Prodromal Parkinson's disease and the catecholaldehyde hypothesis: Insight from olfactory bulb organotypic cultures.

Parkinson's disease (PD) is a progressive, neurodegenerative disorder with an increasing incidence, unknown etiology, and is currently incurable. Advances in understanding the pathological mechanisms at a molecular level have been slow, with little attention focused on the early prodromal phase of the disease. Consequently, the development of early-acting disease-modifying therapies has been hindered. The olfactory bulb (OB), the brain region responsible for initial processing of olfactory information, is particularly affected early in PD at both functional and molecular levels but there is little information on how the cells in this region are affected by disease. Organotypic and primary OB cultures were developed and characterized. These platforms were then used to assess the effects of 3,4-dihydroxyphenylacetylaldehyde (DOPAL), a metabolite of dopamine present in increased levels in post-mortem PD tissue and which is thought to contribute to PD pathogenesis. Our findings showed that DOPAL exposure can recapitulate many aspects of PD pathology. Oxidative stress, depolarization of mitochondrial membranes, and neurodegeneration were all induced by DOPAL addition, as were measured transcriptomic changes consistent with those reported in PD clinical studies. These olfactory models of prodromal disease lend credence to the catecholaldehyde hypothesis of PD and provide insight into the mechanisms by which the OB may be involved in disease progression.

A Predictive and an Optimization Mathematical Model for Device Design in Cardiac Pulsed Field Ablation Using Design of Experiments.

Cardiac catheter ablation (CCA) is a common method used to correct cardiac arrhythmia. Pulsed Field Ablation (PFA) is a recently-adapted CCA technology whose ablation is dependent on electrode and waveform parameters (factors). In this work, the use of the Design of Experiments (DoE) methodology is investigated for the design and optimization of a PFA device. The effects of the four factors (input voltage, electrode spacing, electrode width, and on-time) and their interactions are analyzed. An empirical model is formed to predict and optimize the ablation size responses. Based on the ranges tested, the significant factors were the input voltage, the electrode spacing, and the on time, which is in line with the literature. Two-factor interactions were found to be significant and need to be considered in the model. The resulting empirical model was found to predict ablation sizes with less than 2.1% error in the measured area and was used for optimization. The findings and the strong predictive model developed highlight that the DoE approach can be used to help determine PFA device design, to optimize for certain ablation zone sizes, and to help inform device design to tackle specific cardiac arrhythmias.

In Vitro Models for Improved Therapeutic Interventions in Atrial Fibrillation.

Atrial fibrillation is the most common type of cardiac arrhythmias in humans, mostly caused by hyper excitation of specific areas in the atrium resulting in dyssynchronous atrial contractions, leading to severe consequences such as heart failure and stroke. Current therapeutics aim to target this condition through both pharmacological and non-pharmacological approaches. To test and validate any of these treatments, an appropriate preclinical model must be carefully chosen to refine and optimise the therapy features to correctly reverse this condition. A broad range of preclinical models have been developed over the years, with specific features and advantages to closely mimic the pathophysiology of atrial fibrillation. In this review, currently available models are described, from traditional animal models and in vitro cell cultures to state-of-the-art organoids and organs-on-a-chip. The advantages, applications and limitations of each model are discussed, providing the information to select the appropriate model for each research application.

A novel protocol to derive cervical motor neurons from induced pluripotent stem cells for amyotrophic lateral sclerosis.

Sporadic amyotrophic lateral sclerosis (sALS) is the majority of ALS, and the lack of appropriate disease models has hindered its research. Induced pluripotent stem cell (iPSC) technology now permits derivation of iPSCs from somatic cells of sALS patients to investigate disease phenotypes and mechanisms. Most existing differentiation protocols are time-consuming or low efficient in generating motor neurons (MNs). Here we report a rapid and simple protocol to differentiate MNs in monolayer culture using small molecules, which led to nearly pure neural stem cells in 6 days, robust OLIG2+ pMNs (73%-91%) in 12 days, enriched CHAT+ cervical spinal MNs (sMNs) (88%-97%) in 18 days, and functionally mature sMNs in 28 days. This simple and reproducible protocol permitted the identification of hyperexcitability phenotypes in our sALS iPSC-derived sMNs, and its application in neurodegenerative diseases should facilitate in vitro disease modeling, drug screening, and the development of cell therapy.

