Phytic Acid Maintains Peripheral Neuron Integrity and Enhances Survivability against Platinum-Induced Degeneration via Reducing Reactive Oxygen Species and Enhancing Mitochondrial Membrane Potential.

Phytic acid (PA) has been reported to possess anti-inflammatory and antioxidant properties that are critical for neuroprotection in neuronal disorders. This raises the question of whether PA can effectively protect sensory neurons against chemotherapy-induced peripheral neuropathy (CIPN). Peripheral neuropathy is a dose-limiting side effect of chemotherapy treatment often characterized by severe and abnormal pain in hands and feet resulting from peripheral nerve degeneration. Currently, there are no effective treatments available that can prevent or cure peripheral neuropathies other than symptomatic management. Herein, we aim to demonstrate the neuroprotective effects of PA against the neurodegeneration induced by the chemotherapeutics cisplatin (CDDP) and oxaliplatin. Further aims of this study are to provide the proposed mechanism of PA-mediated neuroprotection. The neuronal protection and survivability against CDDP were characterized by axon length measurements and cell body counting of the dorsal root ganglia (DRG) neurons. A cellular phenotype study was conducted microscopically. Intracellular reactive oxygen species (ROS) was estimated by fluorogenic probe dichlorofluorescein. Likewise, mitochondrial membrane potential (MMP) was assessed by fluorescent MitoTracker Orange CMTMRos. Similarly, the mitochondria-localized superoxide anion radical in response to CDDP with and without PA was evaluated. The culture of primary DRG neurons with CDDP reduced axon length and overall neuronal survival. However, cotreatment with PA demonstrated that axons were completely protected and showed increased stability up to the 45-day test duration, which is comparable to samples treated with PA alone and control. Notably, PA treatment scavenged the mitochondria-specific superoxide radicals and overall intracellular ROS that were largely induced by CDDP and simultaneously restored MMP. These results are credited to the underlying neuroprotection of PA in a platinum-treated condition. The results also exhibited that PA had a synergistic anticancer effect with CDDP in ovarian cancer in vitro models. For the first time, PA's potency against CDDP-induced PN is demonstrated systematically. The overall findings of this study suggest the application of PA in CIPN prevention and therapeutic purposes.

Electrical stimulation enhances mitochondrial trafficking as a neuroprotective mechanism against chemotherapy-induced peripheral neuropathy.

Electrical stimulation (ESTIM) has shown to be an effective symptomatic treatment to treat pain associated with peripheral nerve damage. However, the neuroprotective mechanism of ESTIM on peripheral neuropathies is still unknown. In this study, we identified that ESTIM has the ability to enhance mitochondrial trafficking as a neuroprotective mechanism against chemotherapy-induced peripheral neuropathies (CIPNs). CIPN is a debilitating and painful sequalae of anti-cancer chemotherapy treatment which results in degeneration of peripheral nerves. Mitochondrial dynamics were analyzed within axons in response to two different antineoplastic mechanisms by chemotherapy drug treatments paclitaxel and oxaliplatin in vitro. Mitochondrial trafficking response to chemotherapy drug treatment was observed to decrease in conjunction with degeneration of distal axons. Using low-frequency ESTIM, we observed enhanced mitochondrial trafficking to be a neuroprotective mechanism against CIPN. This study confirms ESTIM enhances regeneration of peripheral nerves by increased mitochondrial trafficking.

Mitochondrial trafficking as a protective mechanism against chemotherapy drug-induced peripheral neuropathy: Identifying the key site of action.

