Photodynamic Activity of Protoporphyrin IX-Immobilized Cellulose Monolith for Nerve Tissue Regeneration.

The development of nerve conduits with a three-dimensional porous structure has attracted great attention as they closely mimic the major features of the natural extracellular matrix of the nerve tissue. As low levels of reactive oxygen species (ROS) function as signaling molecules to promote cell proliferation and growth, this study aimed to fabricate protoporphyrin IX (PpIX)-immobilized cellulose (CEPP) monoliths as a means to both guide and stimulate nerve regeneration. CEPP monoliths can be fabricated via a simple thermally induced phase separation method and surface modification. The improved nerve tissue regeneration of CEPP monoliths was achieved by the activation of mitogen-activated protein kinases, such as extracellular signal-regulated kinases (ERKs). The resulting CEPP monoliths exhibited interconnected microporous structures and uniform morphology. The results of in vitro bioactivity assays demonstrated that the CEPP monoliths with under 0.54 ± 0.07 µmol/g PpIX exhibited enhanced photodynamic activity on Schwann cells via the generation of low levels of ROS. This photodynamic activation of the CEPP monoliths is a cell-safe process to stimulate cell proliferation without cytotoxic side effects. In addition, the protein expression of phospho-ERK increased considerably after the laser irradiation on the CEPP monoliths with low content of PpIX. Therefore, the CEPP monoliths have a potential application in nerve tissue regeneration as new nerve conduits.

Transcriptomic Profile Reveals Deregulation of Hearing-Loss Related Genes in Vestibular Schwannoma Cells Following Electromagnetic Field Exposure.

Hearing loss (HL) is the most common sensory disorder in the world population. One common cause of HL is the presence of vestibular schwannoma (VS), a benign tumor of the VIII cranial nerve, arising from Schwann cell (SC) transformation. In the last decade, the increasing incidence of VS has been correlated to electromagnetic field (EMF) exposure, which might be considered a pathogenic cause of VS development and HL. Here, we explore the molecular mechanisms underlying the biologic changes of human SCs and/or their oncogenic transformation following EMF exposure. Through NGS technology and RNA-Seq transcriptomic analysis, we investigated the genomic profile and the differential display of HL-related genes after chronic EMF. We found that chronic EMF exposure modified the cell proliferation, in parallel with intracellular signaling and metabolic pathways changes, mostly related to translation and mitochondrial activities. Importantly, the expression of HL-related genes such as NEFL, TPRN, OTOGL, GJB2, and REST appeared to be deregulated in chronic EMF exposure. In conclusion, we suggest that, at a preclinical stage, EMF exposure might promote the transformation of VS cells and contribute to HL.

Light irradiation of peripheral nerve cells: Wavelength impacts primary sensory neuron outgrowth in vitro.

The expansion of optogenetics via the development and application of new opsins has opened a new world of possibilities as a research and therapeutic tool. Nevertheless, it has also raised questions about the innocuity of using light irradiation on tissues and cells such as those from the Peripheral Nervous System (PNS). Thus, to investigate the potential of PNS being affected by optogenetic light irradiation, rat dorsal root ganglion neurons and Schwann cells were isolated and their response to light irradiation examined in vitro. Light irradiation was delivered as millisecond pulses at wavelengths in the visible spectrum between 627 and 470 nm, with doses ranging between 4.5 and 18 J/cm2 at an irradiance value of 1 mW/mm2. Results show that compared to cultures kept in dark conditions, light irradiation at 470 nm reduced neurite outgrowth in dissociated dorsal root neurons in a dose dependent manner while higher wavelengths had no effect on neuron morphology. Although neurite outgrowth was limited by light irradiation, no signs of cell death or apoptosis were found. On the other hand, peripheral glia, Schwann cells, were insensitive to light irradiation with metabolism, proliferation, and RNA levels of transcription factors c-Jun and krox-20 remaining unaltered following stimulation. As the fields of photostimulation and optogenetics expand, these results indicate the need for consideration to cell type response and stimulation parameters for applications in vitro and further investigation on specific mechanisms driving response.