Extracellular Vesicles, Cell-Penetrating Peptides and miRNAs as Future Novel Therapeutic Interventions for Parkinson's and Alzheimer's Disease.

Neurodegeneration is hallmarked by the progressive loss of dopaminergic neurons and/or a significant increase in protein aggregates in the brain. Neurodegenerative diseases are a leading cause of death worldwide with over 15 million people currently suffering from either Parkinson's disease (PD) or Alzheimer's disease (AD). PD is often characterized by both motor and non-motor symptoms, including muscle rigidity, tremors and bradykinesia, with AD displaying symptoms of confusion and dementia. The current mainstay of therapeutics includes pharmacological approaches such as levodopa to replace dopamine in PD patients, deep brain stimulation in affected regions of the brain and physical therapy. However, these treatments are typically not disease-modifying, though they do help at least for some time with symptom management. These treatments often also fail due to their inability to cross the blood-brain barrier. There is a need to develop new strategies to target neurodegeneration in an ever-ageing population. First, we review the current PD and AD treatments and their limitations. Second, we review the current use of extracellular vesicles (EVs), cell-penetrating peptides (CPPs) and miRNAs as neuroprotective agents. Finally, we discuss the possibility of exploiting these as a combinatory therapeutic, alongside some potential drawbacks.

The development and preliminary evaluation of a clinician e-learning training platform for a neonatal sepsis risk monitor for use in ICU settings.

BACKGROUND: Training clinicians on the use of hospital-based patient monitoring systems (PMS) is vital to mitigate the risk of use errors and of frustration using these devices, especially when used in ICU settings. PMS training is typically delivered through face-to-face training sessions in the hospital. However, it is not always feasible to deliver training in this format to all clinical staff given some constraints (e.g., availability of staff and trainers to attend in-person training sessions and the costs associated with face-to-face training). OBJECTIVE: The literature indicates that E-learning has the potential to mitigate barriers associated with time restrictions for trainers and trainees and evidence shows it to be more flexible, and convenient for learners in healthcare settings. This study aimed to develop and carry out a preliminary evaluation via a case study of an e-learning training platform designed for a novel neonatal sepsis risk monitor system (Digi-NewB). METHODS: A multi-modal qualitative research case study approach was used, including the analysis of three qualitative data sources: (i) audio/video recordings of simulation sessions in which participants were asked to operate the system as intended (e.g., update the clinical observations and monitor the sepsis risk), (ii) interviews with the simulation participants and an attending key opinion leader (KOL), who observed all simulation sessions, and (iii) post-simulation survey. RESULTS: After receiving ethical approval for the study, nine neonatal intensive care unit (NICU) nurses completed the online training and participated in the simulation and follow-up interview sessions. The KOL was also interviewed, and seven out of the nine NICU nurses answered the post-simulation survey. The video/audio analysis of the simulations revealed that participants were able to use and interpret the Digi-NewB interface. Interviews with simulation participants and the KOL, and feedback extracted from the survey, revealed that participants were overall satisfied with the training platform and perceived it as an efficient and effective method to deliver medical device training. CONCLUSIONS: This study developed an online training platform to train clinicians in the use of a critical care medical device and carried out a preliminary evaluation of the platform via a case study. The e-learning platform was designed to supplement and enhance other training approaches. Further research is required to evaluate the effectiveness of this approach.

Electroporation Parameters for Human Cardiomyocyte Ablation In Vitro.

