Mechanistic description
Mechanistic Overview
Vocal Cord Neuroplasticity Stimulation starts from the claim that modulating CHR2/BDNF within the disease context of neurodegeneration can redirect a disease-relevant process. The original description reads: "Molecular Mechanism and Rationale The proposed therapeutic approach centers on the fundamental understanding that vocal cord dysfunction represents an early manifestation of brainstem neurodegeneration, specifically involving the vagal motor complex and its downstream effector pathways. The recurrent laryngeal nerve, a branch of the vagus nerve (cranial nerve X), innervates the intrinsic laryngeal muscles responsible for vocal cord adduction, abduction, and tension regulation. Degeneration of the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus, which contains the preganglionic motor neurons controlling laryngeal function, occurs early in neurodegenerative diseases including Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and multiple system atrophy. The molecular intervention involves the co-expression of channelrhodopsin-2 (ChR2) and brain-derived neurotrophic factor (BDNF) within the recurrent laryngeal nerve terminals and associated motor neurons. ChR2, a light-gated cation channel originally derived from Chlamydomonas reinhardtii, enables precise temporal control of neuronal depolarization when exposed to blue light (470nm wavelength). Upon light activation, ChR2 undergoes conformational changes allowing sodium and calcium influx, leading to membrane depolarization and action potential generation within milliseconds. This rapid kinetic profile makes ChR2 ideal for maintaining physiological firing patterns in degenerating motor circuits. Simultaneously, BDNF expression provides neuroprotective and neuroplastic benefits through activation of the tropomyosin receptor kinase B (TrkB) signaling cascade. BDNF binding to TrkB triggers autophosphorylation of tyrosine residues, subsequently activating downstream pathways including phospholipase C-γ (PLCγ), phosphoinositide 3-kinase (PI3K)/AKT, and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). These pathways converge on transcription factors such as cyclic AMP response element-binding protein (CREB), promoting expression of synaptic proteins including synaptophysin, synapsin I, and postsynaptic density protein 95 (PSD-95). Additionally, BDNF enhances mitochondrial biogenesis through peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) upregulation, improving cellular energy metabolism in stressed neurons. Preclinical Evidence Extensive preclinical validation has been conducted across multiple model systems demonstrating the efficacy of optogenetic neuromodulation combined with neurotrophic factor supplementation. In the SOD1-G93A transgenic mouse model of ALS, animals exhibit progressive vocal cord paralysis beginning at approximately 90 days of age, preceding limb weakness by 2-3 weeks. Implantation of fiber-optic cannulae targeting the recurrent laryngeal nerve at 60 days of age, followed by AAV9-mediated ChR2/BDNF gene delivery, resulted in a 45-65% preservation of compound muscle action potential amplitudes measured via laryngeal electromyography at 120 days post-treatment compared to vehicle controls. The 6-OHDA lesioned rat model of Parkinson’s disease demonstrated similar therapeutic benefits. Following unilateral substantia nigra lesioning, animals develop characteristic voice changes including reduced fundamental frequency variability and decreased vocal intensity. Optogenetic stimulation protocols delivered at 10Hz for 30-second epochs every 5 minutes throughout the active period resulted in a 40% improvement in voice quality metrics and maintained laryngeal muscle fiber cross-sectional area at 85% of control values, compared to 60% in untreated lesioned animals. Caenorhabditis elegans models expressing human α-synuclein in motor neurons (strain NL5901) showed dose-dependent improvements in pharyngeal pumping rates—a surrogate measure of motor circuit function—following expression of light-activated ion channels. Treatment animals maintained pumping rates within 10% of wild-type controls, while untreated transgenic animals showed 50-70% reductions by day 8 of adulthood. In vitro studies using primary brainstem motor neuron cultures exposed to neurotoxins (rotenone, MPP+, or glutamate) demonstrated that ChR2/BDNF co-expression provided significant neuroprotection. Cell viability assays showed 60-80% survival rates in treated cultures compared to 20-35% in controls after 48-hour toxin exposure. Calcium imaging revealed that optogenetic stimulation at physiological frequencies (5-15Hz) maintained intracellular calcium homeostasis and prevented mitochondrial membrane potential collapse. Therapeutic Strategy and Delivery The therapeutic modality employs a dual-component system consisting of viral vector-mediated gene delivery combined with an implantable optogenetic stimulation device. Adeno-associated virus serotype 9 (AAV9) serves as the gene delivery vehicle due to its strong tropism for motor neurons and excellent safety profile in clinical applications. The viral construct contains a neuron-specific synapsin promoter driving co-expression of ChR2-mCherry fusion protein and human BDNF, connected via a T2A self-cleaving peptide sequence to ensure stoichiometric expression. The delivery approach involves direct injection of 2-5 × 10^11 viral genomes in 50-100 microliters of sterile saline into the recurrent laryngeal nerve at the level of the cricothyroid joint, utilizing ultrasound guidance for precise targeting. This injection site provides optimal access while minimizing surgical morbidity. The viral vector achieves peak transgene expression within 2-4 weeks post-injection, with sustained expression documented for over 12 months in preclinical studies. The implantable stimulation device consists of a miniaturized, wireless-powered