The LCHADD Mouse Model Recapitulates Early-Stage Chorioretinopathy in LCHADD Patients.

Purpose: Recent studies have shown that the retinal pigment epithelium (RPE) relies on fatty acid oxidation (FAO) for energy, however, its role in overall retinal health is unknown. The only FAO disorder that presents with chorioretinopathy is long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD). Studying the molecular mechanisms can lead to new treatments for patients and elucidate the role of FAO in the RPE. This paper characterizes the chorioretinopathy progression in a recently reported LCHADD mouse model. Methods: Visual assessments, such as optokinetic tracking and fundus imaging, were performed in wildtype (WT) and LCHADD mice at 3, 6, 10, and 12 months of age. Retinal morphology was analyzed in 12-month retinal cross-sections using hematoxylin and eosin (H&E), RPE65, CD68, and TUNEL staining, whereas RPE structure was assessed using transmission electron microscopy (TEM). Acylcarnitine profiles were measured in isolated RPE/sclera samples to determine if FAO was blocked. Bulk RNA-sequencing of 12 month old male WT mice and LCHADD RPE/sclera samples assessed gene expression changes. Results: LCHADD RPE/sclera samples had a 5- to 7-fold increase in long-chain hydroxyacylcarnitines compared to WT, suggesting an impaired LCHAD step in long-chain FAO. LCHADD mice have progressively decreased visual performance and increased RPE degeneration starting at 6 months. LCHADD RPE have an altered structure and a two-fold increase in macrophages in the subretinal space. Finally, LCHADD RPE/sclera have differentially expressed genes compared to WT, including downregulation of genes important for RPE function and angiogenesis. Conclusions: Overall, this LCHADD mouse model recapitulates early-stage chorioretinopathy seen in patients with LCHADD and is a useful model for studying LCHADD chorioretinopathy.

Preformed Vesicle Approach to LNP Manufacturing Enhances Retinal mRNA Delivery.

Complete encapsulation of nucleic acids by lipid-based nanoparticles (LNPs) is often thought to be one of the main prerequisites for successful nucleic acid delivery, as the lipid environment protects mRNA from degradation by external nucleases and assists in initiating delivery processes. However, delivery of mRNA via a preformed vesicle approach (PFV-LNPs) defies this precondition. Unlike traditional LNPs, PFV-LNPs are formed via a solvent-free mixing process, leading to a superficial mRNA localization. While demonstrating low encapsulation efficiency in the RiboGreen assay, PFV-LNPs improved delivery of mRNA to the retina by up to 50% compared to the LNP analogs across several benchmark formulations, suggesting the utility of this approach regardless of the lipid composition. Successful mRNA and gene editors' delivery is observed in the retinal pigment epithelium and photoreceptors and validated in mice, non-human primates, and human retinal organoids. Deploying PFV-LNPs in gene editing experiments result in a similar extent of gene editing compared to analogous LNP (up to 3% on genomic level) in the Ai9 reporter mouse model; but, remarkably, retinal tolerability is significantly improved for PFV-LNP treatment. The study findings indicate that the LNP formulation process can greatly influence mRNA transfection and gene editing outcomes, improving LNP treatment safety without sacrificing efficacy.

Lipid nanoparticles with PEG-variant surface modifications mediate genome editing in the mouse retina.

Ocular delivery of lipid nanoparticle (LNPs) packaged mRNA can enable efficient gene delivery and editing. We generated LNP variants through the inclusion of positively charged-amine-modified polyethylene glycol (PEG)-lipids (LNPa), negatively charged-carboxyl-(LNPz) and carboxy-ester (LNPx) modified PEG-lipids, and neutral unmodified PEG-lipids (LNP). Subretinal injections of LNPa containing Cre mRNA in the mouse show tdTomato signal in the retinal pigmented epithelium (RPE) like conventional LNPs. Unexpectedly, LNPx and LNPz show 27% and 16% photoreceptor transfection, respectively, with striking localization extending from the photoreceptor synaptic pedicle to the outer segments, displaying pan-retinal distribution in the photoreceptors and RPE. LNPx containing Cas9 mRNA and sgAi9 leads to the formation of an oval elongated structure with a neutral charge resulting in 16.4% editing restricted to RPE. Surface modifications of LNPs with PEG variants can alter cellular tropism of mRNA. LNPs enable genome editing in the retina and in the future can be used to correct genetic mutations that lead to blindness.

Spontaneous allelic variant in deafness-blindness gene Ush1g resulting in an expanded phenotype.

Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1gbw/bw null mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell's kinocilial links. Consistent with other Usher type 1 models, Ush1gbw/bw mice had no substantial retinal degeneration compared with Ush1gbw /+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene.

Cell patterning by surface tension pinning in microfluidic channels.

We present a simple method to pattern multiple cell populations inside a microfluidic channel. The microchannel is partially filled with a cell suspension, and the position of the liquid boundary remains pinned by surface tension. Cells then adhere only in the filled portion of the channel, producing a very sharp boundary. The process can be performed in an unmodified microfluidic channel with only a manual syringe and can be repeated multiple times to pattern cocultures or tricultures. We demonstrate the patterning method with two different mammalian cell types, 3T3 fibroblasts and NMuMG epithelial cells, and channel heights of 1.5 mm and 0.5 mm. We anticipate that this method will be useful for studies of cell-cell interactions where precise control of the fluidic microenvironment is required.

