitochondrial dysfunction can lead to a variety of renal manifestations. Examples of tubular manifestations are renal Fanconi Syndrome, which is often found in patients diagnosed with Kearns-Sayre and Pearson's marrow-pancreas syndrome, and distal tubulopathies, which result in electrolyte disturbances such as hypomagnesemia. Nephrotic syndrome can be a glomerular manifestation of mitochondrial dysfunction and is typically associated with focal segmental glomerular sclerosis on histology. Tubulointerstitial nephritis can also be seen in mitochondrial cytopathies and may lead to end-stage renal disease. The underlying mechanisms of these cytopathies remain incompletely understood; therefore, current therapies focus mainly on symptom relief. A better understanding of the molecular disease mechanisms is critical in order to improve treatments.

Photoacoustic Imaging as a Tool for Assessing Hair Follicular Organization.

Follicular unit extraction (FUE) and follicular unit transplantation (FUT) account for 99% of hair transplant procedures. In both cases, it is important for clinicians to characterize follicle density for treatment planning and evaluation. The existing gold-standard is photographic examination. However, this approach is insensitive to subdermal hair and cannot identify follicle orientation. Here, we introduce a fast and non-invasive imaging technique to measure follicle density and angles across regions of varying density. We first showed that hair is a significant source of photoacoustic signal. We then selected regions of low, medium, and high follicle density and showed that photoacoustic imaging can measure the density of follicles even when they are not visible by eye. We performed handheld imaging by sweeping the transducer across the imaging area to generate 3D images via maximum intensity projection. Background signal from the dermis was removed using a skin tracing method. Measurement of follicle density using photoacoustic imaging was highly correlated with photographic determination (R2 = 0.96). Finally, we measured subdermal follicular angles-a key parameter influencing transection rates in FUE.

Drug Development in Kidney Disease: Proceedings From a Multistakeholder Conference.

Occasional bursts of discovery and innovation have appeared during the otherwise stagnant past several decades of drug development in nephrology. Among other recent drug discoveries, the unexpected kidney benefits observed with sodium/glucose cotransporter 2 inhibitors may herald a renaissance of drug development in kidney disease. This recent progress highlights the need to further promote and stimulate research and development of promising therapies that may ameliorate the morbidity and mortality associated with kidney disease. To help identify and address barriers to drug development in nephrology, the Duke Clinical Research Institute convened a conference in April 2019 that included stakeholders from academia, industry, government agencies, and patient advocacy. From these discussions, several opportunities were identified to improve every stage of drug development for kidney disease from early discovery to implementation into practice. Key topics reviewed in this article are the utility of interconnected data and site research networks, surrogate end points, pragmatic and adaptive trial designs, the promising uses of real-world data, and methods to improve the generalizability of trial results and uptake of approved drugs for kidney-related diseases.

Deep learning improves contrast in low-fluence photoacoustic imaging.

Low fluence illumination sources can facilitate clinical transition of photoacoustic imaging because they are rugged, portable, affordable, and safe. However, these sources also decrease image quality due to their low fluence. Here, we propose a denoising method using a multi-level wavelet-convolutional neural network to map low fluence illumination source images to its corresponding high fluence excitation map. Quantitative and qualitative results show a significant potential to remove the background noise and preserve the structures of target. Substantial improvements up to 2.20, 2.25, and 4.3-fold for PSNR, SSIM, and CNR metrics were observed, respectively. We also observed enhanced contrast (up to 1.76-fold) in an in vivo application using our proposed methods. We suggest that this tool can improve the value of such sources in photoacoustic imaging.

Dictionary learning technique enhances signal in LED-based photoacoustic imaging.

There has been growing interest in low-cost light sources such as light-emitting diodes (LEDs) as an excitation source in photoacoustic imaging. However, LED-based photoacoustic imaging is limited by low signal due to low energy per pulse-the signal is easily buried in noise leading to low quality images. Here, we describe a signal de-noising approach for LED-based photoacoustic signals based on dictionary learning with an alternating direction method of multipliers. This signal enhancement method is then followed by a simple reconstruction approach delay and sum. This approach leads to sparse representation of the main components of the signal. The main improvements of this approach are a 38% higher contrast ratio and a 43% higher axial resolution versus the averaging method but with only 4% of the frames and consequently 49.5% less computational time. This makes it an appropriate option for real-time LED-based photoacoustic imaging.

