Lycium barbarum polysaccharides attenuate nonylphenol and octylphenol-induced oxidative stress and neurotransmitter disorders in PC-12 cells.

Nonylphenol (NP) and octylphenol (OP) are environmental contaminants with potential endocrine disrupting effects. However, there is limited research on the mechanisms and intervention of combined NP and OP exposure-induced neurotoxicity. This study aims to explore the cytotoxicity of combined NP and OP exposure and evaluate the potential of Lycium barbarum polysaccharides (LBP) in mitigating the aforementioned toxicity. In present study, LBP (62.5, 125 and 250 µg/mL) were applied to intervene rat adrenal pheochromocytoma (PC-12) cells treated with combined NP and OP (NP: OP = 4:1, w/w; 1, 2, 4 and 8 µg/mL). The results showed that NP and OP induced oxidative stress, disrupted the 5-hydroxytryptamine (5-HT) and cholinergic systems in PC-12 cells. Additionally, they activated the p38 protein kinase (p38) and suppressed the expression of silent information regulation type 1 (SIRT1), monoamine oxidase A (MAOA), phosphorylated cyclic-AMP response binding protein (p-CREB), brain-derived neurotrophic factor (BDNF) and phosphorylated tropomyosin-related kinase receptor type B (p-TrkB). However, N-acetyl-L-cysteine (NAC) treatment counteracted the changes of signalling molecule p38, SIRT1/MAOA and CREB/BDNF/TrkB pathways-related proteins induced by NP and OP. LBP pretreatment ameliorated combined NP and OP exposure-induced oxidative stress and neurotransmitter imbalances. Furthermore, the application of LBP and administration of a p38 inhibitor both reversed the alterations in the signaling molecule p38, as well as the proteins associated to the SIRT1/MAOA and CREB/BDNF/TrkB pathways. These results implied that LBP may have neuroprotective effects via p38-mediated SIRT1/MAOA and CREB/BDNF/TrkB pathways.

EGLN1: A Biomarker of Poor Prognosis of Cervical Cancer and a Target of Treatment.

Objective: To conduct bioinformatics analysis on the prognostic effect, mechanism of action, and drug sensitivity of Egl-9 family hypoxia-inducible factor 1 (EGLN1) expression on cervical cancer. Methods: Bioinformatics were obtained from Gene Expression Profiling Interactive Analysis (GEPIA), Tumor Immune Estimation Resource (TIMER), and the human cancer metastasis database (HCMDB), and the effect of EGLN1 expression level on the prognosis of cervical cancer was comprehensively analyzed. The protein-protein interaction network was constructed by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), and the possible mechanism of EGLN1 affecting the prognosis of cervical cancer was discussed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In addition, Gene Set Cancer Analysis (GSCALite) was used to predict sensitive drugs online. Results: The higher the expression level of EGLN1, the shorter the tumor-free survival time and overall survival time of cervical cancer. The higher the stage of cervical cancer, the higher the expression level of EGLN1. The expression of EGLN1 affects the degree of immune infiltration, the variation of somatic copy number, and the level of N6-methyladenosine (m6A) modification in cervical cancer. COX regression model suggested that EGLN1 was an independent prognostic factor of cervical cancer. Conclusions: The high expression of EGLN1 in cervical cancer is an independent risk factor for the prognosis of cervical cancer, which affects the prognosis of cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) through different signal pathways. It is expected to be used to predict the sensitive anticancer drugs for the treatment of cervical cancer.

Lycium barbarum polysaccharides attenuate oxidative stress and mitochondrial toxicity induced by mixed plasticizers in HepG2 cells through activation of Nrf2.