Patterning of sharp cellular interfaces with a reconfigurable elastic substrate.

Micropatterned cocultures are a useful experimental tool for the study of cell-cell interactions. Patterning methods often rely on sequential seeding of different cell types or removal of a barrier separating two populations, but it is difficult to pattern sharp interfaces between pure populations with low cross-contamination when using these approaches. Patterning by the use of reconfigurable substrates can overcome these limitations, but such methods can be costly and challenging to employ in a typical biology laboratory. Here, we describe a low-cost and simple-to-use reconfigurable substrate comprised of a transparent elastic material that is partially cut to form a slit that opens when the device is stretched. The slit seals back up when released, allowing two initially separate, adherent cell populations to be brought together to form a contact interface. Fluorescent imaging of patterned cocultures demonstrates the early establishment of a sharp cellular interface. As a proof of principle, we demonstrate the use of this device to study competition at the interface of two stem cell populations.

Apical pressure created during irrigation with the GentleWave™ system compared to conventional syringe irrigation.

OBJECTIVES: The purpose of this study is to compare pressures at the apical foramen created by conventional syringe irrigation and the GentleWave™ System, which releases high-velocity degassed irrigants to the pulp chamber and uses broad-spectrum sound energy for cleaning. MATERIALS AND METHODS: The apical pressure generated during irrigation was measured for palatal and distobuccal root canals of four extracted maxillary molars after no instrumentation, minimal instrumentation to a size #15/.04, instrumentation to a size #40/.04 taper, and after perforating the apical foramen to size #40. The root canals opened into an air-tight custom fixture coupled to a piezoresistive pressure transducer. Apical pressures were measured for the GentleWave™ System and syringe-needle irrigation at different irrigant flow rates, with the needle tip at 1 and 3 mm from the apical foramen using 30-gauge (G) open-ended or side-vented safety tip needles. RESULTS: The GentleWave™ System generated negative apical pressures (P < 0.001 compared with syringe irrigation); the mean pressures were between -13.07 and -17.19 mmHg. The 30 G needles could not reach the 1 and 3 mm from the working length in uninstrumented and 1 mm in minimally instrumented canals. The mean positive pressures between 6.46 and 110.34 mmHg were measured with needle irrigation depending on the flow rate, needle insertion depth, and size of the root canal. CONCLUSIONS: The GentleWave™ System creates negative pressure at the apical foramen during root canal cleaning irrespective of the size of canal instrumentation. Positive apical pressures were measured for syringe irrigation. CLINICAL RELEVANCE: Negative pressure during irrigation contributes to improved safety as compared to high-positive pressure.

In vitro study of calcium hydroxide removal from mandibular molar root canals.

INTRODUCTION: Previous studies have shown the difficulty in removing calcium hydroxide (Ca[OH]2) paste from the root canals before root filling. METHODS: Mesial and distal canals of 30 mandibular molars were prepared with the WaveOne Primary (25/.08) and Large file (40/.08) (Dentsply Tulsa Dental Specialties, Tulsa, OK), respectively. All canals were then filled with Ca(OH)2. The teeth were divided into the following 3 treatment groups (each with n = 10): (1) instrumentation with needle irrigation, (2) instrumentation with irrigation and passive ultrasonic activation (PUI), and (3) the GentleWave system (Sonendo, Inc, Laguna Hills, CA) without instrumentation. The irrigation time in each group was 7.5 minutes. To further test the efficiency of the GentleWave system, shorter times of 90 seconds were tested using water alone. Reconstructed micro-computed tomographic scans were used to measure the volume of the canals and Ca(OH)2 after instrumentation, initial filling of Ca(OH)2, and after its removal. The percentage of Ca(OH)2 remaining in the canals was calculated. RESULTS: None of the 10 teeth (30 canals) in the conventional irrigation and PUI groups were completely cleaned of Ca(OH)2 in 7.5 minutes. In the apical third of mesial and distal canals, respectively, conventional irrigation removed 47.82% ± 16.36% and 77.68% ± 12.82%, PUI removed 61.66% ± 25.54% and 88.85 ± 12.06%, and the GentleWave system removed significantly more Ca(OH)2 (P < .05) with 100% and 98.78% ± 3.84%. Additional experiments in 10 teeth, using only water as the irrigant, revealed that the GentleWave system removed 99.85% and 99.97% of Ca(OH)2 within 90 seconds without the use of any instruments in the mesial and distal canals, respectively. CONCLUSIONS: The study confirms the difficulty to remove Ca(OH)2 from root canals using conventional methods. The GentleWave system removed the paste within 90 seconds using water irrigation alone.

Tissue dissolution by a novel multisonic ultracleaning system and sodium hypochlorite.