AIMS: Chemotherapy induced peripheral neuropathy (CIPN) is a common side effect seen in patients who have undergone most chemotherapy treatments to which there are currently no treatment methods. CIPN has been shown to cause axonal degeneration leading to Peripheral Neuropathy (PN), which can lead to major dosage reduction and may prevent further chemotherapy treatment due to oftentimes debilitating pain. Previously, we have determined the site-specific action of Paclitaxel (PTX), a microtubule targeting agent, as well as the neuroprotective effect of Fluocinolone Acetonide (FA) against Paclitaxel Induced Peripheral Neuropathy (PIPN). MAIN METHODS: Mitochondrial trafficking analysis was determined for all sample sets, wherein FA showed enhanced anterograde (axonal) mitochondrial trafficking leading to neuroprotective effects for all samples. KEY FINDINGS: Using this system, we demonstrate that PTX, Monomethyl auristatin E (MMAE), and Vincristine (VCR), are toxic at clinically prescribed levels when treated focally to axons. However, Cisplatin (CDDP) was determined to have a higher toxicity when treated to cell bodies. Although having different targeting mechanisms, the administration of FA was determined to have a significant neuroprotective effect for against all chemotherapy drugs tested. SIGNIFICANCE: This study identifies key insights regarding site of action and neuroprotective strategies to further development as potential therapeutics against CIPN. FA was treated alongside each chemotherapy drug to identify the neuroprotective effect against CIPN, where FA was found to be neuroprotective for all drugs tested. This study found that treatment with FA led to an enhancement in the anterograde movement of mitochondria based on fluorescent imaging.

A review on current brain organoid technologies from a biomedical engineering perspective.

Brain organoids are 3D cytoarchitectures resembling the embryonic human brain. This review focuses on current advancements in biomedical engineering methods to develop organoids such as pluripotent stem cells assemblies, quickly aggregated floating culture, hydrogel suspension, microfluidic systems (both photolithography and 3D printing), and brain organoids-on-a-chip. These methods have the potential to create a large impact on neurological disorder studies by creating a model of the human brain investigating pathogenesis and drug screening for individual patients. 3D brain organoid cultures mimic not only features of patients' unknown drug reactions, but also early human brain development at cellular, structural, and functional levels. The challenge of current brain organoids lies in the formation of distinct cortical neuron layers, gyrification, and the establishment of complex neuronal circuitry, as they are critically specialized, developmental aspects. Furthermore, recent advances such as vascularization and genome engineering are in development to overcome the barrier of neuronal complexity. Future technology of brain organoids is needed to improve tissue cross-communication, body axis simulation, cell patterning signals, and spatial-temporal control of differentiation, as engineering methods discussed in this review are rapidly evolving.

Fluocinolone Acetonide Enhances Anterograde Mitochondria Trafficking and Promotes Neuroprotection against Paclitaxel-Induced Peripheral Neuropathy.

Paclitaxel (PTX)-induced peripheral neuropathy (PIPN) is a debilitating health condition which is a result of degeneration of peripheral nerves found in extremities. Currently, there are no established treatment methods that can prevent or protect from PIPN. Fluocinolone acetonide (FA) has been recently identified as a potential candidate for protection from PIPN. However, the fundamental mechanism of action is still unknown. In this study, we showed that enhanced anterograde mitochondrial movement in dorsal root ganglion (DRG) cells has a major role in FA-mediated neuroprotection in PIPN. In this study, cells were treated with PTX or FA along with their combination followed by mitochondrial fluorescence staining. Somal (proximal) and axonal (distal) mitochondria were selectively stained using a microfluidic compartmentalized chamber with different MitoTrackers blue and red, respectively, which we termed, the two-color staining approach. Results revealed that axons were protected from degeneration by the PTX effect when treated along with FA. PTX exposure alone resulted in low mitochondrial mobility in DRG cells. However, cotreatment with PTX and FA showed significant enhancement of anterograde trafficking of somal (proximal) mitochondria to distal axons. Similarly, cotreatment with FA restored mitochondrial mobility significantly. Overall, this study affirms that increasing mitochondrial recruitment into the axon by cotreatment with FA can be a worthwhile strategy to protect or prevent PIPN. The proposed two-color staining approach can be extended to study trafficking for other neuron-specific subcellular organelles.

A Review on the Technological Advances and Future Perspectives of Axon Guidance and Regeneration in Peripheral Nerve Repair.