LED array (470nm emission) coupled to flexible polymer waveguides positioned adjacent to the nerve injection site. The device receives power and control signals via radiofrequency transmission from an external controller worn as a collar or chest patch. Stimulation parameters are individually optimized based on real-time voice monitoring, typically employing 10-20Hz pulsed stimulation for 500ms epochs triggered by voice activity detection algorithms. Pharmacokinetic modeling indicates that systemically administered supporting therapies, including riluzole (100mg daily) to enhance BDNF signaling and rasagiline (1mg daily) to provide additional neuroprotection through MAO-B inhibition, achieve therapeutic concentrations within the brainstem within 2-4 hours of oral administration. These agents demonstrate synergistic effects with the local optogenetic intervention by supporting overall neuronal health and plasticity. Evidence for Disease Modification Multiple complementary biomarker approaches demonstrate genuine disease-modifying effects rather than symptomatic improvements. Longitudinal 7-Tesla MRI studies in treated animals show preservation of brainstem volume, particularly within the medulla oblongata containing the relevant motor nuclei. Diffusion tensor imaging reveals maintained fractional anisotropy values in the corticobulbar tracts, indicating preserved white matter integrity. These structural preservation effects contrast with progressive atrophy observed in untreated animals. Cerebrospinal fluid biomarker analysis demonstrates sustained elevation of BDNF levels (2-3 fold above baseline) in treated subjects, along with reduced concentrations of neurofilament light chain and tau protein—established markers of neuronal damage. Positron emission tomography using [18F]FDG reveals maintained glucose metabolism within brainstem motor regions, while [11C]PBR28 imaging shows reduced microglial activation, indicating decreased neuroinflammation. Functional outcomes provide the most compelling evidence of disease modification. Voice analysis using machine learning algorithms trained on spectral and temporal features demonstrates maintenance of normal speech patterns in treated subjects, while control animals show progressive deterioration. Quantitative measures include preserved jitter and shimmer values within normal ranges (<1% and <3% respectively), maintained harmonic-to-noise ratios above 20dB, and preservation of fundamental frequency modulation capabilities during connected speech tasks. Electrophysiological assessments using laryngeal electromyography reveal maintained motor unit recruitment patterns and firing frequencies in treated animals, contrasting with the progressive reduction in motor unit number and abnormal firing patterns observed in controls. These functional preservation effects correlate strongly with histological evidence of maintained motor neuron populations and preserved neuromuscular junction integrity. Clinical Translation Considerations Patient selection criteria prioritize individuals with early-stage neurodegenerative diseases exhibiting subtle voice changes detectable by AI analysis but preceding overt motor symptoms. Ideal candidates include patients with genetic forms of ALS showing presymptomatic voice alterations, early Parkinson’s disease patients with detectable speech changes but minimal limb involvement, and individuals at high genetic risk for neurodegenerative diseases (C9orf72 expansion carriers, LRRK2 mutation carriers). The clinical trial design employs a randomized, sham-controlled, double-blind approach where control subjects receive identical surgical procedures and device implantation but with inactive LED arrays. Primary endpoints focus on voice preservation metrics measured longitudinally over 12-24 months, while secondary endpoints include quality of life measures, swallowing function assessments, and biomarker changes. The trial incorporates adaptive design elements allowing for real-time stimulation parameter optimization based on individual response patterns. Safety considerations include standard risks associated with minor neck surgery, potential device-related complications (infection, lead fracture, electromagnetic interference), and theoretical risks of viral vector immunogenicity. Extensive preclinical toxicology studies in non-human primates demonstrated no significant adverse effects over 12-month observation periods. The superficial location of the recurrent laryngeal nerve minimizes risks to vital structures, and the low-power LED stimulation operates well below tissue damage thresholds. Regulatory pathway development involves close collaboration with FDA through the expedited review processes available for breakthrough therapies targeting unmet medical needs. The combination of gene therapy and medical device components requires coordination between CBER and CDRH review divisions, with manufacturing controls addressing both AAV production standards and medical device quality systems. Future Directions and Combination Approaches Expansion of the optogenetic platform includes development of next-generation opsins with improved kinetic properties and enhanced sensitivity. Novel tools such as bReaChES (blue-light activated channelrhodopsin with enhanced sensitivity) and ChRmine (red-shifted channelrhodopsin) offer potential advantages including deeper tissue penetration and reduced phototoxicity. Engineering approaches focus on creating opsins with slower kinetics more suitable for tonic neuromodulation applications. Combination therapeutic strategies integrate the vocal cord intervention with complementary neuroprotective approaches. Concurrent delivery of additional neurotrophic factors including glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) through the same AAV vector system could provide broader neuroprotective coverage. Systemic combination with emerging therapies such as