Intravitreal MPTP drives retinal ganglion cell loss with oral nicotinamide treatment providing robust neuroprotection.

Neurodegenerative diseases have common underlying pathological mechanisms including progressive neuronal dysfunction, axonal and dendritic retraction, and mitochondrial dysfunction resulting in neuronal death. The retina is often affected in common neurodegenerative diseases such as Parkinson's and Alzheimer's disease. Studies have demonstrated that the retina in patients with Parkinson's disease undergoes changes that parallel the dysfunction in the brain. These changes classically include decreased levels of dopamine, accumulation of alpha-synuclein in the brain and retina, and death of dopaminergic nigral neurons and retinal amacrine cells leading to gross neuronal loss. Exploring this disease's retinal phenotype and vision-related symptoms is an important window for elucidating its pathophysiology and progression, and identifying novel ways to diagnose and treat Parkinson's disease. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is commonly used to model Parkinson's disease in animal models. MPTP is a neurotoxin converted to its toxic form by astrocytes, transported to neurons through the dopamine transporter, where it causes mitochondrial Complex I inhibition and neuron degeneration. Systemic administration of MPTP induces retinal changes in different animal models. In this study, we assessed the effects of MPTP on the retina directly via intravitreal injection in mice (5 mg/mL and 50 mg/mL to 7, 14 and 21 days post-injection). MPTP treatment induced the reduction of retinal ganglion cells-a sensitive neuron in the retina-at all time points investigated. This occurred without a concomitant loss of dopaminergic amacrine cells or neuroinflammation at any of the time points or concentrations tested. The observed neurodegeneration which initially affected retinal ganglion cells indicated that this method of MPTP administration could yield a fast and straightforward model of retinal ganglion cell neurodegeneration. To assess whether this model could be amenable to neuroprotection, mice were treated orally with nicotinamide (a nicotinamide adenine dinucleotide precursor) which has been demonstrated to be neuroprotective in several retinal ganglion cell injury models. Nicotinamide was strongly protective following intravitreal MPTP administration, further supporting intravitreal MPTP use as a model of retinal ganglion cell injury. As such, this model could be utilized for testing neuroprotective treatments in the context of Parkinson's disease and retinal ganglion cell injury.

Electron microscopic analysis of the influence of iPSC-derived motor neurons on bioengineered human skeletal muscle tissues.

3D bioengineered skeletal muscle macrotissues are increasingly important for studies of cell biology and development of therapeutics. Tissues derived from immortalized cells obtained from patient samples, or from pluripotent stem cells, can be co-cultured with motor-neurons to create models of human neuromuscular junctions in culture. In this study, we present foundational work on 3D cultured muscle ultrastructure, with and without motor neurons, which is enabled by the development of a new co-culture platform. Our results show that tissues from Duchenne muscular dystrophy patients are poorly organized compared to tissues grown from healthy donor and that the presence of motor neurons invariably improves sarcomere organization. Electron micrographs show that in the presence of motor neurons, filament directionality, banding patterns, z-disc continuity, and the appearance of presumptive SSR and T-tubule profiles all improve in healthy, DMD-, and iPSC-derived muscle tissue. Further work to identify the underlying defects of DMD tissue disorganization and the mechanisms by which motor neurons support muscle are likely to yield potential new therapeutic approaches for treating patients suffering from Duchenne muscular dystrophy.

Altered Outer Retinal Structure, Electrophysiology and Visual Perception in Parkinson's Disease.

