Pulsating Extremely Low-Frequency Electromagnetic Fields Influence Differentiation of Mouse Neural Stem Cells towards Astrocyte-like Phenotypes: In Vitro Pilot Study.

Even though electromagnetic fields have been reported to assist endogenous neurogenesis, little is known about the exact mechanisms of their action. In this pilot study, we investigated the effects of pulsating extremely low-frequency electromagnetic fields on neural stem cell differentiation towards specific phenotypes, such as neurons and astrocytes. Neural stem cells isolated from the telencephalic wall of B6(Cg)-Tyrc-2J/J mouse embryos (E14.5) were randomly divided into three experimental groups and three controls. Electromagnetic field application setup included a solenoid placed within an incubator. Each of the experimental groups was exposed to 50Hz ELF-EMFs of varied strengths for 1 h. The expression of each marker (NES, GFAP, ß-3 tubulin) was then assessed by immunocytochemistry. The application of high-strength ELF-EMF significantly increased and low-strength ELF-EMF decreased the expression of GFAP. A similar pattern was observed for ß-3 tubulin, with high-strength ELF-EMFs significantly increasing the immunoreactivity of ß-3 tubulin and medium- and low-strength ELF-EMFs decreasing it. Changes in NES expression were observed for medium-strength ELF-EMFs, with a demonstrated significant upregulation. This suggests that, even though ELF-EMFs appear to inhibit or promote the differentiation of neural stem cells into neurons or astrocytes, this effect highly depends on the strength and frequency of the fields as well as the duration of their application. While numerous studies have demonstrated the capacity of EMFs to guide the differentiation of NSCs into neuron-like cells or ß-3 tubulin+ neurons, this is the first study to suggest that ELF-EMFs may also steer NSC differentiation towards astrocyte-like phenotypes.

Lusca: FIJI (ImageJ) based tool for automated morphological analysis of cellular and subcellular structures.

The human body consists of diverse subcellular, cellular and supracellular structures. Neurons possess varying-sized projections that interact with different cellular structures leading to the development of highly complex morphologies. Aiming to enhance image analysis of complex biological forms including neurons using available FIJI (ImageJ) plugins, Lusca, an advanced open-source tool, was developed. Lusca utilizes machine learning for image segmentation with intensity and size thresholds. It performs particle analysis to ascertain parameters such as area/volume, quantity, and intensity, in addition to skeletonization for determining length, branching, and width. Moreover, in conjunction with colocalization measurements, it provides an extensive set of 29 morphometric parameters for both 2D and 3D analysis. This is a significant enhancement compared to other scripts that offer only 5-15 parameters. Consequently, it ensures quicker and more precise quantification by effectively eliminating noise and discerning subtle details. With three times larger execution speed, fewer false positive and negative results, and the capacity to measure various parameters, Lusca surpasses other existing open-source solutions. Its implementation of machine learning-based segmentation facilitates versatile applications for different cell types and biological structures, including mitochondria, fibres, and vessels. Lusca's automated and precise measurement capability makes it an ideal choice for diverse biological image analyses.

The Oxygen and Glucose Deprivation of Immature Cells of the Nervous System Exerts Distinct Effects on Mitochondria, Mitophagy, and Autophagy, Depending on the Cells' Differentiation Stage.

Perinatal brain damage, one of the most common causes of lifelong impairment, is predominantly caused by a lack of oxygen and glucose during early development. These conditions, in turn, affect cells of the nervous tissue through various stages of their maturation. To quantify the influence of these factors on cell differentiation and mitochondrial parameters, we exposed neural cell precursors to oxygen and glucose deprivation (OGD) during three stages of their differentiation: day 1, day 7, and day 14 (D1, D7, and D14, respectively). The obtained results show that OGD slows down cellular differentiation and causes cell death. Regardless of the level of cell maturity, the overall area of the mitochondria, their length, and the branching of their filaments decreased uniformly when exposed to OGD-related stress. Moreover, the cells in all stages of differentiation exhibited an increase in ROS production, hyperpolarization of the mitochondrial membrane, and autophagy. Interestingly, day 7 was the only stage in which a significant increase in mitochondrial fission, along with measurable instances of mitophagy, were detected. Taken together, the results of this study suggest that, apart from common reactions to a sudden lack of oxygen and glucose, cells in specific stages of neural differentiation can also exhibit increased preferences for mitochondrial fission and mitophagy. Such findings could play a role in guiding the future development of novel therapeutic approaches targeting perinatal brain damage during specific stages of nervous system development.

Mesenchymal stem cell therapy for neurological disorders: The light or the dark side of the force?