AIMS: In daily life, it is common for humans to be exposed to multiple phthalate esters (PAEs). However, there is limited research on the mechanisms and intervention of combined PAEs toxicity. This study aims to explore the cytotoxicity of combined PAEs and evaluate the potential of Lycium barbarum polysaccharides (LBP) in mitigating the aforementioned toxicity. MAIN METHODS: LBP (62.5, 125 and 250 µg/mL) were applied to intervene HepG2 cells treated with DEHP and DBP mixtures (50, 100, 200, 400 and 800 µg/mL). Western Blot and different kits were mainly performed in our study. KEY FINDINGS: DEHP and DBP mixtures suppressed the expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and activated MAPK pathway by increasing ROS. Combined DEHP and DBP exposure reduced ATP content and inhibited the mitochondrial biogenesis pathway in HepG2 cells through oxidative stress, which in turn caused cytotoxicity. LBP reduced oxidative stress and cell death induced by mixed plasticizers, upregulated Nrf2 levels and mitochondrial biogenesis pathway levels and inhibited MAPK pathway activation. Notably, after treating HepG2 cells with Nrf2-specific inhibitor (ML385, 0.5 µM), we found that the activation of Nrf2 played a crucial role on LBP intervention of DEHP and DBP induced HepG2 cytotoxicity. SIGNIFICANCE: This study not only enhances our understanding of the toxicological effects caused by combined PAEs exposure, but also has significant implications in devising strategies to mitigate the toxicological consequences of combined exposure to exogenous chemicals through the investigation of the role of LBP.

The feasibility of using the compound kushen injection to treat cervical cancer based on network pharmacology and transcriptomics.

BACKGROUND: To investigate the molecular targets and mechanisms of compound kushen injection (CKI) in the prevention and treatment of cervical cancer based on network pharmacology and transcriptomics. METHODS: In this study, we used network pharmacology methods to screen for effective compounds, integrated the results of network pharmacology and RNA-seq to comprehensively screen and predict target genes, analyze the biological functions and signaling pathways of target genes, and construct a PPI network to screen for hub genes. The results were further verified by biological experiments, molecular docking, RT-PCR, and western blot analysis. RESULTS: The results showed that the hub genes CXCL2, anti-vascular endothelial growth factor, hexokinase 2 are therapeutic targets of CKI for the treatment of Cervical Cancer. These targets were significantly enriched in pathways mainly including pathways in cancer, cell cycle, MAPK signaling pathways, etc. In vitro cell experiments showed that CKI could effectively inhibit the proliferation of cancer cells, promote apoptosis, and induce cell cycle arrest. RT-PCR and western blot experiments showed that the expression of hub genes was significantly decreased. The compounds have good binding activity to hub genes. CONCLUSION: CKI, based on its active ingredients and through multiple targets and multiple pathways, can stop the growth of cervical cancer cells at a certain phase of the cell cycle and cause apoptosis, which proved CKI's effect in treating cervical cancer.

DBP induced autophagy and necrotic apoptosis in HepG2 cells via the mitochondrial damage pathway.

Phthalate esters (PAEs) are widely present in human tissues and pose significant health risks. In this study, HepG2 cells were treated with 0.0625, 0.125, 0.25, 0.5 and 1 mM Dibutyl phthalate (DBP) for 48 h to investigate mitochondrial toxicity. The results showed that DBP caused mitochondrial damage, autophagy, apoptosis and necroptosis; Transcriptomics analysis identified that MAPK and PI3K were significant factors in the cytotoxic changes induced by DBP; N-Acetyl-L-cysteine (NAC), SIRT1 activator, ERK inhibitor, p38 inhibitor and ERK siRNA treatments counteracted the changes of SIRT1/PGC-1α and Nrf2 pathway-related proteins, autophagy and necroptotic apoptosis proteins induced by DBP. While PI3K and Nrf2 inhibitors exacerbated the changes in SIRT1/PGC-1α, Nrf2-associated proteins and autophagy and necroptosis proteins induced by DBP. In addition, the autophagy inhibitor 3-MA alleviated the increase in DBP-induced necroptosis proteins. These results suggested that DBP-induced oxidative stress activated the MAPK pathway, inhibited the PI3K pathway, which in turn inhibited the SIRT1/PGC-1α pathway and Nrf2 pathway, thereby causing cell autophagy and necroptosis.

Cochlear implant material effects on inflammatory cell function and foreign body response.