Iodide-doped precious metal nanoparticles: measuring oxidative stress in vivo via photoacoustic imaging.

Accumulation of reactive oxygen and nitrogen species (RONS) can induce cell damage and even cell death. RONS are short-lived species, which makes direct, precise, and real-time measurement difficult. Biologically-relevant RONS levels are in the nM-µM scale; hence, there is a need for highly sensitive RONS probes. We previously used hybrid gold-core silver-shell nanoparticles with mM sensitivity to H2O2. These particles reported the presence of RONS via spectral shifts which could easily be quantified via photoacoustic imaging. Here, we used halide doping to tune the electrochemical properties of these materials to better match the oxidation potential of RONS. This work describes the synthesis, characterization, and application of these AgI-coated gold nanorods (AgI/AuNR). The I : Ag molar ratio, pH, and initial Ag shell thickness were optimized for good RONS detection limits. Halide doping lowers the reduction potential of Ag from to resulting in a 1000-fold increase in H2O2 and 100 000-fold increase in ONOO- sensitivity. The AgI/AuNR system also etches 45-times faster than undoped Ag/AuNR. The AgI/AuNR easily reported the endogenously produced RONS in established cells lines as well as murine models.

Molecular imaging of oxidative stress using an LED-based photoacoustic imaging system.

LED-based photoacoustic imaging has practical value in that it is affordable and rugged; however, this technology has largely been confined to anatomic imaging with limited applications into functional or molecular imaging. Here, we report molecular imaging reactive oxygen and nitrogen species (RONS) with a near-infrared (NIR) absorbing small molecule (CyBA) and LED-based photoacoustic imaging equipment. CyBA produces increasing photoacoustic signal in response to peroxynitrite (ONOO-) and hydrogen peroxide (H2O2) with photoacoustic signal increases of 3.54 and 4.23-fold at 50 µM of RONS at 700 nm, respectively. CyBA is insensitive to OCl-, ˙NO, NO2-, NO3-, tBuOOH, O2-, C4H9O˙, HNO, and ˙OH, but can detect ONOO- in whole blood and plasma. CyBA was then used to detect endogenous RONS in macrophage RAW 246.7 cells as well as a rodent model; these results were confirmed with fluorescence microscopy. Importantly, CyB suffers photobleaching under a Nd:YAG laser but the signal decrease is <2% with the low-power LED-based photoacoustic system and the same radiant exposure time. To the best of our knowledge, this is the first report to describe molecular imaging with an LED-based photoacoustic scanner. This study not only reveals the sensitive photoacoustic detection of RONS but also highlights the utility of LED-based photoacoustic imaging.

Tuning the ultrasonic and photoacoustic response of polydopamine-stabilized perfluorocarbon contrast agents.

Contrast-enhanced ultrasound (CEUS) offers the exciting prospect of retaining the ease of ultrasound imaging while enhancing imaging clarity, diagnostic specificity, and theranostic capability. To advance the capabilities of CEUS, the synthesis and understanding of new ultrasound contrast agents (UCAs) is a necessity. Many UCAs are nano- or micro-scale materials composed of a perfluorocarbon (PFC) and stabilizer that synergistically induce an ultrasound response that is both information-rich and easily differentiated from natural tissue. In this work, we probe the extent to which CEUS is modulated through variation in a PFC stabilized with fluorine-modified polydopamine nanoparticles (PDA NPs). The high level of synthetic tunability in this system allows us to study signal as a function of particle aggregation and PFC volatility in a systematic manner. Separation of aggregated and non-aggregated nanoparticles lead to a fundamentally different signal response, and for this system, PFC volatility has little effect on CEUS intensity despite a range of over 50 °C in boiling point. To further explore the imaging tunability and multimodality, Fe3+-chelation was employed to generate an enhanced photoacoustic (PA) signal in addition to the US signal. In vitro and in vivo results demonstrate that PFC-loaded PDA NPs show stronger PA signal than the non-PFC ones, indicating that the PA signal can be used for in situ differentiation between PFC-loading levels. In sum, these data evince the rich role synthetic chemistry can play in guiding new directions of development for UCAs.