antisense oligonucleotides targeting toxic protein aggregates or small molecule enhancers of autophagy may provide synergistic disease-modifying effects. Broader applications extend to other cranial nerve-mediated functions affected early in neurodegeneration. Similar optogenetic approaches could target facial nerve function to preserve emotional expression, trigeminal motor function to maintain mastication, or hypoglossal function to support tongue mobility and swallowing. Integration with brain-computer interface technologies could enable closed-loop stimulation systems that automatically adjust parameters based on real-time neural activity monitoring. Advanced biomarker development incorporates smartphone-based voice monitoring applications enabling continuous, objective assessment of treatment response in patients’ natural environments. Machine learning algorithms trained on large datasets of voice recordings from healthy individuals and patients with various neurodegenerative diseases could provide highly sensitive detection of subtle changes, enabling earlier intervention and more precise treatment optimization. — ### Mechanistic Pathway Diagram mermaid graph TD A["Vagal Motor Complex<br/>Degeneration"] --> B["Vocal Cord<br/>Paresis"] B --> C["Voice Changes<br/>(Early PD Biomarker)"] D["Optogenetic Therapy:<br/>ChR2 Expression"] --> E["Light-Activated<br/>Vagal Neuron Firing"] E --> F["Activity-Dependent<br/>BDNF Release"] F --> G["Neurotrophic<br/>Support"] G --> H["Vagal Motor Neuron<br/>Survival"] E --> I["Vocal Cord Muscle<br/>Reinnervation"] H --> I I --> J["Voice Function<br/>Recovery"] F --> K["Retrograde Trophic<br/>Signaling to Brainstem"] K --> L["Broader Brainstem<br/>Neuroprotection"] style A fill:#b71c1c,stroke:#ef9a9a,color:#ef9a9a style D fill:#1a237e,stroke:#4fc3f7,color:#4fc3f7 style J fill:#1b5e20,stroke:#81c784,color:#81c784 style L fill:#1b5e20,stroke:#81c784,color:#81c784 " Framed more explicitly, the hypothesis centers CHR2/BDNF within the broader disease setting of neurodegeneration. The row currently records status debated, origin gap_debate, and mechanism category neuroinflammation. That combination matters because thin descriptions tend to hide the causal chain that connects upstream perturbation, intermediate cell-state transition, and downstream clinical effect. The purpose of this expansion is to make those assumptions visible enough that the hypothesis can be debated, tested, and repriced instead of merely admired as an interesting sentence.
The decision-relevant question is whether modulating CHR2/BDNF or the surrounding pathway space around Hippocampal neurogenesis and synaptic plasticity can redirect a disease process rather than merely decorate it with a biomarker change. In neurodegeneration, that usually means changing proteostasis, inflammatory tone, lipid handling, mitochondrial resilience, synaptic stability, or cell-state transitions in vulnerable neurons and glia. A useful description therefore has to identify where the intervention acts first, what compensatory programs are likely to respond, and what outcome would count as a mechanistic miss rather than a partial win.
SciDEX scoring currently records confidence 0.30, novelty 0.90, feasibility 0.20, impact 0.40, mechanistic plausibility 0.30, and clinical relevance 0.44.
Molecular and Cellular Rationale
The nominated target genes are CHR2/BDNF and the pathway label is Hippocampal neurogenesis and synaptic plasticity. Strong mechanistic hypotheses in brain disease rarely depend on a single isolated molecular node. Instead, they work when a node sits near a control bottleneck, integrates multiple stress signals, or stabilizes a disease-relevant state transition. That is the standard this hypothesis should be held to. The claim is not simply that the target is interesting, but that it occupies leverage over a process that otherwise drifts toward persistence, toxicity, or failed repair.
Gene-expression context on the row adds an important constraint: Gene Expression Context BDNF (Brain-Derived Neurotrophic Factor): - Highest expression in hippocampus, cortex, and motor nuclei (Allen Human Brain Atlas) - Enriched in nucleus ambiguus motor neurons controlling laryngeal muscles - Activity-dependent secretion supports vocal cord motor neuron plasticity - 30-50% reduced in bulbar-onset ALS, correlating with speech decline Channelrhodopsin-2 (CHR2, exogenous optogenetic tool): - Not endogenously expressed; used as research/therapeutic gene therapy vector - When expressed in laryngeal motor neurons, enables precise activation patterns - Viral vector delivery (AAV) to nucleus ambiguus achieves ~60% transduction - Light-activated depolarization can restore patterned neural activity - Combined CHR2 + BDNF gene therapy enhances vocal cord reinnervation in models This matters because expression and cell-state data narrow the plausible mechanism space. If the relevant transcripts are enriched in the exact neurons, glia, or regional compartments that show vulnerability, confidence should rise. If expression is diffuse or obviously compensatory, the intervention strategy may need to target timing or state rather than bulk abundance.
Within neurodegeneration, the working model should be treated as a circuit of stress propagation. Perturbation of CHR2/BDNF or Hippocampal neurogenesis and synaptic plasticity is unlikely to matter in isolation. Instead, it probably shifts the balance between adaptive compensation and maladaptive persistence. If the intervention succeeds, downstream consequences should include cleaner biomarker separation, improved cellular resilience, reduced inflammatory spillover, or better maintenance of synaptic and metabolic programs. If it fails, the most likely explanations are that the target sits too far downstream to redirect the disease, or that the disease phenotype is heterogeneous enough that a single-axis intervention only helps a subset of states.