OBJECTIVES: The objectives of this study were to assess the effects of cochlear implant (CI) biomaterials on the function of macrophages and fibroblasts, two key mediators of the foreign body response (FBR) and to determine how these materials influence fibrous tissue growth and new bone formation within the cochlea. METHODS: Macrophages and fibroblasts were cultured on polydimethylsiloxane (PDMS) and platinum substrates and human CI electrodes in vitro. Cell count, cell proliferation, cytokine production, and cell adhesion were measured. CI electrodes were implanted into murine cochleae for three weeks without electrical stimulation. Implanted cochleae were harvested for 3D X-ray microscopy with the CI left in-situ. The location of new bone growth within the scala tympani (ST) with reference to different portions of the implant (PDMS vs platinum) was quantified. RESULTS: Cell counts of macrophages and fibroblasts were significantly higher on platinum substrates and platinum contacts of CI electrodes. Fibroblast proliferation was greater on platinum relative to PDMS, and cells grown on platinum formed more/larger focal adhesions. 3D X-ray microscopy showed neo-ossification in the peri­implant areas of the ST. Volumetric quantification of neo-ossification showed a trend toward greater bone formation adjacent to the platinum electrodes compared to areas opposite or away from the platinum electrode bearing surfaces. CONCLUSIONS: Fibrotic reactions are biomaterial specific, as demonstrated by the differences in cell adhesion, proliferation, and fibrosis on platinum and PDMS. The inflammatory reaction to platinum contacts on CI electrodes likely contributes to fibrosis to a greater degree than PDMS, and platinum contacts may influence the deposition of new bone, as demonstrated in the in vivo data. This information can potentially be used to influence the design of future generations of neural prostheses.

Latent Classes of Circadian Type and Presenteeism and Work-Related Flow Differences Among Clinical Nurses: A Cross-Sectional Study.

OBJECTIVE: To classify the characteristics of circadian type among clinical nurses and examine their relationships with presenteeism and work-related flow. METHODS: Using a cross-sectional design, 568 nurses were recruited through convenience sampling in January 2021 from three hospitals in Shandong Province, China. The data were collected using self-report measures, including the 11-item Circadian Type Inventory, Stanford Presenteeism Scale-6, and Work-Related Flow Inventory. Latent class analysis was performed to identify any clustering of circadian types. One-way analysis was performed to compare the differences between presenteeism and work-related flow in different circadian types. RESULTS: Four latent classes were identified, including high response class (14.4%), high flexible class (20.1%), high languid class (51.1%), and low response class (14.4%). Regarding presenteeism, the high languid class had higher scores than others. Regarding work-related flow, the scores of high flexible class were higher than those of high languid class, while the differences in all three dimensions were statistically significant. CONCLUSION: Although the shift work mode is not expected to change, nursing managers could use circadian type as a predictive index to select and employ individuals for shift work to enhance work performance and provide sufficient support to staff who are intolerant to shift work.

Antifouling and Mechanical Properties of Photografted Zwitterionic Hydrogel Thin-Film Coatings Depend on the Cross-Link Density.

Zwitterionic polymer networks have shown promise in reducing the short- and long-term inflammatory foreign body response to implanted biomaterials by combining the antifouling properties of zwitterionic polymers with the mechanical stability provided by cross-linking. Cross-link density directly modulates mechanical properties (i.e., swelling behavior, resistance to stress and strain, and lubricity) but theoretically could reduce desirable biological properties (i.e., antifouling) of zwitterionic materials. This work examined the effect of varying poly(ethylene glycol) dimethacrylate cross-linker concentration on protein adsorption, cell adhesion, equilibrium swelling, compressive modulus, and lubricity of zwitterionic thin films. Furthermore, this work aimed to determine the appropriate balance among each of these mechanical and biologic properties to produce thin films that are strong, durable, and lubricious, yet also able to resist biofouling. The results demonstrated nearly a 20-fold reduction in fibrinogen adsorption on zwitterionic thin films photografted on polydimethylsiloxane (PDMS) across a wide range of cross-link densities. Interestingly, either at high or low cross-link densities, increased levels of protein adsorption were observed. In addition to fibrinogen, macrophage and fibroblast cell adhesion was reduced significantly on zwitterionic thin films, with a large range of cross-link densities, resulting in low cell counts. The macrophage count was reduced by 30-fold, while the fibroblast count was reduced nearly 10-fold on grafted zwitterionic films relative to uncoated films. Increasing degrees of cell adhesion were noted as the cross-linker concentration exceeded 50%. As expected, increased cross-link density resulted in a reduced swelling but greater compressive modulus. Notably, the coefficient of friction was dramatically reduced for zwitterionic thin films compared to uncoated PDMS across a broad range of cross-link densities, an attractive property for insertional implants. This work identified a broad range of cross-link densities that provide desirable antifouling effects while also maintaining the mechanical functionality of the thin films.