Cardiac ablation with irreversible electroporation (IRE) is quickly being established as a modality of choice for atrial fibrillation treatment. While it has not yet been optimised, IRE has the potential to significantly limit collateral damage and improve cell-specific targeting associated with other energy sources. However, more tissue and cell-specific evidence is required to demonstrate the selective threshold parameters for human cells. The aim here is to determine the optimal ablation threshold parameters related to lesion size for human cardiomyocytes in 2D culture. Conventional biphasic pulses of different field strengths and on-times were delivered in a monolayer culture system of human AC16 cardiomyocytes. The dynamics of cell death and lesion dimensions were examined at different time points. Human cardiomyocytes are susceptible to significant electroporation and cell death at a field strength of 750 V/cm or higher with 100 µs pulses. Increasing the IRE on-time from 3 ms to 60 ms reduces the effective field threshold to 250 V/cm. Using very short pulses of 2 µs and 5 µs also causes significant cell death, but only at fields higher than 1000 V/cm. A longer on-time results in more cell death and induced greater lesion area in 2D models. In addition, different forms of cell death are predicted based on the evolution of cell death over time. This study presents important findings on the ability of different IRE parameters to induce human cardiomyocyte cell death. Lesion size can be tuned by appropriate choice of IRE parameters and cardiomyocytes display an upregulation of delayed cell death 24 h after electroporation, which is an important consideration for clinical practice.

Establishing electroporation thresholds for targeted cell specific cardiac ablation in a 2D culture model.

BACKGROUND: Irreversible electroporation has emerged as a new modality to overcome issues associated with other energy sources for cardiac ablation. Strong evidence on the optimal, effective, and selective voltage threshold is lacking for both in vitro and preclinical in vivo studies. The aim of this study is to examine the optimal threshold for selective cell ablation on cardiac associated cell types. METHODS: Conventional monophasic and biphasic pulses of different field strength were delivered in a monolayer culture system of cardiomyocytes, neurons, and adipocytes. The dynamics of cell death mechanisms were examined at different time points. RESULTS: Neurons exhibit higher susceptibility to electroporation and cell death at higher field strength of 1250 V/cm in comparison to cardiomyocytes. Cardiac adipocytes showed lower susceptibility to electroporation in comparison to other cell types. A significant proportion of cardiomyocytes recovered after 24 h postelectroporation, while neuronal cell death remained consistent but with a significant delayed cell death at a higher voltage threshold. Caspase 3/7 activity was observed in both cardiomyocytes and neurons, with a higher level of activity in cardiomyocytes in response to electroporation. Biphasic and monophasic pulses showed no significant difference in both cell types, and significantly lower cell death in neurons when inter pulse interval was reduced. CONCLUSIONS: This study presents important findings on the differences in the susceptibility of neurons and cardiomyocytes to irreversible electroporation. Cell type alone yielded selective and different dynamics in terms of the evolution and signaling mechanism of cell death in response to electroporation.

Design of a Planner-Based Intervention to Facilitate Diet Behaviour Change in Type 2 Diabetes.

Diet behaviour is influenced by the interplay of the physical and social environment as well as macro-level and individual factors. In this study, we focus on diet behaviour at an individual level and describe the design of a behaviour change artefact to support diet behaviour change in persons with type 2 diabetes. This artefact was designed using a human-centred design methodology and the Behaviour Change Wheel framework. The designed artefact sought to support diet behaviour change through the addition of healthy foods and the reduction or removal of unhealthy foods over a 12-week period. These targeted behaviours were supported by the enabling behaviours of water consumption and mindfulness practice. The artefact created was a behaviour change planner in calendar format, that incorporated behaviour change techniques and which focused on changing diet behaviour gradually over the 12-week period. The behaviour change planner forms part of a behaviour change intervention which also includes a preparatory workbook exercise and one-to-one action planning sessions and can be customised for each participant.

Establishing Irreversible Electroporation Electric Field Potential Threshold in A Suspension In Vitro Model for Cardiac and Neuronal Cells.