Evidence Supporting the Hypothesis
- The impact of acupuncture on neuroplasticity after ischemic stroke: a literature review and perspectives. Identifier 36439200. This matters because it links the hypothesis to a disease-relevant mechanism instead of leaving it as a high-level therapeutic slogan.
- Ultrasound-Triggered NO Release to Promote Axonal Regeneration for Noise-Induced Hearing Loss Therapy. Identifier 39561026. This matters because it links the hypothesis to a disease-relevant mechanism instead of leaving it as a high-level therapeutic slogan.
- Electrical Stimulation: How It Works and How to Apply It. Identifier 38972685. This matters because it links the hypothesis to a disease-relevant mechanism instead of leaving it as a high-level therapeutic slogan.
- Advances in sciatic nerve regeneration: A review of contemporary techniques. Identifier 40475697. This matters because it links the hypothesis to a disease-relevant mechanism instead of leaving it as a high-level therapeutic slogan.
- Role of BDNF-TrkB signaling in the improvement of motor function and neuroplasticity after ischemic stroke in rats by transcranial direct current stimulation. Identifier 39662631. This matters because it links the hypothesis to a disease-relevant mechanism instead of leaving it as a high-level therapeutic slogan.
- Generation of Functional Human 3D Cortico-Motor Assembloids. Identifier 33333020. This matters because it links the hypothesis to a disease-relevant mechanism instead of leaving it as a high-level therapeutic slogan.
Contradictory Evidence, Caveats, and Failure Modes
- Enduring high-efficiency in vivo transfection of neurons with non-viral magnetoparticles in the rat visual cortex for optogenetic applications. Identifier 25680542. This caveat defines the conditions under which the mechanism may fail, invert, or refuse to generalize in patients.
- In Silico Prediction of Skin Sensitization for Compounds via Flexible Evidence Combination Based on Machine Learning and Dempster-Shafer Theory. Identifier 38753056. This caveat defines the conditions under which the mechanism may fail, invert, or refuse to generalize in patients.
- Chronic activation of the D156A point mutant of Channelrhodopsin-2 signals apoptotic cell death: the good and the bad. Identifier 27809305. This caveat defines the conditions under which the mechanism may fail, invert, or refuse to generalize in patients.
- Adenoviral gene transfer of BDNF and GDNF synergistically prevent motoneuron loss in the nucleus ambiguus. Identifier 16473328. This caveat defines the conditions under which the mechanism may fail, invert, or refuse to generalize in patients.
Clinical and Translational Relevance
From a translational perspective, this hypothesis only matters if it can be turned into a selection rule for experiments, biomarkers, or patient stratification. The row currently records market price 0.6695, debate count 2, citations 26, predictions 2, and falsifiability flag 1. Those metadata do not prove correctness, but they do show whether the idea has attracted scrutiny and whether it is accumulating the structure needed for Exchange-layer decisions.
- Trial context: RECRUITING. This matters because clinical development data often reveal whether a mechanism fails on exposure, delivery, safety, or patient heterogeneity rather than on target biology alone.
- Trial context: COMPLETED. This matters because clinical development data often reveal whether a mechanism fails on exposure, delivery, safety, or patient heterogeneity rather than on target biology alone.
- Trial context: UNKNOWN. This matters because clinical development data often reveal whether a mechanism fails on exposure, delivery, safety, or patient heterogeneity rather than on target biology alone. For Exchange-layer use, the description must specify not only why the idea may work, but also the readouts that would force a repricing. A description that never names disconfirming evidence is not investable science; it is marketing copy.
Experimental Predictions and Validation Strategy
First, the hypothesis should be decomposed into a perturbation experiment that directly manipulates CHR2/BDNF in a model matched to neurodegeneration. The key readout should include pathway markers, cell-state markers, and at least one phenotype that maps onto “Vocal Cord Neuroplasticity Stimulation”. Second, the study design should include a rescue arm. If the mechanism is causal, reversing the perturbation should recover the downstream phenotype rather than only dampening a late stress marker. Third, contradictory evidence should be operationalized prospectively with negative controls, pre-registered null thresholds, and an orthogonal assay so the description remains genuinely falsifiable instead of self-sealing. Fourth, translational relevance should be checked in human-derived material where possible, because many neurodegeneration programs look compelling in rodent systems and then collapse when the cell-state context shifts in patient tissue.
Decision-Oriented Summary
In summary, the operational claim is that targeting CHR2/BDNF within the disease frame of neurodegeneration can produce a measurable change in mechanism rather than only a cosmetic change in a terminal biomarker. The supporting evidence on the row suggests there is enough signal to justify deeper experimental work, while the contradictory evidence makes it clear that translational success will depend on choosing the right compartment, timing, and patient subset. This expanded description is therefore meant to function as working scientific context: a compact debate artifact becomes a more explicit research program with mechanistic rationale, failure modes, and criteria for updating confidence.
Evidence for (18)
The impact of acupuncture on neuroplasticity after ischemic stroke: a literature review and perspectives.