Effects of 660-nm and 780-nm Laser Therapy on ST88-14 Schwann Cells.

The aim of the present study was to evaluate the comparative effects of red (660-nm) and near-infrared (780-nm) low-level laser therapy (LLLT) on viability, mitochondrial activity, morphology and gene expression of growth factors on Schwann cells (SC). ST88-14 cells were grown in RPMI 1640 with 10 mM of HEPES, 2 mM of glutamine, 10% fetal bovine serum and 1% antibiotic-antimycotic solution at 37°C in humidified atmosphere of 5% CO2 . Cells were detached with trypsin and centrifugated at 231 g for 5 min at 10°C, and the pellet (8 × 104  cells/tube) was irradiated at the bottom of 50 ml polypropylene tube with a Twin-Laser system (660 and 780 nm, 40 mW, 1 mW cm-2 , 3.2 and 6.4 J, 80 and 160 J cm-2 with 80 and 160 s). After 1, 3 and 7 days, the analysis was performed. After irradiation, the SC increase mitochondrial activity, gene expression of the neural growth factors NGF and BDNF, and cell migration and increase the G2/M cells. SC showed neuronal morphology, normal F-actin cytoskeleton organization and positive labeling for S100. PBM increased metabolic activity, mitosis and gene expression when irradiated with red and infrared LLLT. An increase in cell migration was obtained when irradiated with infrared LLLT.

The effects of low intensity focused ultrasonic stimulation on dorsal root ganglion neurons and Schwann cells in vitro.

Satisfactory treatment of peripheral nerve injury (PNI) faces difficulties owing to the intrinsic biological barriers in larger injuries and invasive surgical interventions. Injury gaps >3 cm have low chances of full motor and sensory recovery, and the unmet need for PNI repair techniques which increase the likelihood of functional recovery while limiting invasiveness motivate this work. Building upon prior work in ultrasound stimulation (US) of dorsal root ganglion (DRG) neurons, the effects of US on DRG neuron and Schwann cell (SC) cocultures were investigated to uncover the role of SCs in mediating the neuronal response to US in vitro. Acoustic intensity-dependent alteration in selected neuromorphometrics of DRG neurons in coculture with SCs was observed in total outgrowth, primary neurites, and length compared to previously reported DRG monoculture in a calcium-independent manner. SC viability and proliferation were not impacted by US. Conditioned medium studies suggest secreted factors from SCs subjected to US impact DRG neuron morphology. These findings advance the current understanding of mechanisms by which these cell types respond to US, which may lead to new noninvasive US therapies for treating PNI.

Low-intensity pulsed ultrasound for regenerating peripheral nerves: potential for penile nerve.

Peripheral nerve damage, such as that found after surgery or trauma, is a substantial clinical challenge. Much research continues in attempts to improve outcomes after peripheral nerve damage and to promote nerve repair after injury. In recent years, low-intensity pulsed ultrasound (LIPUS) has been studied as a potential method of stimulating peripheral nerve regeneration. In this review, the physiology of peripheral nerve regeneration is reviewed, and the experiments employing LIPUS to improve peripheral nerve regeneration are discussed. Application of LIPUS following nerve surgery may promote nerve regeneration and improve functional outcomes through a variety of proposed mechanisms. These include an increase of neurotrophic factors, Schwann cell (SC) activation, cellular signaling activations, and induction of mitosis. We searched PubMed for articles related to these topics in both in vitro and in vivo animal research models. We found numerous studies, suggesting that LIPUS following nerve surgery promotes nerve regeneration and improves functional outcomes. Based on these findings, LIPUS could be a novel and valuable treatment for nerve injury-induced erectile dysfunction.

Low-Intensity Extracorporeal Shock Wave Therapy Enhances Brain-Derived Neurotrophic Factor Expression through PERK/ATF4 Signaling Pathway.