Interaction of micropatterned topographical and biochemical cues to direct neurite growth from spiral ganglion neurons.

Functional outcomes with neural prosthetic devices, such as cochlear implants, are limited in part due to physical separation between the stimulating elements and the neurons they stimulate. One strategy to close this gap aims to precisely guide neurite regeneration to position the neurites in closer proximity to electrode arrays. Here, we explore the ability of micropatterned biochemical and topographic guidance cues, singly and in combination, to direct the growth of spiral ganglion neuron (SGN) neurites, the neurons targeted by cochlear implants. Photopolymerization of methacrylate monomers was used to form unidirectional topographical features of ridges and grooves in addition to multidirectional patterns with 90o angle turns. Microcontact printing was also used to create similar uni- and multi-directional patterns of peptides on polymer surfaces. Biochemical cues included peptides that facilitate (laminin, LN) or repel (EphA4-Fc) neurite growth. On flat surfaces, SGN neurites preferentially grew on LN-coated stripes and avoided EphA4-Fc-coated stripes. LN or EphA4-Fc was selectively adsorbed onto the ridges or grooves to test the neurite response to a combination of topographical and biochemical cues. Coating the ridges with EphA4-Fc and grooves with LN lead to enhanced SGN alignment to topographical patterns. Conversely, EphA4-Fc coating on the grooves or LN coating on the ridges tended to disrupt alignment to topographical patterns. SGN neurites respond to combinations of topographical and biochemical cues and surface patterning that leverages both cues enhance guided neurite growth.

Effects of Neurod1 Expression on Mouse and Human Schwannoma Cells.

OBJECTIVES: The objective was to explore the effect of the proneuronal transcription factor neurogenic differentiation 1 (Neurod1, ND1) on Schwann cells (SC) and schwannoma cell proliferation. METHODS: Using a variety of transgenic mouse lines, we investigated how expression of Neurod1 effects medulloblastoma (MB) growth, schwannoma tumor progression, vestibular function, and SC cell proliferation. Primary human vestibular schwannoma (VS) cell cultures were transduced with adenoviral vectors expressing Neurod1. Cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine (EdU) uptake. STUDY DESIGN: Basic science investigation. RESULTS: Expression of Neurod1 reduced the growth of slow-growing but not fast-growing MB models. Gene transfer of Neurod1 in human schwannoma cultures significantly reduced cell proliferation in dose-dependent way. Deletion of the neurofibromatosis type 2 (Nf2) tumor-suppressor gene via Cre expression in SCs led to increased intraganglionic SC proliferation and mildly reduced vestibular sensory-evoked potentials (VsEP) responses compared to age-matched wild-type littermates. The effect of Neurod1-induced expression on intraganglionic SC proliferation in animals lacking Nf2 was mild and highly variable. Sciatic nerve axotomy significantly increased SC proliferation in wild-type and Nf2-null animals, and expression of Neurod1 reduced the proliferative capacity of both wild-type and Nf2-null SCs following nerve injury. CONCLUSION: Expression of Neurod1 reduces slow-growing MB progression and reduces human SC proliferation in primary VS cultures. In a genetic mouse model of schwannomas, we find some effects of Neurod1 expression; however, the high variability indicates that more tightly regulated Neurod1 expression levels that mimic our in vitro data are needed to fully validate Neurod1 effects on schwannoma progression. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E259-E270, 2021.