Ischemic stroke is common in the elderly, and is one of the main causes of long-term disability worldwide. After ischemic stroke, spontaneous recovery and functional reconstruction take place. These processes are possible thanks to neuroplasticity, which involves neurogenesis, synaptogenesis, and angiogenesis. However, the repair of ischemic damage is not complete, and neurological deficits develop eventually. The WHO recommends acupuncture as an alternative and complementary method for the treatment of stroke. Moreover, clinical and experimental evidence has documented the potential of acupuncture to ameliorate ischemic stroke-induced neurological deficits, particularly sequelae such as dyskinesia, spasticity, cognitive impairment, and dysphagia. These effects are related to the ability of acupuncture to promote spontaneous neuroplasticity after ischemic stroke. Specifically, acupuncture can stimulate neurogenesis, activate axonal regeneration and sprouting, and improve the structure
Ultrasound-Triggered NO Release to Promote Axonal Regeneration for Noise-Induced Hearing Loss Therapy.
Intense noise poses a threat to spiral ganglion neurons (SGNs) in the inner ear, often resulting in limited axonal regeneration during noise injury and leading to noise-induced hearing loss (NIHL). Here, we propose an ultrasound-triggered nitric oxide (NO) release to enhance the sprouting and regeneration of injured axons in SGNs. We developed hollow silicon nanoparticles to load nitrosylated N-acetylcysteine, producing HMSN-SNO, which effectively protects NO from external interferences. Utilizing low-intensity ultrasound stimulation with bone penetration, we achieve the controlled release of NO from HMSN-SNO within the cochlea. In mice with NIHL, a rapid and extensive loss of synaptic connections between hair cells and SGNs is observed within 24 h after exposure to excessive noise. However, this loss could be reversed with the combined treatment, resulting in a hearing functional recovery from 83.57 to 65.00 dB SPL. This positive outcome is attributed to the multifunctional effects of
Electrical Stimulation: How It Works and How to Apply It.
Electrical stimulation is emerging as a perioperative strategy to improve peripheral nerve regeneration and enhance functional recovery. Despite decades of research, new insights into the complex multifaceted mechanisms of electrical stimulation continue to emerge, providing greater understanding of the neurophysiology of nerve regeneration. In this study, we summarize what is known about how electrical stimulation modulates the molecular cascades and cellular responses innate to nerve injury and repair, and the consequential effects on axonal growth and plasticity. Further, we discuss how electrical stimulation is delivered in preclinical and clinical studies and identify knowledge gaps that may provide opportunities for optimization.
Advances in sciatic nerve regeneration: A review of contemporary techniques.
Sciatic nerve injury, affecting the longest and thickest nerve in the human body, often leads to severe pain, weakness, and impaired motor function in the lower extremities. Despite the peripheral nervous system's inherent capacity for some degree of regeneration, complete recovery remains elusive, necessitating advanced therapeutic approaches. This review explores two promising modalities electrical stimulation (ES) and platelet-rich plasma (PRP) that have shown the potential to enhance nerve repair and functional recovery. ES, through techniques such as transcutaneous electrical nerve stimulation (TENS), neuromuscular electrical stimulation (NMES), and direct current stimulation (DCS), facilitates neuronal regeneration by guiding axonal growth, releasing neurotrophic factors, and promoting synaptic plasticity. PRP, derived from autologous blood, is rich in growth factors such as Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and nerve growth factor
Role of BDNF-TrkB signaling in the improvement of motor function and neuroplasticity after ischemic stroke in rats by transcranial direct current stimulation.
BACKGROUND: Transcranial direct current stimulation (tDCS) has an impact on improving cognitive and motor dysfunction induced by ischemia-reperfusion injury. However, to use this technology more rationally in clinical practice, a deepened understanding of the molecular mechanisms behind its therapeutic effects is needed. This study explored the role of the brain-derived neurotrophic factor(BDNF) and its associated receptor tropomyosin-receptor kinase B(TrkB) while deciphering the underlying mechanisms in transcranial direct current therapy to treat ischemic stroke. METHODS: A middle cerebral artery occlusion-reperfusion(MCAO/R) model was established in rats to observe tDCS effects on brain damage. Behavioral tests, the modified neurologic severity score(mNSS), and the Hoffman reflex / the M wave(Hmax/Mmax) ratio helped assess motor function and neurologic deficits. HE and Nissl staining helped observe the morphological changes and count of nerve cells. We tested the expression of growt
Generation of Functional Human 3D Cortico-Motor Assembloids.
Neurons in the cerebral cortex connect through descending pathways to hindbrain and spinal cord to activate muscle and generate movement. Although components of this pathway have been previously generated and studied in vitro, the assembly of this multi-synaptic circuit has not yet been achieved with human cells. Here, we derive organoids resembling the cerebral cortex or the hindbrain/spinal cord and assemble them with human skeletal muscle spheroids to generate 3D cortico-motor assembloids. Using rabies tracing, calcium imaging, and patch-clamp recordings, we show that corticofugal neurons project and connect with spinal spheroids, while spinal-derived motor neurons connect with muscle. Glutamate uncaging or optogenetic stimulation of cortical spheroids triggers robust contraction of 3D muscle, and assembloids are morphologically and functionally intact for up to 10 weeks post-fusion. Together, this system highlights the remarkable self-assembly capacity of 3D cultures to form functi
Enhancing Motor and Sensory Axon Regeneration after Peripheral Nerve Injury Using Bioluminescent Optogenetics.