AIMS: Irreversible electroporation is an ablation technique being adapted for the treatment of atrial fibrillation. Currently, there are many differences reported in the in vitro and pre-clinical literature for the effective voltage threshold for ablation. The aim of this study is a direct comparison of different cell types within the cardiovascular system and identification of optimal voltage thresholds for selective cell ablation. METHODS: Monophasic voltage pulses were delivered in a cuvette suspension model. Cell viability and live-dead measurements of three different neuronal lines, cardiomyocytes, and cardiac fibroblasts were assessed under different voltage conditions. The immediate effects of voltage and the evolution of cell death was measured at three different time points post ablation. RESULTS: All neuronal and atrial cardiomyocyte lines showed cell viability of less than 20% at an electric field of 1000 V/cm when at least 30 pulses were applied with no significant difference amongst them. In contrast, cardiac fibroblasts showed an optimal threshold at 1250 V/cm with a minimum of 50 pulses. Cell death overtime showed an immediate or delayed cell death with a proportion of cell membranes re-sealing after three hours but no significant difference was observed between treatments after 24 h. CONCLUSIONS: The present data suggest that understanding the optimal threshold of irreversible electroporation is vital for achieving a safe ablation modality without any side-effect in nearby cells. Moreover, the evolution of cell death post electroporation is key to obtaining a full understanding of the effects of IRE and selection of an optimal ablation threshold.

In Vitro Model to Investigate Communication between Dorsal Root Ganglion and Spinal Cord Glia.

Chronic discogenic back pain is associated with increased inflammatory cytokine levels that can influence the proximal peripheral nervous system, namely the dorsal root ganglion (DRG). However, transition to chronic pain is widely thought to involve glial activation in the spinal cord. In this study, an in vitro model was used to evaluate the communication between DRG and spinal cord glia. Primary neonatal rat DRG cells were treated with/without inflammatory cytokines (TNF-α, IL-1ß, and IL-6). The conditioned media were collected at two time points (12 and 24 h) and applied to spinal cord mixed glial culture (MGC) for 24 h. Adult bovine DRG and spinal cord cell cultures were also tested, as an alternative large animal model, and results were compared with the neonatal rat findings. Compared with untreated DRG-conditioned medium, the second cytokine-treated DRG-conditioned medium (following medium change, thus containing solely DRG-derived molecules) elevated CD11b expression and calcium signal in neonatal rat microglia and enhanced Iba1 expression in adult bovine microglia. Cytokine treatment induced a DRG-mediated microgliosis. The described in vitro model allows the use of cells from large species and may represent an alternative to animal pain models (3R principles).

NRXN1α+/- is associated with increased excitability in ASD iPSC-derived neurons.

BACKGROUND: NRXN1 deletions are identified as one of major rare risk factors for autism spectrum disorder (ASD) and other neurodevelopmental disorders. ASD has 30% co-morbidity with epilepsy, and the latter is associated with excessive neuronal firing. NRXN1 encodes hundreds of presynaptic neuro-adhesion proteins categorized as NRXN1α/ß/γ. Previous studies on cultured cells show that the short NRXN1ß primarily exerts excitation effect, whereas the long NRXN1α which is more commonly deleted in patients involves in both excitation and inhibition. However, patient-derived models are essential for understanding functional consequences of NRXN1α deletions in human neurons. We recently derived induced pluripotent stem cells (iPSCs) from five controls and three ASD patients carrying NRXN1α+/- and showed increased calcium transients in patient neurons. METHODS: In this study we investigated the electrophysiological properties of iPSC-derived cortical neurons in control and ASD patients carrying NRXN1α+/- using patch clamping. Whole genome RNA sequencing was carried out to further understand the potential underlying molecular mechanism. RESULTS: NRXN1α+/- cortical neurons were shown to display larger sodium currents, higher AP amplitude and accelerated depolarization time. RNASeq analyses revealed transcriptomic changes with significant upregulation glutamatergic synapse and ion channels/transporter activity including voltage-gated potassium channels (GRIN1, GRIN3B, SLC17A6, CACNG3, CACNA1A, SHANK1), which are likely to couple with the increased excitability in NRXN1α+/- cortical neurons. CONCLUSIONS: Together with recent evidence of increased calcium transients, our results showed that human NRXN1α+/- isoform deletions altered neuronal excitability and non-synaptic function, and NRXN1α+/- patient iPSCs may be used as an ASD model for therapeutic development with calcium transients and excitability as readouts.

α-synuclein pathogenesis in hiPSC models of Parkinson's disease.