BACKGROUND: Visual biomarkers of Parkinson's disease (PD) are attractive as the retina is an outpouching of the brain. Although inner retinal neurodegeneration in PD is well-established this has overlap with other neurodegenerative diseases and thus outer retinal (photoreceptor) measures warrant further investigation. OBJECTIVE: To examine in a cross-sectional study whether clinically implementable measures targeting outer retinal function and structure can differentiate PD from healthy ageing and whether these are sensitive to intraday levodopa (L-DOPA) dosing. METHODS: Centre-surround perceptual contrast suppression, macular visual field sensitivity, colour discrimination, light-adapted electroretinography and optical coherence tomography (OCT) were tested in PD participants (n = 16) and controls (n = 21). Electroretinography and OCT were conducted before and after midday L-DOPA in PD participants, or repeated after ∼2 hours in controls. RESULTS: PD participants had decreased center-surround contrast suppression (p < 0.01), reduced macular visual field sensitivity (p < 0.05), color vision impairment (p < 0.01) photoreceptor dysfunction (a-wave, p < 0.01) and photoreceptor neurodegeneration (outer nuclear layer thinning, p < 0.05), relative to controls. Effect size comparison between inner and outer retinal parameters showed that photoreceptor metrics were similarly robust in differentiating the PD group from age-matched controls as inner retinal changes. Electroretinography and OCT were unaffected by L-DOPA treatment or time. CONCLUSIONS: We show that outer retinal outcomes of photoreceptoral dysfunction (decreased cone function and impaired color vision) and degeneration (i.e., outer nuclear layer thinning) were equivalent to inner retinal metrics at differentiating PD from healthy age-matched adults. These findings suggest outer retinal metrics may serve as useful biomarkers for PD.

Levodopa Rescues Retinal Function in the Transgenic A53T Alpha-Synuclein Model of Parkinson's Disease.

BACKGROUND: Loss of substantia nigra dopaminergic cells and alpha-synuclein (α-syn)-rich intraneuronal deposits within the central nervous system are key hallmarks of Parkinson's disease (PD). Levodopa (L-DOPA) is the current gold-standard treatment for PD. This study aimed to evaluate in vivo retinal changes in a transgenic PD model of α-syn overexpression and the effect of acute levodopa (L-DOPA) treatment. METHODS: Anaesthetised 6-month-old mice expressing human A53T alpha-synuclein (HOM) and wildtype (WT) control littermates were intraperitoneally given 20 mg/kg L-DOPA (50 mg levodopa, 2.5 mg benserazide) or vehicle saline (n = 11-18 per group). In vivo retinal function (dark-adapted full-field ERG) and structure (optical coherence tomography, OCT) were recorded before and after drug treatment for 30 min. Ex vivo immunohistochemistry (IHC) on flat-mounted retina was conducted to assess tyrosine hydroxylase (TH) positive cell counts (n = 7-8 per group). RESULTS: We found that photoreceptor (a-wave) and bipolar cell (b-wave) ERG responses (p < 0.01) in A53T HOM mice treated with L-DOPA grew in amplitude more (47 ± 9%) than WT mice (16 ± 9%) treated with L-DOPA, which was similar to the vehicle group (A53T HOM 25 ± 9%; WT 19 ± 7%). While outer retinal thinning (outer nuclear layer, ONL, and outer plexiform layer, OPL) was confirmed in A53T HOM mice (p < 0.01), L-DOPA did not have an ameliorative effect on retinal layer thickness. These findings were observed in the absence of changes to the number of TH-positive amacrine cells across experiment groups. Acute L-DOPA treatment transiently improves visual dysfunction caused by abnormal alpha-synuclein accumulation. CONCLUSIONS: These findings deepen our understanding of dopamine and alpha-synuclein interactions in the retina and provide a high-throughput preclinical framework, primed for translation, through which novel therapeutic compounds can be objectively screened and assessed for fast-tracking PD drug discovery.

Amyloid-beta and tau protein beyond Alzheimer's disease.

ABSTRACT: The aggregation of amyloid-beta peptide and tau protein dysregulation are implicated to play key roles in Alzheimer's disease pathogenesis and are considered the main pathological hallmarks of this devastating disease. Physiologically, these two proteins are produced and expressed within the normal human body. However, under pathological conditions, abnormal expression, post-translational modifications, conformational changes, and truncation can make these proteins prone to aggregation, triggering specific disease-related cascades. Recent studies have indicated associations between aberrant behavior of amyloid-beta and tau proteins and various neurological diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, as well as retinal neurodegenerative diseases like Glaucoma and age-related macular degeneration. Additionally, these proteins have been linked to cardiovascular disease, cancer, traumatic brain injury, and diabetes, which are all leading causes of morbidity and mortality. In this comprehensive review, we provide an overview of the connections between amyloid-beta and tau proteins and a spectrum of disorders.

Measuring the Full-Field Electroretinogram in Rodents.