Low-intensity extracorporeal shock wave therapy (Li-ESWT) is used in the treatment of erectile dysfunction, but its mechanisms are not well understood. Previously, we found that Li-ESWT increased the expression of brain-derived neurotrophic factor (BDNF). Here we assessed the underlying signaling pathways in Schwann cells in vitro and in penis tissue in vivo after nerve injury. The result indicated that BDNF were significantly increased by the Li-ESWT after nerve injury, as well as the expression of BDNF in Schwann cells (SCs, RT4-D6P2T) in vitro. Li-ESWT activated the protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) pathway by increasing the phosphorylation levels of PERK and eukaryotic initiation factor 2a (eIF2α), and enhanced activating transcription factor 4 (ATF4) in an energy-dependent manner. In addition, GSK2656157-an inhibitor of PERK-effectively inhibited the effect of Li-ESWT on the phosphorylation of PERK, eIF2α, and the expression of ATF4. Furthermore, silencing ATF4 dramatically attenuated the effect of Li-ESWT on the expression of BDNF, but had no effect on hypoxia-inducible factor (HIF)1α or glial cell-derived neurotrophic factor (GDNF) in Schwann cells. In conclusion, our findings shed new light on the underlying mechanisms by which Li-ESWT may stimulate the expression of BDNF through activation of PERK/ATF4 signaling pathway. This information may help to refine the use of Li-ESWT to further improve its clinical efficacy.

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 implementing optogenetics to drive nerve growth in specific cell populations.

In vitro responses of neurofibroma fibroblasts, mast cells and Schwann cells obtained from patients with neurofibromatosis 1 to 308-nm excimer light and/or vitamin D3.

Fibroblasts, mast cells and Schwann cells were isolated from neurofibromas of patients with neurofibromatosis 1, and their responses to 308-nm excimer light irradiation and/or vitamin D3 or an analog (tacalcitol; 1,24-dihydroxyvitamin D3 ) were examined in vitro. Excimer light irradiation (300 mJ/cm(2) ) suppressed the growth of all three cell types. Exposure to 10(-7)  mol/L of 1α,25(OH)2 D3 (VD3 ) or tacalcitol suppressed the growth of fibroblasts and mast cells, but not Schwann cells. These results suggest that the different neurofibroma cell types show different responses to VD3 . A combination of excimer light irradiation and VD3 is necessary to suppress the growth of neurofibroma cells in vivo.

Electric field stimulation through a substrate influences Schwann cell and extracellular matrix structure.

OBJECTIVE: Electric field (EF) stimulation has been used to cue cell growth for tissue engineering applications. In this study, we explore the electrical parameters and extracellular mechanisms that elicit changes in cell behavior when stimulated through the substrate. APPROACH: Rat Schwann cell morphology was compared when exposed to EF through the media or a conductive indium tin oxide substrate. Ionic and structural effects were then analyzed on Matrigel and collagen I, respectively. MAIN RESULTS: When stimulating through media, cells had greater alignment perpendicular to the EF with higher current densities (106 mA cm(-2) at 245 mV mm(-1)), and reached maximum alignment within 8 h. Stimulation through the substrate with EF (up to 110 mV mm(-1)) did not affect Schwann cell orientation, however the EF caused extracellular matrix (ECM) coatings on substrates to peel away, suggesting EF can physically change the ECM. Applying alternating current (ac) 2-1000 Hz signals through the media or substrate both caused cells to flatten and protrude many processes, without preferential alignment. Matrigel exposed to a substrate EF of 10 mV mm(-1) for 2 h had a greater calcium concentration near the cathode, but quickly dissipated when the EF was removed. Schwann cells seeded 7 d after gels were exposed to substrate EF still aligned perpendicular to the EF direction. Microscopy of collagen I exposed to substrate EF shows alignment and bundling of fibrils. SIGNIFICANCE: These findings demonstrate EF exposure can control Schwann cell alignment and morphology, change ECM bulk/surface architecture, and align ECM structures.