INTRODUCTION: This study aimed to evaluate the effectiveness of a novel Multisonic Ultracleaning System (Sonendo Inc, Laguna Hills, CA) in tissue dissolution in comparison with conventional irrigation devices. METHODS: Pieces of bovine muscle tissue (68 ± 2 mg) were placed in 0.7-mL test tubes (height: 23.60 mm, inner diameter: 6.00 mm, outer diameter: 7.75 mm) and exposed to 5 minutes of irrigation by different devices. Endodontic devices included the Multisonic Ultracleaning System, the Piezon Master 700 (EMS, Dallas, TX) ultrasonic system with agitation, the EndoVac negative-pressure irrigation system (SybronEndo, Orange, CA), and a conventional positive-pressure 27-G irrigation needle at a flow rate of 10 mL/min. The systems were tested with 0.5%, 3%, and 6% sodium hypochlorite (NaOCl) at room temperature (21°C) as well as 40°C. Irrigation with sterile water was used as a control. The mass of tissue specimens was measured and recorded before and after the use of each device, and if the specimen was completely dissolved visually within 5 minutes, the dissolution time was recorded. The rate of tissue dissolution (%/s) was then calculated. RESULTS: The Multisonic Ultracleaning System had the fastest rate of tissue dissolution (P < .05), at 1.0% ± 0.1% per second using 0.5% NaOCl, 2.3% ± 0.9% per second using 3% NaOCl, and 2.9% ± 0.7% per second using 6% NaOCl. This tissue dissolution rate was more than 8 times greater than the second fastest device tested (P < .01), the Piezon Master 700 ultrasonic system, which resulted in a tissue dissolution rate of 0.328% ± 0.002% per second using 6% NaOCl at 40°C. For all irrigation devices tested, the rate of tissue dissolution increased with a higher concentration and temperature of the NaOCl solution. CONCLUSIONS: The novel Multisonic Ultracleaning System achieved a significantly faster tissue dissolution rate when compared with the other systems examined in vitro.

Case of paradoxical embolic transient ischemic attack from an unusual extra cardiac shunt detected on a contrast-enhanced echocardiogram.

Paradoxical embolism due to extracardiac right to left shunts (RLSs) manifesting as stroke remains anecdotal. We describe a case of 63-year-old female who presented with a transient ischemic attack and at agitated saline contrast echocardiogram was found to have an unusual type of an extracardiac RLS. Further evaluation leads to diagnosis of superior vena cava (SVC) thrombosis from a prior indwelling central venous catheter. The SVC thrombosis induced systemic-pulmonary venous collaterals were responsible for this unusual extracardiac RLS and paradoxical transient ischemic attack. We discuss the diagnosis and management of this rare clinical case.

Gender variations in the optical properties of skin in murine animal models.

Gender is identified as a significant source of variation in optical reflectance measurements on mouse skin, with variation in the thickness of the dermal layer being the key explanatory variable. For three different mouse strains, the thickness values of the epidermis, dermis, and hypodermis layers, as measured by histology, are correlated to optical reflectance measurements collected with elastic scattering spectroscopy (ESS). In all three strains, males are found to have up to a 50% increase in dermal thickness, resulting in increases of up to 80% in reflectance values and higher observed scattering coefficients, as compared to females. Collagen in the dermis is identified as the primary source of these differences due to its strong scattering nature; increased dermal thickness leads to a greater photon path length through the collagen, as compared to other layers, resulting in a larger scattering signal. A related increase in the observed absorption coefficient in females is also observed. These results emphasize the importance of considering gender during experimental design in studies that involve photon interaction with mouse skin. The results also elucidate the significant impact that relatively small thickness changes can have on observed optical measurements in layered tissue.

Temporal variations of skin pigmentation in C57BL/6 mice affect optical bioluminescence quantitation.

PURPOSE: Depilation-induced skin pigmentation in C57Bl/6 mice is a known occurrence, and presents a unique problem for quantitative optical imaging of small animals, especially for bioluminescence. The work reported here quantitatively investigated the optical attenuation of bioluminescent light due to melanin pigmentation in the skin of transgenic C57Bl/6 mice, modified such that luciferase expression is under the transcription control of a physiologically and pharmacologically inducible gene. PROCEDURE: Both in vivo and ex vivo experiments were performed to track bioluminescence signal attenuation through different stages of the mouse hair growth cycle. Simultaneous reflectance measurements were collected in vivo to estimate melanin levels. RESULTS: Biological variability of skin pigmentation was found to dramatically affect collected bioluminescent signal emerging through the skin of the mice. When compared to signal through skin with no pigmentation, the signal through highly pigmented skin was attenuated an average of 90%. Positive correlation was found between reflectance measurements and bioluminescence signal loss. A correction scheme is proposed based on this correlation, but signal variation due to non-melanin scattering and absorption sources introduce significant errors. Advanced spectral reflectance analysis will be necessary to develop a more reliable correction method in the future. CONCLUSION: Skin pigmentation is a significant variable in bioluminescent imaging, and should be considered in experimental design and implementation for longitudinal studies, and especially when sensitivity to small signal changes, or differences among animals, is required.