Electroretinography allows for noninvasive functional assessment of the retina and is a mainstay for preclinical studies of retinal function in health and disease. The full-field electroretinogram is useful for a variety of applications as it returns a functional readout from each of the major cell classes within the retina: photoreceptors, bipolar cells, amacrine cells, and retinal ganglion cells. Rodent models are commonly employed in ocular degeneration studies due to the fast throughput of these mammalian species and the conservation of the electroretinogram from the preclinic to the clinic. Here we describe approaches for in vivo electroretinography in rodent models.

Age-related deficits in retinal autophagy following intraocular pressure elevation in autophagy reporter mouse model.

This study quantified age-related changes to retinal autophagy using the CAG-RFP-EGFP-LC3 autophagy reporter mice and considered how aging impacts autophagic responses to acute intraocular pressure (IOP) stress. IOP was elevated to 50 mm Hg for 30 minutes in 3-month-old and 12-month-old CAG-RFP-EGFP-LC3 (n = 7 per age group) and Thy1-YFPh transgenic mice (n = 3 per age group). Compared with younger eyes, older eyes showed diminished basal autophagy in the outer retina, while the inner retina was unaffected. Autophagic flux (red:yellow puncta ratio) was elevated in the inner plexiform layer. Three days following IOP elevation, older eyes showed poorer functional recovery, most notably in ganglion cell responses compared to younger eyes (12 months old: -33.4 ±â€¯5.3% vs. 3 months mice: -13.4 ±â€¯4.5%). This paralleled a reduced capacity to upregulate autophagic puncta volume in the inner retina in older eyes, a response that was seen in younger eyes. Age-related decline in basal and stress-induced autophagy in the retina is associated with greater retinal ganglion cells' susceptibility to IOP elevation.

Retinal Assessment Using In Vivo Electroretinography and Optical Coherence Tomography in Rodent Models of Diabetes.

Electroretinography and optical coherence tomography imaging allow for non-invasive quantitative assessment of the retina. These approaches have become mainstays for identifying the very earliest impact of hyperglycemia on retinal function and structure in animal models of diabetic eye disease. Moreover, they are essential for assessing the safety and efficacy of novel treatment approaches for diabetic retinopathy. Here, we describe approaches for in vivo electroretinography and optical coherence tomography imaging in rodent models of diabetes.

Retinal alpha-synuclein accumulation correlates with retinal dysfunction and structural thinning in the A53T mouse model of Parkinson's disease.

Abnormal alpha-synuclein (α-SYN) protein deposition has long been recognized as one of the pathological hallmarks of Parkinson's disease's (PD). This study considers the potential utility of PD retinal biomarkers by investigating retinal changes in a well characterized PD model of α-SYN overexpression and how these correspond to the presence of retinal α-SYN. Transgenic A53T homozygous (HOM) mice overexpressing human α-SYN and wildtype (WT) control littermates were assessed at 4, 6, and 14 months of age (male and female, n = 15-29 per group). In vivo retinal function (electroretinography, ERG) and structure (optical coherence tomography, OCT) were recorded, and retinal immunohistochemistry and western blot assays were performed to examine retinal α-SYN and tyrosine hydroxylase. Compared to WT controls, A53T mice exhibited reduced light-adapted (cone photoreceptor and bipolar cell amplitude, p < 0.0001) ERG responses and outer retinal thinning (outer plexiform layer, outer nuclear layer, p < 0.0001) which correlated with elevated levels of α-SYN. These retinal signatures provide a high throughput means to study α-SYN induced neurodegeneration and may be useful in vivo endpoints for PD drug discovery.

Retinal Dysfunction in a Mouse Model of HCN1 Genetic Epilepsy.