Photograftable Zwitterionic Coatings Prevent Staphylococcus aureus and Staphylococcus epidermidis Adhesion to PDMS Surfaces.

Due to its attractive mechanical properties and biocompatibility, poly(dimethyl)siloxane (PDMS) is widely used in the fabrication of biomedical materials. On the other hand, PDMS is also prone to adsorption of both proteins and bacteria, making PDMS implants susceptible to infection. Herein, we examine the use of durably cross-linked zwitterionic coatings for PDMS surfaces to mitigate bacterial adhesion. Using a single-step photografting technique, poly(sulfobetaine methacrylate) (pSBMA) and poly(carboxybetaine methacrylate) (pCBMA) thin films were covalently attached to PDMS substrates. The abilities of these coatings to resist the adhesion of Staphylococcus aureus and Staphylococcus epidermidis were tested in vitro under both wet and droplet conditions, as well as in subcutaneous and transcutaneous implantation models using Sprague-Dawley rats. Zwitterionic thin films effectively reduced bacterial adhesion in both in vitro and in vivo conditions. This was particularly true for pCBMA-coated materials, which exhibited significant reduction in bacterial adhesion and growth with respect to S. aureus and S. epidermidis for all in vitro conditions as well as the ability to resist bacterial growth on PDMS implants. The results of this study suggest that a simple and durable photografting process can be used to produce polymer thin films capable of preventing infection of implantable medical devices.

Antifouling Photograftable Zwitterionic Coatings on PDMS Substrates.

The foreign body response (FBR) to implantable materials can negatively impact performance of medical devices such as the cochlear implant. Engineering surfaces that resist the FBR could lead to enhanced functionality including potentially improving outcomes for cochlear implant recipients through reduction in fibrosis. In this work, we coat poly(dimethylsiloxane) (PDMS) surfaces with two zwitterionic polymers, poly(sulfobetaine methacrylate) (pSBMA) and poly(carboxybetaine methacrylate) (pCBMA), using a simultaneous photografting/photo-cross-linking process to produce a robust grafted zwitterionic hydrogel. reduce nonspecific protein adsorption, the first step of the FBR. The coating process uses benzophenone, a photografting agent and type II photoinitiator, to covalently link the cross-linked zwitterionic thin film to the PDMS surface. As the concentration of benzophenone on the surface increases, the adhesive strength of the zwitterionic thin films to PDMS surfaces increases as determined by shear adhesion. Additionally, with increased concentration of the adsorbed benzophenone, failure of the system changes from adhesive delamination to cohesive failure within the hydrogel, demonstrating that durable adhesive bonds are formed from the photografting process. Interestingly, antifouling properties of the zwitterionic polymers are preserved with significantly lower levels of nonspecific protein adsorption on zwitterion hydrogel-coated samples compared to uncoated controls. Fibroblast adhesion is also dramatically reduced on coated substrates. These results show that cross-linked pSBMA and pCBMA hydrogels can be readily photografted to PDMS substrates and show promise in potentially changing the fibrotic response to implanted biomaterials.

Photopolymerized micropatterns with high feature frequencies overcome chemorepulsive borders to direct neurite growth.