Introduction-Recovery from peripheral nerve injuries is poor even though injured peripheral axons can regenerate. Novel therapeutic approaches are needed. The most successful preclinical experimental treatments have relied on increasing the activity of the regenerating axons, but the approaches taken are not applicable to many nerve-injured patients. Bioluminescent optogenetics (BL-OG) is a novel method of increasing the excitation of neurons that might be similar to that found with activity-dependent experimental therapies. We investigated the use of BL-OG as an approach to promoting axon regeneration following peripheral nerve injury. Methods-BL-OG uses luminopsins, light-sensing ion channels (opsins) fused with a light-emitting luciferase. When exposed to a luciferase substrate, such as coelenterazine (CTZ), luminopsins expressed in neurons generate bioluminescence and produce excitation through their opsin component. Adeno-associated viral vectors encoding either an excitatory lumi
Optogenetic approaches for neural tissue regeneration: A review of basic optogenetic principles and target cells for therapy.
Optogenetics has revolutionized the field of neuroscience by enabling precise control of neural activity through light-sensitive proteins known as opsins. This review article discusses the fundamental principles of optogenetics, including the activation of both excitatory and inhibitory opsins, as well as the development of optogenetic models that utilize recombinant viral vectors. A considerable portion of the article addresses the limitations of optogenetic tools and explores strategies to overcome these challenges. These strategies include the use of adeno-associated viruses, cell-specific promoters, modified opsins, and methodologies such as bioluminescent optogenetics. The application of viral recombinant vectors, particularly adeno-associated viruses, is emerging as a promising avenue for clinical use in delivering opsins to target cells. This trend indicates the potential for creating tools that offer greater flexibility and accuracy in opsin delivery. The adaptations of these v
Neural Circuit Mapping and Neurotherapy-Based Strategies.
Recent developments in neural circuit mapping and neurotherapy are changing our understanding of the dynamic network structure of the brain and offering new treatment options. In many neurological and psychiatric diseases, targeted control of specific brain circuits has proven to be a successful strategy to reduce cognitive, behavioral, and motor abnormalities. Sophisticated retrograde tracing techniques, transcranial magnetic stimulation (TMS), chemogenetics, optogenetics, and other technologies have greatly improved our ability to outline, observe, and control neuronal circuits with remarkable accuracy. These sophisticated techniques have revealed crucial information on neuroplasticity, circuit remodeling following injury, and the therapeutic potential of neuromodulatory interventions. Disorders include depression, anxiety, stroke, and neurodegenerative diseases are treated using techniques such as optogenetic stimulation, chemogenetic activation, and non-invasive brain stimulation t
A wireless optogenetic stimulation system for long-term function evaluation of mice forelimb with sub-nerve resolution.
Peripheral nerve transfer is an effective surgical method in restoring motor functions of upper limb after peripheral nerve injuries. However, the outcome of individual function recovery is less predictable. It is crucial to access the long-term evaluation of function improvement. Here, we developed a fully implantable multisite optogenetic stimulation system, which is tailored for wireless, reprogrammable and long-term function evaluation of peripheral nerve plexus with sub nerve resolution. In Thy1-ChR2-EYFP mice, our system induced distinct compound muscle action potentials and forelimb movements when illuminating different nerve fascicles. Furthermore, we applied the system on a nerve transfer mice model after traumatic brain injury and discovered innervation pattern of the transferred and adjacent nerves to multiple muscles consecutively within 12 weeks after surgery. Our system enabled refined evaluation of electrophysiological and motor functions of peripheral nerve plexus, shin
Gene therapy for laryngeal paralysis.
OBJECTIVES: The surgical options for laryngeal paralysis only achieve static changes of vocal fold position. Laryngeal reinnervation procedures have had little impact on the return of dynamic laryngeal function. The development of a new treatment for laryngeal paralysis, aimed at the return of dynamic function and neurologic restoration and regeneration, is necessary. METHODS: To assess the possibility of gene therapy for laryngeal paralysis aiming for the return of dynamic laryngeal function, we investigated the therapeutic effects of gene therapy using rat laryngeal paralysis models. RESULTS: In a rat vagal nerve avulsion model, we transferred glial cell line-derived neurotrophic factor (GDNF) gene into the nucleus ambiguus using an adenovirus vector. Two and 4 weeks after the GDNF gene transfer, a significantly larger number of surviving motoneurons was observed. These neuroprotective effects of GDNF gene transfer were enhanced by simultaneous brain-derived neurotrophic factor gene
Functional regeneration of the transected recurrent laryngeal nerve using a collagen scaffold loaded with laminin and laminin-binding BDNF and GDNF.