Effects of low level laser therapy on proliferation and neurotrophic factor gene expression of human schwann cells in vitro.

Previous studies have been proposed that proliferation and release of certain growth factors by different types of cells can be modulated by low level laser therapy. We aimed to demonstrate the effect of laser irradiation on human schwann cell proliferation and neurotrophic factor gene expression in vitro. Human schwann cells (SCs) were harvested from sural nerve that was obtained from organ donor followed by treatment with an 810 nm, 50 mW diode laser (two different energies: 1 J/cm(2) and 4 J/cm(2)) in three consecutive days. SC proliferation was measured, after first irradiation on days 1, 4 and 7 by the MTT assay. Real time PCR analysis was utilized on days 5 and 20 to evaluate the expression of key genes involved in nerve regeneration consist of NGF, BDNF and GDNF. Evaluation of cellular proliferation following one day after laser treatment revealed significant decrease in cell proliferation compared to control group. However on day 7, significant increase in proliferation was found in both the irradiated groups in comparison with the control group. No significant difference was found between the laser treated groups. Treatment of SCs with laser resulted in significant increase in NGF gene expression on day 20. Difference between two treated groups and control group was not significant for BDNF and GDNF gene expression. Our results demonstrate that low level laser therapy stimulate human schwann cell proliferation and NGF gene expression in vitro.

Ultrasound-stimulated peripheral nerve regeneration within asymmetrically porous PLGA/Pluronic F127 nerve guide conduit.

Recently, we developed a novel method to fabricate a nerve guide conduit (NGC) with asymmetrical pore structure and hydrophilicity using poly(lactic-co-glycolic acid) (PLGA) and Pluronic F127 by a modified immersion precipitation method. From the animal study using a rat model (sciatic nerve defect of rat), we recognized that the unique PLGA/Pluronic F127 tube provided good environments for nerve regeneration. In this study, we applied low-intensity pulsed ultrasound as a simple and noninvasive stimulus at the PLGA/F127 NGC-implanted site transcutaneously in rats to investigate the feasibility of ultrasound for the enhanced nerve regeneration through the tube. The nerve regeneration behaviors within the ultrasound-stimulated PLGA/Pluronic F127 NGCs were compared with the NGCs without the ultrasound treatment as well as normal nerve by histological and immunohistochemical observations. It was observed that the PLGA/Pluronic F127 tube-implanted group applied with the ultrasound had more rapid nerve regeneration behavior (approximately 0.71 mm/day) than the tube-implanted group without the ultrasound treatment (approximately 0.48 mm/day). The ultrasound-treated tube group also showed greater neural tissue area as well as larger axon diameter and thicker myelin sheath than the tube group without the ultrasound treatment, indicating better nerve regeneration. The better nerve regeneration behavior in the our NGC/ultrasound system may be caused by the synergistic effect of the asymmetrically porous PLGA/Pluronic F127 tube with unique properties (selective permeability, hydrophilicity, and structural stability, which can provide good environment for nerve regeneration) and physical stimulus (stimulation of the Schwann cells and activation of the neurotrophic factors).

Absence of relevant effects of 5 mT static magnetic field on morphology, orientation and growth of a rat Schwann cell line in culture.

The aim of this study is to observe possible changes in the morphology, orientation or cell growth of an in vitro cultured Schwann cell line by 24 h exposure to 5 mT static magnetic fields. The magnetic field generator basically consists of a pair of circular coils in a Helmholtz arrangement and enables temperature to be controlled (37+/-0.1 degrees C). We did not find any statistically significant differences in the cell growth rate between control and exposed cells, nor did we observe any differences in cell morphology or orientation.

Experimental strategies to promote functional recovery after peripheral nerve injuries.