Pathogenic variants in HCN1 are associated with a range of epilepsy syndromes including a developmental and epileptic encephalopathy. The recurrent de novo HCN1 pathogenic variant (M305L) results in a cation leak, allowing the flux of excitatory ions at potentials where the wild-type channels are closed. The Hcn1M294L mouse recapitulates patient seizure and behavioral phenotypes. As HCN1 channels are highly expressed in rod and cone photoreceptor inner segments, where they shape the light response, mutated channels are likely to impact visual function. Electroretinogram (ERG) recordings from male and female mice Hcn1M294L mice revealed a significant decrease in the photoreceptor sensitivity to light, as well as attenuated bipolar cell (P2) and retinal ganglion cell responses. Hcn1M294L mice also showed attenuated ERG responses to flickering lights. ERG abnormalities are consistent with the response recorded from a single female human subject. There was no impact of the variant on the structure or expression of the Hcn1 protein in the retina. In silico modeling of photoreceptors revealed that the mutated HCN1 channel dramatically reduced light-induced hyperpolarization, resulting in more Ca2+ flux during the response when compared with the wild-type situation. We propose that the light-induced change in glutamate release from photoreceptors during a stimulus will be diminished, significantly blunting the dynamic range of this response. Our data highlight the importance of HCN1 channels to retinal function and suggest that patients with HCN1 pathogenic variants are likely to have a dramatically reduced sensitivity to light and a limited ability to process temporal information.SIGNIFICANCE STATEMENT Pathogenic variants in HCN1 are emerging as an important cause of catastrophic epilepsy. HCN1 channels are ubiquitously expressed throughout the body, including the retina. Electroretinogram recordings from a mouse model of HCN1 genetic epilepsy showed a marked decrease in the photoreceptor sensitivity to light and a reduced ability to respond to high rates of light flicker. No morphologic deficits were noted. Simulation data suggest that the mutated HCN1 channel blunts light-induced hyperpolarization and consequently limits the dynamic range of this response. Our results provide insights into the role HCN1 channels play in retinal function as well as highlighting the need to consider retinal dysfunction in disease caused by HCN1 variants. The characteristic changes in the electroretinogram open the possibility of using this tool as a biomarker for this HCN1 epilepsy variant and to facilitate development of treatments.

Increased disparities associated with black women and abnormal cervical cancer screening follow-up.

Background: To determine whether race and ethnicity impacts patient adherence to follow-up for colposcopy after abnormal cervical cancer screening. Methods: This retrospective chart review included women that were randomly selected from patients presenting to our colposcopy clinic from 1/2019 to 12/2019. Inclusion criteria were females age ≥21 years-old and appropriate referral for colposcopy. Patients were grouped into three categories: (1) ADHERENT to follow-up if they came to their first scheduled appointment; (2) DELAYED if they presented more than three months from their original referral (usually missing 1-3 appointments); and (3) NOT ADHERENT if they did not show for their appointment after referral. Analysis was performed using SPSS v.26. Results: 284 women met inclusion criteria for the study. The majority of women were Black (65.2 %) followed by non-Hispanic Whites (20.0 %) and Latinx (14.8 %). Overall, 39.1 % were ADHERENT, 18.6 % were DELAYED, and 42.3 % were NOT ADHERENT. When compared with non-Hispanic White women, there was a significant difference between race/ethnicity and timing of follow-up (p = 0.03). Blacks were more likely to be NOT ADHERENT (45.9 %; p = 0.03), and Latinx and Blacks were the most likely to be DELAYED (35.7 % and 21.1 %; p = 0.03). Private insurance patients were more likely to be ADHERENT for care compared with un-/underinsured patients (78.9 vs 27.8 %, p = 0.0001). Conclusion: There is inadequate follow-up after abnormal cervical cancer screening across all races/ethnicities; however, lack of adherence is higher in Black patients. Moreover, 25% of Hispanic and Black women present in a delayed fashion. Culturally relevant assessments and interventions are needed to understand and address these gaps.

Characterization of retinal function and structure in the MPTP murine model of Parkinson's disease.