α-synuclein is an increasingly prominent player in the pathology of a variety of neurodegenerative conditions. Parkinson's disease (PD) is a neurodegenerative disorder that affects mainly the dopaminergic (DA) neurons in the substantia nigra of the brain. Typical of PD pathology is the finding of protein aggregations termed 'Lewy bodies' in the brain regions affected. α-synuclein is implicated in many disease states including dementia with Lewy bodies (DLB) and Alzheimer's disease. However, PD is the most common synucleinopathy and continues to be a significant focus of PD research in terms of the α-synuclein Lewy body pathology. Mutations in several genes are associated with PD development including SNCA, which encodes α-synuclein. A variety of model systems have been employed to study α-synuclein physiology and pathophysiology in an attempt to relate more closely to PD pathology. These models include cellular and animal system exploring transgenic technologies, viral vector expression and knockdown approaches, and models to study the potential prion protein-like effects of α-synuclein. The current review focuses on human induced pluripotent stem cell (iPSC) models with a specific focus on mutations or multiplications of the SNCA gene. iPSCs are a rapidly evolving technology with huge promise in the study of normal physiology and disease modeling in vitro. The ability to maintain a patient's genetic background and replicate similar cell phenotypes make iPSCs a powerful tool in the study of neurological diseases. This review focuses on the current knowledge about α-synuclein physiological function as well as its role in PD pathogenesis based on human iPSC models.

Ablation Modalities for Therapeutic Intervention in Arrhythmia-Related Cardiovascular Disease: Focus on Electroporation.

Targeted cellular ablation is being increasingly used in the treatment of arrhythmias and structural heart disease. Catheter-based ablation for atrial fibrillation (AF) is considered a safe and effective approach for patients who are medication refractory. Electroporation (EPo) employs electrical energy to disrupt cell membranes which has a minimally thermal effect. The nanopores that arise from EPo can be temporary or permanent. Reversible electroporation is transitory in nature and cell viability is maintained, whereas irreversible electroporation causes permanent pore formation, leading to loss of cellular homeostasis and cell death. Several studies report that EPo displays a degree of specificity in terms of the lethal threshold required to induce cell death in different tissues. However, significantly more research is required to scope the profile of EPo thresholds for specific cell types within complex tissues. Irreversible electroporation (IRE) as an ablative approach appears to overcome the significant negative effects associated with thermal based techniques, particularly collateral damage to surrounding structures. With further fine-tuning of parameters and longer and larger clinical trials, EPo may lead the way of adapting a safer and efficient ablation modality for the treatment of persistent AF.

Modelling Parkinson's Disease: iPSCs towards Better Understanding of Human Pathology.

Parkinson's Disease (PD) is a chronic neurodegenerative disorder characterized by motor and non-motor symptoms, among which are bradykinesia, rigidity, tremor as well as mental symptoms such as dementia. The underlying cause of Parkinson disease is degeneration of dopaminergic neurons. It has been challenging to develop an efficient animal model to accurately represent the complex phenotypes found with PD. However, it has become possible to recapitulate the myriad of phenotypes underlying the PD pathology by using human induced pluripotent stem cell (iPSC) technology. Patient-specific iPSC-derived dopaminergic neurons are available and present an opportunity to study many aspects of the PD phenotypes in a dish. In this review, we report the available data on iPSC-derived neurons derived from PD patients with identified gene mutations. Specifically, we will report on the key phenotypes of the generated iPSC-derived neurons from PD patients with different genetic background. Furthermore, we discuss the relationship these cellular phenotypes have to PD pathology and future challenges and prospects for iPSC modelling and understanding of the pathogenesis of PD.

A robust platform for high-throughput screening of therapeutic strategies for acute and chronic spinal cord injury.

Astrocytes and microglia are critical regulators of inflammatory cascade after spinal cord injury (SCI). Existing glial in vitro studies do not replicate inflammatory phases associated with SCI. Here, we report an in vitro model of mixed glial culture where inflammation is induced by the administration of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1ß, and interleukin-6) to promote pathologically relevant "acute" and "chronic" inflammatory phases. We observed SCI relevant differential modulation of inflammatory pathways, cytokines, chemokines, and growth factors over 21 days. Mitochondrial dysfunction was associated with a cytokine combination treatment. Highly expressed cytokine induced neutrophil chemoattractant (CINC-3) chemokine was used as a biomarker to establish an enzyme-linked immunosorbent assay-based high-throughput screening (HTS) platform. We screened a 786-compound drug library to demonstrate the efficacy of the HTS platform. The developed model is robust and will facilitate in vitro screening of anti-reactive glial therapeutics for the treatment of SCI.