Neurological disorders are recognized as major causes of death and disability worldwide. Because of this, they represent one of the largest public health challenges. With awareness of the massive burden associated with these disorders, came the recognition that treatment options were disproportionately scarce and, oftentimes, ineffective. To address these problems, modern research is increasingly looking into novel, more effective methods to treat neurological patients; one of which is cell-based therapies. In this review, we present a critical analysis of the features, challenges, and prospects of one of the stem cell types that can be employed to treat numerous neurological disorders-mesenchymal stem cells (MSCs). Despite the fact that several studies have already established the safety of MSC-based treatment approaches, there are still some reservations within the field regarding their immunocompatibility, heterogeneity, stemness stability, and a range of adverse effects-one of which is their tumor-promoting ability. We additionally examine MSCs' mechanisms of action with respect to in vitro and in vivo research as well as detail the findings of past and ongoing clinical trials for Parkinson's and Alzheimer's disease, ischemic stroke, glioblastoma multiforme, and multiple sclerosis. Finally, this review discusses prospects for MSC-based therapeutics in the form of biomaterials, as well as the use of electromagnetic fields to enhance MSCs' proliferation and differentiation into neuronal cells.

Critical points for optimizing long-term culture and neural differentiation capacity of rodent and human neural stem cells to facilitate translation into clinical settings.

Despite several decades of research on the nature and functional properties of neural stem cells, which brought great advances in regenerative medicine, there is still a plethora of ambiguous protocols and interpretations linked to their applications. Here, we present a whole spectrum of protocol elements that should be standardized in order to obtain viable cell cultures and facilitate their translation into clinical settings. Additionally, this review also presents outstanding limitations and possible problems to be encountered when dealing with protocol optimization. Most importantly, we also outline the critical points that should be considered before starting any experiments utilizing neural stem cells or interpreting their results.

Overview of Neural Tube Defects: Gene-Environment Interactions, Preventative Approaches and Future Perspectives.

Neural tube defects (NTDs) are the second most common congenital malformations of humans, characterized by impaired development of the central nervous system. Even though the etiology of most birth defects remains undetermined, genetic and environmental risk factors in the background of NTDs have been identified and extensively reported. On top of genetic and nutritional risks which include mutations in both coding and non-coding regions and maternal folate status, respectively, recent years have seen a rise in the identification of a variety of teratogens that could be implicated in NTD development. These include polycyclic aromatic hydrocarbons, arsenic, pesticides, maternal hyperthermia and antibiotics as well as pain and seizure medication. With an increase in understanding of teratogens leading to NTD formation, preventative and treatment approaches have witnessed great advances throughout the years. While the most common preventative approach includes folic acid food fortification as well as suggested inositol supplementation, treatment and management approaches differ greatly depending on the developmental stage and the site of the lesion and include prenatal surgery, stem cell transplantation and postnatal surgery. Because NTDs still represent a large health and financial burden for the patient and society as a whole, it is crucial to investigate potential risk factors and develop novel approaches in order to fully prevent this category of disorders.

Regenerative Neurology and Regenerative Cardiology: Shared Hurdles and Achievements.

From the first success in cultivation of cells in vitro, it became clear that developing cell and/or tissue specific cultures would open a myriad of new opportunities for medical research. Expertise in various in vitro models has been developing over decades, so nowadays we benefit from highly specific in vitro systems imitating every organ of the human body. Moreover, obtaining sufficient number of standardized cells allows for cell transplantation approach with the goal of improving the regeneration of injured/disease affected tissue. However, different cell types bring different needs and place various types of hurdles on the path of regenerative neurology and regenerative cardiology. In this review, written by European experts gathered in Cost European action dedicated to neurology and cardiology-Bioneca, we present the experience acquired by working on two rather different organs: the brain and the heart. When taken into account that diseases of these two organs, mostly ischemic in their nature (stroke and heart infarction), bring by far the largest burden of the medical systems around Europe, it is not surprising that in vitro models of nervous and heart muscle tissue were in the focus of biomedical research in the last decades. In this review we describe and discuss hurdles which still impair further progress of regenerative neurology and cardiology and we detect those ones which are common to both fields and some, which are field-specific. With the goal to elucidate strategies which might be shared between regenerative neurology and cardiology we discuss methodological solutions which can help each of the fields to accelerate their development.

Ozone influences migration and proliferation of neural stem cells in vitro.

Ozone (O3) is a short-lived molecule which can be produced in a controlled reaction when oxygen is exposed to electric discharge. In the last few decades, many publications dealing both with animals and humans reported beneficial effects of ozone administration linked to its immunomodulatory and protective role against cellular damage. This is the first work which brings insight into how ozone influences cells of neural lineage in vitro and hypothesizes the potential molecular and novel electromagnetic mechanisms behind its action. By using neural stem cells, we show that ozone, especially in concentrations of around 11 µg/mL, significantly increases the speed of neural cell migration. With much lower effects, it also increases cell proliferation and cytokine production. Results of this study, at least partly, explain the observed beneficial effects of ozone in diseases of the nervous system tested on animal models and in human clinical trials. Therefore, here described effects of ozone on cellular level represent a firm basis for further investigation of possible applications of ozone in regeneration of the nervous system.