In addition to well characterized motor symptoms, visual disturbances are increasingly recognized as an early manifestation in Parkinson's disease (PD). A better understanding of the mechanisms underlying these changes would facilitate the development of vision tests which can be used as preclinical biomarkers to support the development of novel therapeutics for PD. This study aims to characterize the retinal phenotype of a mouse model of dopaminergic dysfunction and to examine whether these changes are reversible with levodopa treatment. We use a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD to characterize the neurotoxic effects of MPTP on in vivo retinal function (electroretinography, ERG), retinal structure (optical coherence tomography, OCT) and retinal dopaminergic cell number (tyrosine hydroxylase immunohistochemistry, IHC) at two time points (21 and 45 days) post MPTP model induction. We also investigate the effect of levodopa (L-DOPA) as a proof-of-principle chronic intervention against MPTP-induced changes in the retina. We show that MPTP decreases dopaminergic amacrine cell number (9%, p < 0.05) and that a component of the ERG that involves these cells, in particular oscillatory potential (OP) peak timing, was significantly delayed at Day 45 (7-13%, p < 0.01). This functional deficit was paralleled by outer plexiform layer (OPL) thinning (p < 0.05). L-DOPA treatment ameliorated oscillatory potential deficits (7-13%, p < 0.001) in MPTP animals. Our data suggest that the MPTP toxin slows the timing of inner retinal feedback circuits related to retinal dopaminergic pathways which mirrors findings from humans with PD. It also indicates that the MPTP model causes structural thinning of the outer retinal layer on OCT imaging that is not ameliorated with L-DOPA treatment. Together, these non-invasive measures serve as effective biomarkers for PD diagnosis as well as for quantifying the effect of therapy.

White matter tract conductivity is resistant to wide variations in paranodal structure and myelin thickness accompanying the loss of Tyro3: an experimental and simulated analysis.

Myelination within the central nervous system (CNS) is crucial for the conduction of action potentials by neurons. Variation in compact myelin morphology and the structure of the paranode are hypothesised to have significant impact on the speed of action potentials. There are, however, limited experimental data investigating the impact of changes in myelin structure upon conductivity in the central nervous system. We have used a genetic model in which myelin thickness is reduced to investigate the effect of myelin alterations upon action potential velocity. A detailed examination of the myelin ultrastructure of mice in which the receptor tyrosine kinase Tyro3 has been deleted showed that, in addition to thinner myelin, these mice have significantly disrupted paranodes. Despite these alterations to myelin and paranodal structure, we did not identify a reduction in conductivity in either the corpus callosum or the optic nerve. Exploration of these results using a mathematical model of neuronal conductivity predicts that the absence of Tyro3 would lead to reduced conductivity in single fibres, but would not affect the compound action potential of multiple myelinated neurons as seen in neuronal tracts. Our data highlight the importance of experimental assessment of conductivity and suggests that simple assessment of structural changes to myelin is a poor predictor of neural functional outcomes.

Comprehensiveness of Online Information in Gender-Affirming Surgery: Current Trends and Future Directions in Academic Plastic Surgery.

BACKGROUND: Despite high rates of online misinformation, transgender and gender diverse (TGD) patients frequently utilize online resources to identify suitable providers of gender-affirming surgical care. AIM: The objective of this study was to analyze the webpages of United States academic plastic surgery programs for the types of gender-affirming surgery (GAS) procedures offered and to determine how this correlates with the presence of an institutional transgender health program and geographic region in order to identify potential gaps for improvement. METHODS: Online institutional webpages of 82 accredited academic plastic surgery programs were analyzed for the presence of the following: GAS services, specification of type of GAS by facial, chest, body and genital surgery, and presence of a concomitant institutional transgender health program. This data was analyzed for correlations with geographic region and assessed for any significant associations. OUTCOMES: Frequencies of GAS services, specification of the type of GAS by facial, chest, body and genital surgery, presence of a concomitant institutional transgender health program, and statistical correlations between these items are the primary outcomes. RESULTS: Overall, 43 of 82 (52%) academic institutions offered GAS. Whether an institution offered GAS varied significantly with the presence of an institutional transgender health program (P < .001) but not with geographic region (P = .329). Whether institutions that offer GAS specified which anatomic category of GAS procedures were offered varied significantly with the presence of an institutional transgender health program (P < .001) but not with geographic region (P = .235). CLINICAL IMPLICATIONS: This identifies gaps for improved transparency in the practice of communication around GAS for both physicians and academic medical institutions. STRENGTHS & LIMITATIONS: This is the first study analyzing the quality, content, and accessibility of online information pertaining to GAS in academic institutions. The primary limitation of this study is the nature and accuracy of online information, as current data may be outdated and not reflect actuality. CONCLUSION: Based on our analysis of online information, many gaps currently exist in information pertaining to GAS in academic settings, and with a clear and expanding need, increased representation and online availability of information regarding all GAS procedure types, as well as coordination with comprehensive transgender healthcare programs, is ideal. Aryanpour Z, Nguyen CT, Blunck CK, et al., Comprehensiveness of Online Information in Gender-Affirming Surgery: Current Trends and Future Directions in Academic Plastic Surgery. J Sex Med 2022;19:846-851.