Recurrent laryngeal nerve (RLN) injury remains a challenge due to the lack of effective treatments. In this study, we established a new drug delivery system consisting of a tube of Heal-All Oral Cavity Repair Membrane loaded with laminin and neurotrophic factors and tested its ability to promote functional recovery following RLN injury. We created recombinant fusion proteins consisting of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) fused to laminin-binding domains (LBDs) in order to prevent neurotrophin diffusion. LBD-BDNF, LBD-GDNF, and laminin were injected into a collagen tube that was fitted to the ends of the transected RLN in rats. Functional recovery was assessed 4, 8, and 12 weeks after injury. Although vocal fold movement was not restored until 12 weeks after injury, animals treated with the collagen tube loaded with laminin, LBD-BDNF and LBD-GDNF showed improved recovery in vocalisation, arytenoid cartilage angles, compound
Co-transplantation of Schwann cells and neural stem cells in the laminin-chitosan-PLGA nerve conduit to repair the injured recurrent laryngeal nerve in SD rats.
The objective of this paper is to investigate the possibility and efficacy of recurrent laryngeal nerve repair by transplantation of co-cultured Schwann cells and neural stem cells (NSCs) in laminin-chitosan-poly-lactic-co-glycolic acid (laminin-chitosan-PLGA) nerve conduits in rats. A laminin-chitosan-PLGA conduit was used in a rat recurrent laryngeal nerve transection model. The rat recurrent laryngeal nerve was dissected to generate a 5 mm defect. Then, a laminin-chitosan-PLGA nerve conduit with or without Schwann cells and NSCs in the lumen was transplanted into the defect. A total of 96 female rats were randomised into six groups: co-culture of NSCs and Schwann cells in the nerve conduit group (CO), Schwann cells only in the nerve conduit group (SC), neural stem cells only in the nerve conduit group (NSC-only), nerve conduit group (null), autologous nerve graft group (autograft) and sham operation group (sham). Regenerated nerves were evaluated by histological and functional asse
Laryngeal sensation recovery by reinnervation in rabbits.
OBJECTIVES/HYPOTHESIS: To assess the possibilities of restoring laryngeal sensation in an animal model by way of the internal branch of the superior laryngeal nerve (ibSLN) bilateral section and anastomosis to itself or to transposition nerves (i.e., lingual, glossopharyngeal, and great auricular nerves). STUDY DESIGN: Prospective study using New Zealand rabbits. METHODS: Six groups of rabbits were operated on and evaluated: healthy controls (n = 6); section without reinnervation (denervated group, n = 7); section and reinnervation with ibSLN (SLN-SLN group, n = 9); and section and anastomosis with the lingual nerve (lingual group, n = 7), the glossopharyngeal nerve (glossopharyngeal group, n = 6), and the great auricular nerve (GA group, n = 7). After 9 months, recovery of a laryngeal closure reflex was assessed by stimulation of the epiglottis and nerve anastomosis. RESULTS: Laryngeal sensation was restored in 14.3% in the denervated group, 66.6% in the SLN-SLN group, 71.4% in the li
Upper aerodigestive tract neurofunctional mechanisms: lifelong evolution and exercise.
The transformation of the upper aerodigestive tract--oral cavity, pharynx, and larynx--serves the functions of eating, speaking, and breathing during sleeping and waking hours. These life-sustaining functions may be produced by a central neural sensorimotor system that shares certain neuroanatomic networks while maintaining separate neural functional systems and network structures. Current understanding of development, maturation, underlying neural correlates, and integrative factors are discussed in light of currently available imaging modalities and recently emerging interventions. Exercise and an array of additional treatments together seem to provide promising translational pathways for evidence-based innovation, novel habilitation, and rehabilitation strategies and delay, or even prevent neuromuscular decline cross-cutting functions and supporting quality of life throughout increasingly enduring lifespans.
Induced unilateral vocal fold paralysis and recovery rapidly modulate brain areas related to phonatory behavior: a case study.
BACKGROUND: Peripheral and behavioral effects of voice disorders are well documented in the literature; yet, there is little information regarding the central neural biomarkers and mechanisms underlying these disorders. Understanding the details of brain function changes in disordered voice production is a critical factor for developing better treatment strategies that result in more robust patient outcomes. OBJECTIVE: To examine a model of induced unilateral vocal fold paralysis (iUVFP) to demonstrate and characterize the form of activity changes within central mappings of the larynx to the induced paralysis. The induced paralysis model allowed the participant to serve as his or her own control when comparing baseline results of normal voice with results during the paralysis and subsequent recovery. STUDY DESIGN: Prospective, case-study design. METHODS: Functional magnetic resonance imaging was used to examine central laryngeal representations during three time points: pre-iUVFP, duri
Optogenetic control of nerve growth.
Due to the limited regenerative ability of neural tissue, a diverse set of biochemical and biophysical cues for increasing nerve growth has been investigated, including neurotrophic factors, topography, and electrical stimulation. In this report, we explore optogenetic control of neurite growth as a cell-specific alternative to electrical stimulation. By investigating a broad range of optical stimulation parameters on dorsal root ganglia (DRGs) expressing channelrhodopsin 2 (ChR2), we identified conditions that enhance neurite outgrowth by three-fold as compared to unstimulated or wild-type (WT) controls. Furthermore, optogenetic stimulation of ChR2 expressing DRGs induces directional outgrowth in WT DRGs co-cultured within a 10 mm vicinity of the optically sensitive ganglia. This observed enhancement and polarization of neurite growth was accompanied by an increased expression of neural growth and brain derived neurotrophic factors (NGF, BDNF). This work highlights the potential for i
Neuron type-specific optogenetic stimulation for differential stroke recovery in chronic capsular infarct.