Despite a significant advance in the pathophysiological understanding of peripheral nerve damage, the successful treatment of large nerve defects remains an unmet medical need. In this article, axon growth guidance for peripheral nerve regeneration was systematically reviewed and discussed mainly from the engineering perspective. In addition, the common approaches to surgery, bioengineering approaches to emerging technologies such as optogenetic stimulation and magnetic stimulation for functional recovery were discussed, along with their pros and cons. Additionally, clear future perspectives of axon guidance and nerve regeneration were addressed.

Therapeutic potential of neuromodulation for demyelinating diseases.

Neuromodulation represents a cutting edge class of both invasive and non-invasive therapeutic methods which alter the activity of neurons. Currently, several different techniques have been developed - or are currently being investigated - to treat a wide variety of neurological and neuropsychiatric disorders. Recently, in vivo and in vitro studies have revealed that neuromodulation can also induce myelination, meaning that it could hold potential as a therapy for various demyelinating diseases including multiple sclerosis and progressive multifocal leukencepalopathy. These findings come on the heels of a paradigm shift in the view of myelin's role within the nervous system from a static structure to an active co-regulator of central nervous system plasticity and participant in neuron-mediated modulation. In the present review, we highlight several of the recent findings regarding the role of neural activity in altering myelination including several soluble and contact-dependent factors that seem to mediate neural activity-dependent myelination. We also highlight several considerations for neuromodulatory techniques, including the need for further research into spatiotemporal precision, dosage, and the safety and efficacy of transcranial focused ultrasound stimulation, an emerging neuromodulation technology. As the field of neuromodulation continues to evolve, it could potentially bring forth methods for the treatment of demyelinating diseases, and as such, further investigation into the mechanisms of neuron-dependent myelination as well as neuro-imaging modalities that can monitor myelination activity is warranted.

Development of an Axon-Guiding Aligned Nanofiber-Integrated Compartmentalized Microfluidic Neuron Culture System.

Microfluidic-based neuron cell culture systems have recently gained a lot of attention due to their efficiency in supporting the spatial and temporal control of cellular microenvironments. However, the lack of axon guidance is the key limitation in current culture systems. To combat this, we have developed electrospun aligned nanofiber-integrated compartmentalized microfluidic neuron culture systems (NIMSs), where the nanofibers have enabled axonal guidance and stability. The resulting platform significantly improved axon alignment, length, and stability for both rat primary embryonic motor neurons (MNs) and dorsal root ganglia (DRG) neurons compared to the conventional glass-based microfluidic systems (GMSs). The results showed that axonal growth covered more than two times the area on the axonal chamber of NIMSs compared to the area covered for GMSs. Overall, this platform can be used as a valuable tool for fundamental neuroscience research, drug screening, and biomaterial testing.

Modulation of Neural Activity for Myelination in the Central Nervous System.

Electrical stimulation has been playing a significant role in revealing various functions and mechanisms of the nervous system. It is no different for myelination, a process in which oligodendrocytes in the central nervous system (CNS) or Schwann Cells in the peripheral nerve system (PNS) wrap around axons to provide an insulating layer in vitro and in vivo. It has been widely recognized that the myelin sheath accelerates axon signal conduction and provides neuroprotection. Recent studies have begun to reveal its role in plasticity. The major mechanism that enables this process is activity-dependent myelination - the phenomenon where neuronal activity supports oligodendrocyte maturation and myelin sheath formation. In light of recent discoveries, a better understanding of this phenomenon has a potential to provide therapeutic targets for not only demyelinating diseases, but also psychiatric disorders. There is a growing need for experimental platforms capable of dissecting the effect of neural activity on myelination in health and disease. The effect of neural activity is commonly studied by comparing the myelination levels in cultures with neurons of low and high activity. Electrical stimulation is particularly well suited as a method of inducing neural activity in these systems. In this review, we describe in vitro platforms for studying activity-dependent myelination, which utilize neuron stimulation via electrical field. We also discuss stimulation profiles, as well as the alternatives to electrical stimulation in the context of regular, compartmentalized, and organotypic co-cultures.