Progressive impairments in executive function in the APP/PS1 model of Alzheimer's disease as measured by translatable touchscreen testing.

Executive function deficits in Alzheimer's disease (AD) occur early in disease progression and may be predictive of cognitive decline. However, no preclinical studies have identified deficits in rewarded executive function in the commonly used APPSwe/PS1∆E9 (APP/PS1) mouse model. To address this, we assessed 12-26 month old APP/PS1 mice on rewarded reversal and/or extinction tasks. 16-month-old, but not 13- or 26-month-old, APP/PS1 mice showed an attenuated rate of extinction. Reversal deficits were seen in 22-month-old, but not 13-month-old APP/PS1 animals. We then confirmed that impairments in reversal were unrelated to previously reported visual impairments in both AD mouse models and humans. Age, but not genotype, had a significant effect on markers of retinal health, indicating the deficits seen in APP/PS1 mice were directly related to cognition. This is the first characterisation of rewarded executive function in APP/PS1 mice, and has great potential to facilitate translation from preclinical models to the clinic.

Electrophysiological analysis of healthy and dystrophic 3-D bioengineered skeletal muscle tissues.

Recently, methods for creating three-dimensional (3-D) human skeletal muscle tissues from myogenic cell lines have been reported. Bioengineered muscle tissues are contractile and respond to electrical and chemical stimulation. In this study, we provide an electrophysiological analysis of healthy and dystrophic 3-D bioengineered skeletal muscle tissues, focusing on Duchenne muscular dystrophy (DMD). We enlist the 3-D in vitro model of DMD muscle tissue to evaluate muscle cell electrical properties uncoupled from presynaptic neural inputs, an understudied aspect of DMD. Our data show that previously reported electrophysiological aspects of DMD, including effects on membrane potential and membrane resistance, are replicated in the 3-D muscle tissue model. Furthermore, we test a potential therapeutic compound, poloxamer 188, and demonstrate capacity for improving the membrane potential in DMD muscle. Therefore, this study serves as a baseline for a new in vitro method to examine potential therapies for muscular disorders.

Disruption of Drosophila larval muscle structure and function by UNC45 knockdown.

BACKGROUND: Proper muscle function is heavily dependent on highly ordered protein complexes. UNC45 is a USC (named since this region is shared by three proteins UNC45/CRO1/She4P) chaperone that is necessary for myosin incorporation into the thick filaments. UNC45 is expressed throughout the entire Drosophila life cycle and it has been shown to be important during late embryogenesis when initial muscle development occurs. However, the effects of UNC45 manipulation at later developmental times, after muscle development, have not yet been studied. MAIN RESULTS: UNC45 was knocked down with RNAi in a manner that permitted survival to the pupal stage, allowing for characterization of sarcomere organization in the well-studied third instar larvae. Second harmonic generation (SHG) microscopy revealed changes in the striated pattern of body wall muscles as well as a reduction of signal intensity. This observation was confirmed with immunofluorescence and electron microscopy imaging, showing diminished UNC45 signal and disorganization of myosin and z-disk proteins. Concomitant alterations in both synaptic physiology and locomotor function were also found. Both nerve-stimulated response and spontaneous vesicle release were negatively affected, while larval movement was impaired. CONCLUSIONS: This study highlights the dependency of normal sarcomere structure on UNC45 expression. We confirm the known role of UNC45 for myosin localization and further show the I-Z-I complex is also disrupted. This suggests a broad need for UNC45 to maintain sarcomere integrity. Newly discovered changes in synaptic physiology reveal the likely presence of a homeostatic response to partially maintain synaptic strength and muscle function.

De novo revertant fiber formation and therapy testing in a 3D culture model of Duchenne muscular dystrophy skeletal muscle.