Identifying Barriers and Facilitators to Diet and Physical Activity Behaviour Change in Type 2 Diabetes Using a Design Probe Methodology.

Treatment of Type 2 Diabetes (T2D) typically involves pharmacological methods and adjunct behavioural modifications, focused on changing diet and physical activity (PA) behaviours. Changing diet and physical activity behaviours is complex and any behavioural intervention in T2D, to be successful, must use an appropriate suite of behaviour change techniques (BCTs). In this study, we sought to understand the perceived barriers and facilitators to diet and PA behaviour change in persons with T2D, with a view to creating artefacts to facilitate the required behaviour changes. The Design Probe was chosen as the most appropriate design research instrument to capture the required data, as it enabled participants to reflect and self-document, over an extended period of time, on their daily lived experiences and, following this reflection, to identify their barriers and facilitators to diet and PA behaviour change. Design Probes were sent to 21 participants and 13 were fully completed. A reflective thematic analysis was carried out on the data, which identified themes of food environment, mental health, work schedule, planning, social support, cravings, economic circumstances and energy associated with diet behaviour. Similar themes were identified for PA as well as themes of physical health, weather, motivation and the physical environment.

Applicability and tolerability of electrical stimulation applied to the upper and lower leg skin surface for cueing applications in Parkinson's disease.

Due to possible sensory impairments in people with Parkinson's disease, several methodological aspects of electrical stimulation as a potential cueing method remain to be explored. This study aimed to investigate the applicability and tolerability of sensory and motor electrical stimulation in 10 people with Parkinson's disease. The study focused on assessing the electrical stimulation voltages and visual analogue scale discomfort scores at the electrical sensory, motor, discomfort, and pain thresholds. Results show that sensory electrical stimulation at the tibialis anterior, soleus, hamstrings, and quadriceps stimulation sites was applicable and tolerable for 6/10, 10/10, 9/10, and 10/10 participants, respectively. Furthermore, motor electrical stimulation at the tibialis anterior, soleus, hamstrings, and quadriceps stimulation sites were applicable and tolerable for 7/10, 7/10, 7/10, and 8/10 participants, respectively. Interestingly, the thresholds for the lower leg were higher than those of the upper leg. The data presented in this paper indicate that sensory and motor electrical stimulation is applicable and tolerable for cueing applications in people with Parkinson's disease. Sensory electrical stimulation was applicable and tolerable at the soleus and quadriceps sites. Motor electrical stimulation was not tolerable for two participants at any of the proposed stimulation sites. Therefore, future studies investigating motor electrical stimulation cueing, should apply it with caution in people with Parkinson's disease.

A high molecular weight hyaluronic acid biphasic dispersion as potential therapeutics for interstitial cystitis.

Interstitial cystitis (IC) is a progressive bladder disease characterized by increased urothelial permeability, inflammation of the bladder with abdominal pain. While there is no consensus on the etiology of the disease, it was believed that restoring the barrier between urinary solutes and (GAG) urothelium would interrupt the progression of this disease. Currently, several treatment options include intravesical delivery of hyaluronic acid (HA) and/or chondroitin sulfate solutions, through a catheter to restore the urothelial barrier, but have shown limited success in preclinical, clinical trials. Herein we report for the first time successful engineering and characterization of biphasic system developed by combining cross-linked hyaluronic acid and naïve HA solution to decrease inflammation and permeability in an in vitro model of interstitial cystitis. The cross-linking of HA was performed by 4-arm-polyethyeleneamine chemistry. The HA formulations were tested for their viscoelastic properties and the effects on cell metabolism, inflammatory markers, and permeability. Our study demonstrates the therapeutic effects of different ratios of the biphasic system and reports their ability to increase the barrier effect by decreasing the permeability and alteration of cell metabolism with respect to relative controls. Restoring the barrier by using biphasic system of HA therapy may be a promising approach to IC.