Developing and regenerating neurites respond to a variety of biophysical and biochemical cues in their micro-environment to reach target cells and establish appropriate synapses. Defining the hierarchal relationship of both types of cues to direct neurite growth carries broad significance for neural development, regeneration, and, in particular, engineering of neural prostheses that improve tissue integration with native neural networks. In this work, chemorepulsive biochemical borders are established on substrates with a range of surface microfeatures to determine the potential of physical cues to overcome conflicting biochemical cues. Physical micropatterns are fabricated using photomasking techniques to spatially control photoinitiation events of the polymerization. Temporal control of the reaction allows for generation of microfeatures with the same amplitude across a range of feature frequencies or periodicities. The micropatterned substrates are then modified with repulsive chemical borders between laminin and either EphA4-Fc or tenascin C that compete with the surface microfeatures to direct neurite growth. Behaviour of neurites from spiral ganglion and trigeminal neurons is characterized at biochemical borders as cross, turn, stop, or repel events. Both the chemical borders and physical patterns significantly influence neurite pathfinding. On unpatterned surfaces, most neurites that originate on laminin are deterred by the border with tenascin C or EphA4-Fc. Importantly, substrates with frequent micropattern features overcome the influence of the chemorepulsive border to dominate neurite trajectory. Designing prosthesis interfaces with appropriate surface features may allow for spatially organized neurite outgrowth in vivo even in the presence of conflicting biochemical cues in native target tissues.

Photopolymerized Microfeatures Guide Adult Spiral Ganglion and Dorsal Root Ganglion Neurite Growth.

HYPOTHESIS: Microtopographical patterns generated by photopolymerization of methacrylate polymer systems will direct growth of neurites from adult neurons, including spiral ganglion neurons (SGNs). BACKGROUND: Cochlear implants (CIs) provide hearing perception to patients with severe to profound hearing loss. However, their ability to encode complex auditory stimuli is limited due, in part, to poor spatial resolution caused by spread of the electrical currents in the inner ear. Directing the regrowth of SGN peripheral processes towards stimulating electrodes could help reduce current spread and improve spatial resolution provided by the CI. Previous work has demonstrated that micro- and nano-scale patterned surfaces precisely guide the growth of neurites from a variety of neonatal neurons including SGNs. Here, we sought to determine the extent to which adult neurons likewise respond to these topographical surface features. METHODS: Photopolymerization was used to fabricate methacrylate polymer substrates with micropatterned surfaces of varying amplitudes and periodicities. Dissociated adult dorsal root ganglion neurons (DRGNs) and SGNs were cultured on these surfaces and the alignment of the neurite processes to the micropatterns was determined. RESULTS: Neurites from both adult DRGNs and SGNs significantly aligned to the patterned surfaces similar to their neonatal counterparts. Further DRGN and SGN neurite alignment increased as the amplitude of the microfeatures increased. Decreased pattern periodicity also improved neurite alignment. CONCLUSION: Microscale surface topographic features direct the growth of adult SGN neurites. Topographical features could prove useful for guiding growth of SGN peripheral axons towards a CI electrode array.

Photopolymerizable Zwitterionic Polymer Patterns Control Cell Adhesion and Guide Neural Growth.

Developing materials that reduce or eliminate fibrosis encapsulation of neural prosthetic implants could significantly enhance implant fidelity by improving the tissue/electrode array interface. Here, we report on the photografting and patterning of two zwitterionic materials, sulfobetaine methacrylate (SBMA) and carboxybetaine methacrylate (CBMA), for controlling the adhesion and directionality of cells relevant to neural prosthetics. CBMA and SBMA polymers were photopolymerized and grafted on glass surfaces then characterized by X-ray photoelectron spectroscopy, water contact angle, and protein adsorption. Micropatterned surfaces were fabricated with alternating zwitterionic and uncoated bands. Fibroblasts, cells prevalent in fibrotic tissue, almost exclusively migrate and grow on uncoated bands with little to no cells present on zwitterionic bands, especially for CBMA-coated surfaces. Astrocytes and Schwann cells showed similarly low levels of cell adhesion and morphology changes when cultured on zwitterionic surfaces. Additionally, Schwann cells and inner ear spiral ganglion neuron neurites aligned well to zwitterionic patterns.

Intracellular calcium and cyclic nucleotide levels modulate neurite guidance by microtopographical substrate features.