The capacity of Schwann cells (SCs) in the peripheral nervous system to support axonal regeneration, in contrast to the oligodendrocytes in the central nervous system, has led to the misconception that peripheral nerve regeneration always restores function. Here, we consider how prolonged periods of time that injured neurons remain without targets during axonal regeneration (chronic axotomy) and that SCs in the distal nerve stumps remain chronically denervated (chronic denervation) progressively reduce the number of motoneurons that regenerate their axons. We demonstrate the effectiveness of low-dose, brain-derived neurotrophic and glial-derived neurotrophic factors to counteract the effects of chronic axotomy in promoting axonal regeneration. High-dose brain-derived neurotrophic factor (BDNF) on the other hand, acting through the p75 receptor, inhibits axonal regeneration and may be a factor in stopping regenerating axons from forming neuromuscular connections in skeletal muscle. The immunophilin, FK506, is also effective in promoting axonal regeneration after chronic axotomy. Chronic denervation of SCs (>1 month) severely deters axonal regeneration, although the few motor axons that do regenerate to reinnervate muscles become myelinated and form enlarged motor units in the reinnervated muscles. We found that in vitro incubation of chronically denervated SCs with transforming growth factor-beta re-established their growth-supportive phenotype in vivo, consistent with the idea that the interaction between invading macrophages and denervated SCs during Wallerian degeneration is essential to sustain axonal regeneration by promoting the growth-supportive SC phenotype. Finally, we consider the effectiveness of a brief period of 20 Hz electrical stimulation in promoting the regeneration of axons across the surgical gap after nerve repair.

Dysfunctional oligodendrocyte progenitor cell (OPC) populations may inhibit repopulation of OPC depleted tissue.

We have attempted to extend a previously described rat model of focal oligodendrocyte progenitor cell (OPC) depletion, using 40 Gy X-irradiation (Chari and Blakemore [2002] Glia 37:307-313), to the adult mouse spinal cord, to examine the ability of OPCs present in adjacent normal areas to colonise areas of progenitor depletion. In contrast to rat, OPCs in the mouse spinal cord appeared to be a comparatively radiation-resistant population, as 30-35% of OPCs survived in X-irradiated tissue (whereas <1% of OPCs survive in X-irradiated rat spinal cord). The numbers of surviving OPCs remained constant with time indicating that this population was incapable of regenerating itself in response to OPC loss. Additionally, these OPCs did not contribute to remyelination of axons when demyelinating lesions were placed in X-irradiated tissue, suggesting that the surviving cells are functionally impaired. Importantly, the length of the OPC-depleted area did not diminish with time, as would be expected if progressive repopulation of OPC-depleted areas by OPCs from normal areas was occurring. Our findings therefore raise the possibility that the presence of a residual dysfunctional OPC population may inhibit colonisation of such areas by normal OPCs.

Morphological changes in skin nerves caused by electromagnetic radiation of the millimeter range.

The morphological changes in skin nerves of BALB/C mice after millimeter wavelength range electromagnetic exposure at a frequency of 42.25 GHz and power of 50 mW/cm2 were studied. Immediately after 15 minutes of exposure, the destruction of the cytoplasm of myelinated and unmyelinated axons was found.

Schwann cell-induced loss of synapses in the central nervous system.

Synaptophysin immunostaining of areas of spinal gray matter occupied by radiation-induced intraspinal Schwann cells revealed a loss of immunoreactivity from the neuropil. In contrast, synaptophysin immunoreactivity was preserved on the somata and proximal dendrites of motor neurons. The present study extended these observations to the ultrastructural level and confirmed the absence not only of synapses but also of astrocytes and small- and medium-sized dendrites. These neural elements were abundant and appropriately organized in contiguous areas of irradiated neuropil not occupied by Schwann cells.

Function of skeletal muscle tissue formed after myoblast transplantation into irradiated mouse muscles.