The biological basis of Duchenne muscular dystrophy (DMD) pathology is only partially characterized and there are still few disease-modifying therapies available, therein underlying the value of strategies to model and study DMD. Dystrophin, the causative gene of DMD, is responsible for linking the cytoskeleton of muscle fibers to the extracellular matrix beyond the sarcolemma. We posited that disease-associated phenotypes not yet captured by two-dimensional culture methods would arise by generating multinucleated muscle cells within a three-dimensional (3D) extracellular matrix environment. Herein we report methods to produce 3D human skeletal muscle microtissues (hMMTs) using clonal, immortalized myoblast lines established from healthy and DMD donors. We also established protocols to evaluate immortalized hMMT self-organization and myotube maturation, as well as calcium handling, force generation, membrane stability (i.e., creatine kinase activity and Evans blue dye permeability) and contractile apparatus organization following electrical-stimulation. In examining hMMTs generated with a cell line wherein the dystrophin gene possessed a duplication of exon 2, we observed rare dystrophin-positive myotubes, which were not seen in 2D cultures. Further, we show that treating DMD hMMTs with a ß1-integrin activating antibody, improves contractile apparatus maturation and stability. Hence, immortalized myoblast-derived DMD hMMTs offer a pre-clinical system with which to investigate the potential of duplicated exon skipping strategies and those that protect muscle cells from contraction-induced injury. STATEMENT OF SIGNIFICANCE: Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disorder that is caused by mutation of the dystrophin gene. The biological basis of DMD pathology is only partially characterized and there is no cure for this fatal disease. Here we report a method to produce 3D human skeletal muscle microtissues (hMMTs) using immortalized human DMD and healthy myoblasts. Morphological and functional assessment revealed DMD-associated pathophysiology including impaired calcium handling and de novo formation of dystrophin-positive revertant muscle cells in immortalized DMD hMMTs harbouring an exon 2 duplication, a feature of many DMD patients that has not been recapitulated in culture prior to this report. We further demonstrate that this "DMD in a dish" system can be used as a pre-clinical assay to test a putative DMD therapeutic and study the mechanism of action.

Retinal hyperspectral imaging in the 5xFAD mouse model of Alzheimer's disease.

Hyperspectral imaging of the retina has recently been posited as a potentially useful form of spectroscopy of amyloid-beta (Aß) protein in the eyes of those with Alzheimer's disease (AD). The concept of using the retina as a biomarker for AD is an attractive one, as current screening tools for AD are either expensive or inaccessible. Recent studies have investigated hyperspectral imaging in Aß models however these studies have been in younger mice. Here we characterised hyperspectral reflectance profile in 6 to 17 months old 5xFAD mice and compare this to Aß in isolated preparations. Hyperspectral imaging was conducted across two preparations of Aß using a custom built bench ophthalmoscope. In the in vitro condition, 1 mg of purified human Aß42 was solubilised and left to aggregate for 72 h. This soluble/insoluble Aß mixture was then imaged by suspending the solution at a pipette tip and compared against phosphate buffered saline (PBS) control (n = 10 ROIs / group). In the in vivo condition, a 5xFAD transgenic mouse model was used and retinae were imaged at the age of 6 (n = 9), 12 (n = 9) and 17 months (n = 8) with age matched wildtype littermates as control (n = 12, n = 13, n = 15 respectively). In the vitro condition, hyperspectral imaging of the solution showed greater reflectance compared with vehicle (p < 0.01), with the greatest differences occurring in the short visible spectrum (< 500 nm). In the in vivo preparation, 5xFAD showed greater hyperspectral reflectance at all ages (6, 12, 17 months, p < 0.01). These differences were noted most in the short wavelengths at younger ages, with an additional peak appearing at longer wavelengths (~ 550 nm) with advancing age. This study shows that the presence of Aß (soluble/insoluble mixture) can increase the hyperspectral reflectance profile in vitro as well as in vivo. Differences were evident in the short wavelength spectrum (< 500 nm) in vitro and were preserved when imaged through the ocular media in the in vivo conditions. With advancing age a second hump around ~ 550 nm became more apparent. Hyperspectral imaging of the retina does not require the use of contrast agents and is a potentially useful and non-invasive biomarker for AD.