Micro- and nanoscale surface features have emerged as potential tools to direct neurite growth into close proximity with next generation neural prosthesis electrodes. However, the signaling events underlying the ability of growth cones to respond to topographical features remain largely unknown. Accordingly, this study probes the influence of [Ca(2+) ]i and cyclic nucleotide levels on the ability of neurites from spiral ganglion neurons (SGNs) to precisely track topographical micropatterns. Photopolymerization and photomasking were used to generate micropatterned methacrylate polymer substrates. Dissociated SGN cultures were plated on the micropatterned surfaces. Calcium influx and release from internal stores were manipulated by elevating extracellular K(+) , maintenance in calcium-free media, or bath application of various calcium channel blockers. Cyclic nucleotide activity was increased by application of cpt-cAMP or 8-Br-cGMP. Elevation of [Ca(2+) ]i by treatment of cultures with elevated potassium reduced neurite alignment to physical microfeatures. Maintenance of cultures in Ca(2+) -free medium or treatment with the non-selective voltage-gated calcium channel blocker cadmium or L-type Ca(2+) channel blocker nifedipine did not signficantly alter SGN neurite alignment. By contrast, ryanodine or xestospongin C, which block release of internal calcium stores via ryanodine-sensitive channels or inositol-1,4,5-trisphosphate receptors respectively, each significantly decreased neurite alignment. Cpt-cAMP significantly reduced neurite alignment while 8-Br-cGMP significantly enhanced neurite alignment. Manipulation of [Ca(2+) ]i or cAMP levels significantly disrupts neurite guidance while elevation of cGMP levels increases neurite alignment. The results suggest intracellular signaling pathways similar to those recruited by chemotactic cues are involved in neurite guidance by topographical features. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2037-2048, 2016.

Merlin status regulates p75(NTR) expression and apoptotic signaling in Schwann cells following nerve injury.

After nerve injury, Schwann cells (SCs) dedifferentiate, proliferate, and support axon regrowth. If axons fail to regenerate, denervated SCs eventually undergo apoptosis due, in part, to increased expression of the low-affinity neurotrophin receptor, p75(NTR). Merlin is the protein product of the NF2 tumor suppressor gene implicated in SC tumorigenesis. Here we explore the contribution of merlin to SC responses to nerve injury. We find that merlin becomes phosphorylated (growth permissive) in SCs following acute axotomy and following gradual neural degeneration in a deafness model, temporally correlated with increased p75(NTR) expression. p75(NTR) levels are elevated in P0SchΔ39-121 transgenic mice that harbor an Nf2 mutation in SCs relative to wild-type mice before axotomy and remain elevated for a longer period of time following injury. Replacement of wild-type, but not phospho-mimetic (S518D), merlin isoforms suppresses p75(NTR) expression in primary human schwannoma cultures which otherwise lack functional merlin. Despite elevated levels of p75(NTR), SC apoptosis following axotomy is blunted in P0SchΔ39-121 mice relative to wild-type mice suggesting that loss of functional merlin contributes to SC resistance to apoptosis. Further, cultured SCs from mice with a tamoxifen-inducible knock-out of Nf2 confirm that SCs lacking functional merlin are less sensitive to p75(NTR)-mediated cell death. Taken together these results point to a model whereby loss of axonal contact following nerve injury results in merlin phosphorylation leading to increased p75(NTR) expression. Further, they demonstrate that merlin facilitates p75(NTR)-mediated apoptosis in SCs helping to explain how neoplastic SCs that lack functional merlin survive long-term in the absence of axonal contact.

Hearing Loss After Activation of Hearing Preservation Cochlear Implants Might Be Related to Afferent Cochlear Innervation Injury.