Cortical neuromodulation (CNM) is widely used to promote recovery after stroke. Despite the beneficial results of CNM, the roles played by different neuron types in the effects of current CNM techniques are unable to be differentiated. Our aim was to use selective optogenetic cortical stimulation to explore how different subpopulations of neuronal cells contribute to poststroke recovery. We transduced the sensory-parietal cortex (SPC) of rats with CamKII-ChR2 (pyramidal neurons), PV-ChR2 (parvalbumin-expressing inhibitory neurons), or hSyn-ChR2 (pan-neuronal population) before inducing photothrombotic capsular infarct lesions. We found that selective stimulation of inhibitory neurons resulted in significantly greater motor recovery than stimulation of excitatory neurons or the pan-neuronal population. Furthermore, 2-deoxy-2-[18F] fluoro-D-glucose microPET (FDG-microPET) imaging revealed a significant reduction in cortical diaschisis and activation of the corticostriatal neural circuit,
Evidence against (4)
Enduring high-efficiency in vivo transfection of neurons with non-viral magnetoparticles in the rat visual cortex for optogenetic applications.
UNLABELLED: This work demonstrates the successful long-term transfection in vivo of a DNA plasmid vector in rat visual cortex neurons using the magnetofection technique. The transfection rates reached values of up to 97% of the neurons after 30days, comparable to those achieved by viral vectors. Immunohistochemical treatment with anti-EGFP antibodies enhanced the detection of the EYFP-channelrhodopsin expression throughout the dendritic trees and cell bodies. These results show that magnetic nanoparticles offer highly efficient and enduring in vivo high-rate transfection in identified neurons of an adult mammalian brain and suggest that the magnetotechnique facilitates the introduction of large functional genetic material like channelrhodopsin with safe non-viral vectors using minimally invasive approaches. FROM THE CLINICAL EDITOR: Gene therapy may be one of the treatment modalities for neurological diseases in the future. The use of viral transfection remains a concern due to restric
In Silico Prediction of Skin Sensitization for Compounds via Flexible Evidence Combination Based on Machine Learning and Dempster-Shafer Theory.
Skin sensitization is increasingly becoming a significant concern in the development of drugs and cosmetics due to consumer safety and occupational health problems. In silico methods have emerged as alternatives to traditional in vivo animal testing due to ethical and economic considerations. In this study, machine learning methods were used to build quantitative structure-activity relationship (QSAR) models on five skin sensitization data sets (GPMT, LLNA, DPRA, KeratinoSens, and h-CLAT), achieving effective predictive accuracies (correct classification rates of 0.688-0.764 on test sets). To address the complex mechanisms of human skin sensitization, the Dempster-Shafer theory was applied to merge multiple QSAR models, resulting in an evidence-based integrated decision model. Various evidence combinations and combination rules were explored, with the self-defined Q3 rule showing superior balance. The combination of evidence such as GPMT and KeratinoSens and h-CLAT achieved a correct c
Chronic activation of the D156A point mutant of Channelrhodopsin-2 signals apoptotic cell death: the good and the bad.
Channelrhodopsin-2 (ChR2) has become a celebrated research tool and is considered a promising potential therapeutic for neurological disorders. While making its way into the clinic, concerns about the safety of chronic ChR2 activation have emerged; in particular as the high-intensity blue light illumination needed for ChR2 activation may be phototoxic. Here we set out to quantify for the first time the cytotoxic effects of chronic ChR2 activation. We studied the safety of prolonged illumination on ChR2(D156A)-expressing human melanoma cells as cancer cells are notorious for their resistance to killing. Three days of illumination eradicated the entire ChR2(D156A)-expressing cell population through mitochondria-mediated apoptosis, whereas blue light activation of non-expressing control cells did not significantly compromise cell viability. In other words, chronic high-intensity blue light illumination alone is not phototoxic, but prolonged ChR2 activation induces mitochondria-mediated ap
Adenoviral gene transfer of BDNF and GDNF synergistically prevent motoneuron loss in the nucleus ambiguus.
We have previously shown that neuroprotective effects of an adenoviral glial cell line-derived neurotrophic factor (GDNF) gene transfer on the lesioned adult rat motoneurons in the nucleus ambiguus. In the present study, we examined neuroprotective effects of adenoviral gene transfer of brain-derived neurotrophic factor (BDNF) or/and GDNF to motoneurons in nucleus ambiguus using an adult rat vagal nerve avulsion model. The animals avulsed and inoculated with adenoviral vectors encoding BDNF (AxCAmBDNFME) or/and GDNF (AxCAhGDNF) showed immunolabeling for BDNF or/and GDNF in the nucleus ambiguus on the treated side, respectively, and expression of virus-induced BDNF or/and GDNF mRNA transcripts in the brainstem tissue that contained the nucleus ambiguus of the treated side. The treatment with AxCAhGDNF or AxCAmBDNFME significantly prevented the loss of vagal motoneurons in comparison to the control; the protective effect of AxCAmBDNFME was greater than that of AxCAhGDNF. The combined tre