Visualization of Intra-neuronal Motor Protein Transport through Upconversion Microscopy.

Cargo transport along axons, a physiological process mediated by motor proteins, is essential for neuronal function and survival. A current limitation in the study of axonal transport is the lack of a robust imaging technique with a high spatiotemporal resolution to visualize and quantify the movement of motor proteins in real-time and in different depth planes. Herein, we present a dynamic imaging technique that fully exploits the characteristics of upconversion nanoparticles. This technique can be used as a microscopic probe for the quantitative in situ tracking of retrograde transport neurons with single-particle resolution in multilayered cultures. This study may provide a powerful tool to reveal dynamic neuronal activity and intra-axonal transport function as well as any associated neurodegenerative diseases resulting from mutation or impairment in the axonal transport machinery.

Fibro-Neuronal Guidance on Common, 3D-Printed Textured Substrates.

Ability to direct neuronal growth not only carries great potential for treating neural conditions-for example, bridging traumatically shattered connections-but would also be an exquisite tool for bionic applications that require a physical interface between neurons and electronics. A testing platform is needed to better understand axonal guidance in the context of a specific in vivo application. Versatility of 3D printing technology allows tailoring to researcher needs, both in vitro and in vivo. In this paper, we establish a fibro-neuronal co-culture inspired by our neural interface research and demonstrate axon alignment on a textured substrate fabricated with a common, versatile 3D-printing set-up.

Electrospun nanofibers facilitate better alignment, differentiation, and long-term culture in an in vitro model of the neuromuscular junction (NMJ).

The neuromuscular junction (NMJ) is a specialized synapse between motor neurons and the muscle fibers they innervate. Due to the complexity of various signalling molecules and pathways, in vivo NMJs are difficult to study. Therefore, in vitro motor neuron-muscle co-culture plays a pivotal role in studying the mechanisms of NMJ formation associated with neurodegenerative diseases. There is a growing need to develop novel methodologies that can be used to develop long-term cultures of NMJs. To date, there have been few studies on NMJ development and long-term maintenance of the system, which is also the main challenge for the current in vitro models of NMJs. In this study, we demonstrate a long-term co-culture system of primary embryonic motor neurons from Sprague-Dawley rats and C2C12 cells on both random and aligned electrospun polylactic acid (PLA) nanofibrous scaffolds. This is the first study to explore the role of electrospun nanofibers in the long-term maintenance of NMJs. PLA nanofibrous scaffolds provide better contact guidance for C2C12 cells aligning along the fibers, thus guiding myotube formation. We can only maintain the co-culture system on a conventional glass substrate for 2 weeks, whilst 55% and 70% of the cells still survived on random and aligned PLA substrates after 7 weeks. Our nanofiber-based long-term co-culture system is used as an important tool for the fundamental research of NMJs.

Subcellular Optogenetic Stimulation Platform for Studying Activity-Dependent Axon Myelination In Vitro.

Activity-dependent myelination modulates neuron conduction velocity and as such it is essential for a correct wiring of a whole nervous system. Increasing myelination through inducing neuron activity has been proposed as a treatment strategy for demyelination diseases. Yet, the mechanisms and the effects of activity-dependent myelination remain elusive-new tools are needed. In this chapter, we describe a novel compartmentalized device integrated with an optogenetic stimulator for studying activity-dependent myelination in vitro. The platform can be modified to include multiple cell types, stimulation modes, and experimental readouts to answer a specific research question. This versatility combined with a precise control over spatial extent of the stimulation and the stimulation pattern make the proposed platform a valuable tool for molecular myelination studies.

Engineering PCL/lignin nanofibers as an antioxidant scaffold for the growth of neuron and Schwann cell.