How to face the aging world - lessons from dementia research.

A continuous rise in life expectancy has led to an increase in the number of senior citizens, now amounting to a fifth of the global population, and to a dramatic increase in the prevalence of diseases of the elderly. This review discusses the threat of dementia, a disease that imposes enormous financial burden on health systems and warrants efficient therapeutic solutions. What we learned from numerous failed clinical trials is that we have to immediately take into account two major elements: early detection of dementia, much before the onset of symptoms, and personalized (precision) medicine treatment approach. We also discuss some of the most promising therapeutic directions, including stem cells, exosomes, electromagnetic fields, and ozone.

Integrity of White Matter is Compromised in Mice with Hyaluronan Deficiency.

Brain white matter is the means of efficient signal propagation in brain and its dysfunction is associated with many neurological disorders. We studied the effect of hyaluronan deficiency on the integrity of myelin in murine corpus callosum. Conditional knockout mice lacking the hyaluronan synthase 2 were compared with control mice. Ultrastructural analysis by electron microscopy revealed a higher proportion of myelin lamellae intruding into axons of knockout mice, along with significantly slimmer axons (excluding myelin sheath thickness), lower g-ratios, and frequent loosening of the myelin wrappings, even though the myelin thickness was similar across the genotypes. Analysis of extracellular diffusion of a small marker molecule tetramethylammonium (74 MW) in brain slices prepared from corpus callosum showed that the extracellular space volume increased significantly in the knockout animals. Despite this vastly enlarged volume, extracellular diffusion rates were significantly reduced, indicating that the compromised myelin wrappings expose more complex geometric structure than the healthy ones. This finding was confirmed in vivo by diffusion-weighted magnetic resonance imaging. Magnetic resonance spectroscopy suggested that water was released from within the myelin sheaths. Our results indicate that hyaluronan is essential for the correct formation of tight myelin wrappings around the axons in white matter.

Molecular mechanisms of microglia- and astrocyte-driven neurorestoration triggered by application of electromagnetic fields.

AIM: To propose potential mechanisms of action of electromagnetic fields (EMF) on astrocytes and microglia and to elucidate the role of heat shock proteins (HSP), adenosine triphosphate (ATP), calcium ions (Ca2+), and hypoxia-inducible factor 1α (HIF1α) in neurorestoration following the application of EMF. METHODS: We reviewed the existing studies within the public domain and cross-evaluated their results in order to conclude on the molecular mechanisms of microglia-astrocyte crosstalk at work during EMF treatment. RESULTS: The existing studies suggest that EMF induces the increase of HSP70 expression and inhibition of HIF1α, thus decreasing inflammation and allowing the microglia-astrocyte crosstalk to initiate the formation of a glial scar within the central nervous system. Furthermore, by potentially up-regulating A2A and A3 adenosine receptors, EMF increases cAMP accumulation from astrocytes and reduces the expression of inflammatory cytokines TNF α and IL-8, thus initiating neurorestoration. CONCLUSION: The microglia-astrocyte crosstalk during EMF treatment is crucial for the initiation of neurorestoration. Elucidating the exact mechanisms of EMF actions upon microglia and astrocytes, and its role in neurorestoration could be a key step in further research of the therapeutic potential of EMFs in various neurological disorders.

Modeling of inhomogeneous electromagnetic fields in the nervous system: a novel paradigm in understanding cell interactions, disease etiology and therapy.

All major processes in the nervous system depend on interactions between cells and nerve fibers. In this work we present a novel model of inhomogeneous electromagnetic fields originating from nerve fibers and delineate their influence on cells. By expanding Hodgkin-Huxley's applied current into axial current, governed by[Formula: see text], we reveal that cell-with-neuron interactions are regulated by the strength of the electromagnetic fields, which are homogeneous up to 2.066 µm or 6.606 µm away from neurilemma and axolemma, respectively. At the nodes of Ranvier, these fields reach strengths of 3.0 × 10-12T, while at the myelinated segments they only peak at 2.3 × 10-12T. These are the same fields which are, due to inhomogeneity, detected as 1,000 times weaker by magnetoencephalography. Considering the widespread occurrence of neurodegenerative disorders, our model reveals that a 50% demyelination increases the field strength by 0.35 × 10-12T, while a complete demyelination increases it by 0.7 × 10-12T. Since this suggests that the inhomogeneous electromagnetic fields around neurons play a role in physiological and pathological processes, including cell-to-neuron and cell-to-cell communication, their improved understanding opens up new therapeutic strategies based on electromagnetic field modulation or cell's surface charge alteration.