1. Pretreatment of muscles with ionising radiation enhances tissue formation by transplanted myoblasts but little is known about the effects on muscle function. We implanted myoblasts from an expanded, male-donor-derived, culture (i28) into X-ray irradiated (16 Gy) or irradiated and damaged soleus muscles of female syngeneic mice (Balb/c). Three to 6 months later the isometric contractile properties of the muscles were studied in vitro, and donor nuclei were visualised in muscle sections with a Y chromosome-specific DNA probe. 2. Irradiated sham-injected muscles had smaller masses than untreated solei and produced less twitch and tetanic force (all by about 18 %). Injection of 106 myoblasts abolished these deficiencies and innervation appeared normal. 3. Cryodamage of irradiated solei produced muscle remnants with few (1-50) or no fibres. Additional myoblast implantation led to formation of large muscles (25 % above normal) containing numerous small-diameter fibres. Upon direct electrical stimulation, these muscles produced considerable twitch (53 % of normal) and tetanic forces (35 % of normal) but innervation was insufficient as indicated by weak nerve-evoked contractions and elevated ACh sensitivity. 4. In control experiments on irradiated muscles, reinnervation was found to be less complete after botulinum toxin paralysis than after nerve crush indicating that proliferative arrest of irradiated Schwann cells may account for the observed innervation deficits. 5. Irradiation appears to be an effective pretreatment for improving myoblast transplantation. The injected cells can even produce organised contractile tissue replacing whole muscle. However, impaired nerve regeneration limits the functional performance of the new muscle.

Schwann cell invasion of ventral spinal cord: the effect of irradiation on astrocyte barriers.

This study examines a radiation-induced invasion and spread of Schwann cells into ventral gray regions of the lumbar spinal cord. The prevalence of these cells within the gray matter and the time course of their appearance in the ventral spinal cord is quite different from the pattern of Schwann cell development in dorsal spinal cord reported previously. The focus is on 2 possible pathways, each involving astrocytic barriers, by which Schwann cells access the ventral gray matter. The first of these is the glia limitans covering the ventral surface of the spinal cord and the possibility that its integrity has been disrupted by the exposure to x-rays. Comparisons of the glia limitans, including its thickness, between irradiated and nonirradiated rats revealed that exposure to radiation did not result in any morphologically discernible alterations. The second barrier examined was the astrocytic covering of blood vessels. In irradiated animals the astrocyte processes that normally surround blood vessels were missing in some instances, and Schwann cells were observed at these sites. The difference between the dorsal and ventral occurrence of Schwann cells is that, whereas Schwann cells primarily follow axons, specifically dorsal root axons, to access the dorsal spinal cord, it appears that the presence of Schwann cells in the ventral portion of the spinal cord where their location is primarily in the gray matter is associated with the vasculature.

Patterns of Schwann cell myelination of axons within the spinal cord.

Patterns of Schwann cell myelination of long-projecting axons in the spinal cord were studied. The goal was to determine if such axons arising from neurons whose somata and processes are normally confined to the central nervous system can interact effectively with Schwann cells, the myelinating cells of the peripheral nervous system. In one paradigm Schwann cells develop in the dorsal funiculi of the lumbar spinal cord subsequent to radiation-induced alterations in development of the glial populations. Light and electron microscopic evaluations were made in the region of the corticospinal tracts (CSTs), which in the rat occupy the base of the dorsal funiculi. At 90 days following irradiation, larger axons of these tracts (> 1.5 microns in diameter) were myelinated by Schwann cells, and smaller axons were ensheathed by them. In the second paradigm cultured Schwann cells were injected into the medial portions of the ventral funiculi at 13 days post-irradiation when the glial population was markedly reduced. Earlier investigations from this laboratory demonstrated that Schwann cells do not develop in the irradiated ventral funiculi, as they do dorsally. When placed in proximity to long-projecting axons in the medial portion of the ventral funiculi, the Schwann cells either formed compact myelin sheaths or ensheathed axons, depending upon their diameter. Fasciculation and presence of collagen were characteristic of this paradigm but were absent from the Schwann cell-occupied regions of the CSTs. This probably relates to the presence of fibroblasts in the injected cultures.(ABSTRACT TRUNCATED AT 250 WORDS)