Antioxidant is critical for the successful of nerve tissue regeneration, and biomaterials with antioxidant activity might be favorable for peripheral nerve repair. Lignin, a biopolymer from wood with excellent antioxidant properties, is still "unexplored" as biomaterials. To design an antioxidative bioscaffold for nerve regeneration, here we synthesized lignin-polycaprolactone (PCL) copolymers via solvent free ring-opening polymerization (ROP). Then such lignin-PCL copolymers were incorporated with PCL and engineered into nanofibrous scaffolds for supporting the growth of neuron and Schwann cell. Our results showed that the addition of lignin-PCL enhanced the mechanical properties of PCL nanofibers and endowed them with good antioxidant properties (up to 98.3 ±â€¯1.9% free radical inhibition within 4 h). Cell proliferation assay showed that PCL/lignin-PCL nanofibers increased cell viability compared to PCL fibers, especially after an oxidative challenge. Moreover, Schwann cells and dorsal root ganglion (DRG) neurons cultured on the nanofibers to assess their potential for nerve regeneration. These results suggested that nanofibers with lignin copolymers promoted cell proliferation of both BMSCs and Schwann cells, enhanced myelin basic protein expressions of Schwann cells and stimulated neurite outgrowth of DRG neurons. In all, these sustainable, intrinsically antioxidant nanofibers may be a potential candidate for nerve TE applications.

Static Magnetic Field Stimulation Enhances Oligodendrocyte Differentiation and Secretion of Neurotrophic Factors.

The cellular-level effects of low/high frequency oscillating magnetic field on excitable cells such as neurons are well established. In contrast, the effects of a homogeneous, static magnetic field (SMF) on Central Nervous System (CNS) glial cells are less investigated. Here, we have developed an in vitro SMF stimulation set-up to investigate the genomic effects of SMF exposure on oligodendrocyte differentiation and neurotrophic factors secretion. Human oligodendrocytes precursor cells (OPCs) were stimulated with moderate intensity SMF (0.3 T) for a period of two weeks (two hours/day). The differential gene expression of cell activity marker (c-fos), early OPC (Olig1, Olig2. Sox10), and mature oligodendrocyte markers (CNP, MBP) were quantified. The enhanced myelination capacity of the SMF stimulated oligodendrocytes was validated in a dorsal root ganglion microfluidics chamber platform. Additionally, the effects of SMF on the gene expression and secretion of neurotrophic factors- BDNF and NT3 was quantified. We also report that SMF stimulation increases the intracellular calcium influx in OPCs as well as the gene expression of L-type channel subunits-CaV1.2 and CaV1.3. Our findings emphasize the ability of glial cells such as OPCs to positively respond to moderate intensity SMF stimulation by exhibiting enhanced differentiation, functionality as well as neurotrophic factor release.

Subcellular electrical stimulation of neurons enhances the myelination of axons by oligodendrocytes.

Myelin formation has been identified as a modulator of neural plasticity. New tools are required to investigate the mechanisms by which environmental inputs and neural activity regulate myelination patterns. In this study, we demonstrate a microfluidic compartmentalized culture system with integrated electrical stimulation capabilities that can induce neural activity by whole cell and focal stimulation. A set of electric field simulations was performed to confirm spatial restriction of the electrical input in the compartmentalized culture system. We further demonstrate that electrode localization is a key consideration for generating uniform the stimulation of neuron and oligodendrocytes within the compartments. Using three configurations of the electrodes we tested the effects of subcellular activation of neural activity on distal axon myelination with oligodendrocytes. We further investigated if oligodendrocytes have to be exposed to the electrical field to induce axon myelination. An isolated stimulation of cell bodies and proximal axons had the same effect as an isolated stimulation of distal axons co-cultured with oligodendrocytes, and the two modes had a non-different result than whole cell stimulation. Our platform enabled the demonstration that electrical stimulation enhances oligodendrocyte maturation and myelin formation independent of the input localization and oligodendrocyte exposure to the electrical field.

PanIN Neuroendocrine Cells Promote Tumorigenesis via Neuronal Cross-talk.