OBJECTIVE: Characterize hearing loss (HL) after hearing preservation cochlear implantation and determine the association between high charge electrical stimulation (ES) and late loss of acoustic hearing. METHODS: A retrospective cohort analysis of all hearing preservation implantees at our center (n = 42) assayed HL as a function of maximum charge. We analyzed serial audiometry from 85 patients enrolled in the multicenter Hybrid S8 trial to detail the hearing loss greater than 1 month after implantation. Cochleotypic explant cultures were used to assess susceptibility to high levels of ES. RESULTS: Early HL after implantation tends to be mild and averages 12.2 dB. After activation of the Hybrid S8 device, 17 (20%) of 85 patients experienced acceleration of HL. Compared with the majority of patients who did not lose significant hearing after activation, these patients experienced more severe HL at 1 year. Five patients implanted at our center experienced acceleration of HL after high charge exposure. In patients implanted at our center, high charge was associated with late HL (Pearson 0.366, p = 0.016). In rat cochleotypic explants, high voltage ES damaged afferent nerve fibers, reflected by blebbing and a 50% reduction in the number of fibers innervating the organ of Corti. In contrast, hair cells displayed only minor differences in cell number and morphology. CONCLUSIONS: Based on clinical and in vitro data, we theorize that the combination of acoustic amplification and ES in the setting of intact hair cells and neural architecture may contribute, in part, to cochlear toxicity, perhaps by damaging the afferent innervation.

Microtopographical features generated by photopolymerization recruit RhoA/ROCK through TRPV1 to direct cell and neurite growth.

Cell processes, including growth cones, respond to biophysical cues in their microenvironment to establish functional tissue architecture and intercellular networks. The mechanisms by which cells sense and translate biophysical cues into directed growth are unknown. We used photopolymerization to fabricate methacrylate platforms with patterned microtopographical features that precisely guide neurite growth and Schwann cell alignment. Pharmacologic inhibition of the transient receptor potential cation channel subfamily V member 1 (TRPV1) or reduced expression of TRPV1 by RNAi significantly disrupts neurite guidance by these microtopographical features. Exogenous expression of TRPV1 induces alignment of NIH3T3 fibroblasts that fail to align in the absence of TRPV1, further implicating TRPV1 channels as critical mediators of cellular responses to biophysical cues. Microtopographic features increase RhoA activity in growth cones and in TRPV1-expressing NIH3T3 cells. Further, Rho-associated kinase (ROCK) phosphorylation is elevated in growth cones and neurites on micropatterned surfaces. Inhibition of RhoA/ROCK by pharmacological compounds or reduced expression of either ROCKI or ROCKII isoforms by RNAi abolishes neurite and cell alignment, confirming that RhoA/ROCK signaling mediates neurite and cell alignment to microtopographic features. These studies demonstrate that microtopographical cues recruit TRPV1 channels and downstream signaling pathways, including RhoA and ROCK, to direct neurite and cell growth.

Material stiffness effects on neurite alignment to photopolymerized micropatterns.

The ability to direct neurite growth into a close proximity of stimulating elements of a neural prosthesis, such as a retinal or cochlear implant (CI), may enhance device performance and overcome current spatial signal resolution barriers. In this work, spiral ganglion neurons (SGNs), which are the target neurons to be stimulated by CIs, were cultured on photopolymerized micropatterns with varied matrix stiffnesses to determine the effect of rigidity on neurite alignment to physical cues. Micropatterns were generated on methacrylate thin film surfaces in a simple, rapid photopolymerization step by photomasking the prepolymer formulation with parallel line-space gratings. Two methacrylate series, a nonpolar HMA-co-HDDMA series and a polar PEGDMA-co-EGDMA series, with significantly different surface wetting properties were evaluated. Equivalent pattern periodicity was maintained across each methacrylate series based on photomask band spacing, and the feature amplitude was tuned to a depth of 2 µm amplitude for all compositions using the temporal control afforded by the UV curing methodology. The surface morphology was characterized by scanning electron microscopy and white light interferometry. All micropatterned films adsorb similar amounts of laminin from solution, and no significant difference in SGN survival was observed when the substrate compositions were compared. SGN neurite alignment significantly increases with increasing material modulus for both methacrylate series. Interestingly, SGN neurites respond to material stiffness cues that are orders of magnitude higher (GPa) than what is typically ascribed to neural environments (kPa). The ability to understand neurite response to engineered physical cues and mechanical properties such as matrix stiffness will allow the development of advanced biomaterials that direct de novo neurite growth to address the spatial signal resolution limitations of current neural prosthetics.