Nerves are a notable feature of the tumor microenvironment in some epithelial tumors, but their role in the malignant progression of pancreatic ductal adenocarcinoma (PDAC) is uncertain. Here, we identify dense innervation in the microenvironment of precancerous pancreatic lesions, known as pancreatic intraepithelial neoplasms (PanIN), and describe a unique subpopulation of neuroendocrine PanIN cells that express the neuropeptide substance P (SP) receptor neurokinin 1-R (NK1-R). Using organoid culture, we demonstrated that sensory neurons promoted the proliferation of PanIN organoids via SP-NK1-R signaling and STAT3 activation. Nerve-responsive neuroendocrine cells exerted trophic influences and potentiated global PanIN organoid growth. Sensory denervation of a genetically engineered mouse model of PDAC led to loss of STAT3 activation, a decrease in the neoplastic neuroendocrine cell population, and impaired PanIN progression to tumor. Overall, our data provide evidence that nerves of the PanIN microenvironment promote oncogenesis, likely via direct signaling to neoplastic neuroendocrine cells capable of trophic influences. These findings identify neuroepithelial cross-talk as a potential novel target in PDAC treatment. Cancer Res; 77(8); 1868-79. ©2017 AACR.

Subcellular Optogenetic Stimulation for Activity-Dependent Myelination of Axons in a Novel Microfluidic Compartmentalized Platform.

Myelination is governed by neuron-glia communication, which in turn is modulated by neural activity. The exact mechanisms remain elusive. We developed a novel in vitro optogenetic stimulation platform that facilitates subcellular activity induction in hundreds of neurons simultaneously. The light isolation was achieved by creating a biocompatible, light-absorbent, black microfluidic device integrated with a programmable, high-power LED array. The system was applied to a compartmentalized culture of primary neurons whose distal axons were interacting with oligodendrocyte precursor cells. Neural activity was induced along whole neurons or was constrained to cell bodies with proximal axons or distal axons only. All three modes of stimulation promoted oligodendrocyte differentiation and the myelination of axons as evidenced by a decrease in the number of oligodendrocyte precursor cells followed by increases in the number of mature oligodendrocytes and myelin sheath fragments. These results demonstrated the potential of our novel optogenetic stimulation system for the global and focal induction of neural activity in vitro for studying axon myelination.

Identification of fluocinolone acetonide to prevent paclitaxel-induced peripheral neuropathy.

Paclitaxel (PTX) is among the most commonly used cancer drugs that cause chemotherapy-induced peripheral neuropathy (CIPN), a debilitating and serious dose-limiting side effect. Currently, no drugs exist to prevent CIPN, and symptomatic therapy is often ineffective. In order to identify therapeutic candidates to prevent axonal degeneration induced by PTX, we carried out a phenotypic drug screening using primary rodent dorsal root ganglion sensory neurons. We identified fluocinolone acetonide as a neuroprotective compound and verified it through secondary screens. Furthermore, we showed its efficacy in a mouse model of PTX-induced peripheral neuropathy and confirmed with four different cancer cell lines that fluocinolone acetonide does not interfere with PTX's antitumor activity. Our study identifies fluocinolone acetonide as a potential therapy to prevent CIPN caused by PTX.

Novel RNA- and FMRP-binding protein TRF2-S regulates axonal mRNA transport and presynaptic plasticity.

Despite considerable evidence that RNA-binding proteins (RBPs) regulate mRNA transport and local translation in dendrites, roles for axonal RBPs are poorly understood. Here we demonstrate that a non-telomeric isoform of telomere repeat-binding factor 2 (TRF2-S) is a novel RBP that regulates axonal plasticity. TRF2-S interacts directly with target mRNAs to facilitate their axonal delivery. The process is antagonized by fragile X mental retardation protein (FMRP). Distinct from the current RNA-binding model of FMRP, we show that FMRP occupies the GAR domain of TRF2-S protein to block the assembly of TRF2-S-mRNA complexes. Overexpressing TRF2-S and silencing FMRP promotes mRNA entry to axons and enhances axonal outgrowth and neurotransmitter release from presynaptic terminals. Our findings suggest a pivotal role for TRF2-S in an axonal mRNA localization pathway that enhances axon outgrowth and neurotransmitter release.