Oligodendrocyte progenitor cells' fate after neonatal asphyxia-Puzzling implications for the development of hypoxic-ischemic encephalopathy.

Premature birth or complications during labor can cause temporary disruption of cerebral blood flow, often followed by long-term disturbances in brain development called hypoxic-ischemic (HI) encephalopathy. Diffuse damage to the white matter is the most frequently detected pathology in this condition. We hypothesized that oligodendrocyte progenitor cell (OPC) differentiation disturbed by mild neonatal asphyxia may affect the viability, maturation, and physiological functioning of oligodendrocytes. To address this issue, we studied the effect of temporal HI in the in vivo model in P7 rats with magnetic resonance imaging (MRI), microscopy techniques and biochemical analyses. Moreover, we recreated the injury in vitro performing the procedure of oxygen-glucose deprivation on rat neonatal OPCs to determine its effect on cell viability, proliferation, and differentiation. In the in vivo model, MRI evaluation revealed changes in the volume of different brain regions, as well as changes in the directional diffusivity of water in brain tissue that may suggest pathological changes to myelinated neuronal fibers. Hypomyelination was observed in the cortex, striatum, and CA3 region of the hippocampus. Severe changes to myelin ultrastructure were observed, including delamination of myelin sheets. Interestingly, shortly after the injury, an increase in oligodendrocyte proliferation was observed, followed by an overproduction of myelin proteins 4 weeks after HI. Results verified with the in vitro model indicate, that in the first days after damage, OPCs do not show reduced viability, intensively proliferate, and overexpress myelin proteins and oligodendrocyte-specific transcription factors. In conclusion, despite the increase in oligodendrocyte proliferation and myelin protein expression after HI, the production of functional myelin sheaths in brain tissue is impaired. Presented study provides a detailed description of oligodendrocyte pathophysiology developed in an effect of HI injury, resulting in an altered CNS myelination. The described models may serve as useful tools for searching and testing effective of effective myelination-supporting therapies for HI injuries.

Reactive Gliosis in Neonatal Disorders: Friend or Foe for Neuroregeneration?

A developing nervous system is particularly vulnerable to the influence of pathophysiological clues and injuries in the perinatal period. Astrocytes are among the first cells that react to insults against the nervous tissue, the presence of pathogens, misbalance of local tissue homeostasis, and a lack of oxygen and trophic support. Under this background, it remains uncertain if induced astrocyte activation, recognized as astrogliosis, is a friend or foe for progressing neonatal neurodevelopment. Likewise, the state of astrocyte reactivity is considered one of the key factors discriminating between either the initiation of endogenous reparative mechanisms compensating for aberrations in the structures and functions of nervous tissue or the triggering of neurodegeneration. The responses of activated cells are modulated by neighboring neural cells, which exhibit broad immunomodulatory and pro-regenerative properties by secreting a plethora of active compounds (including interleukins and chemokines, neurotrophins, reactive oxygen species, nitric oxide synthase and complement components), which are engaged in cell crosstalk in a paracrine manner. As the developing nervous system is extremely sensitive to the influence of signaling molecules, even subtle changes in the composition or concentration of the cellular secretome can have significant effects on the developing neonatal brain. Thus, modulating the activity of other types of cells and their interactions with overreactive astrocytes might be a promising strategy for controlling neonatal astrogliosis.

Pearls and Pitfalls of Isolating Rat OPCs for In Vitro Culture with Different Methods.

There are several in vitro models to study the biology of oligodendrocyte progenitor cells (OPCs). The use of models based on induced pluripotent stem cells or oligodendrocyte-like cell lines has many advantages but raises significant questions, such as inaccurate reproduction of neural tissue or genetic instability. Moreover, in a specific case of studying the biology of neonatal OPCs, it is particularly difficult to find good representative model, due to the unique metabolism and features of these cells, as well as neonatal brain tissue. The following study evaluates two methods of isolating OPCs from rat pups as a model for in vitro studies. The first protocol is a modification of the classical mixed glial culture with series of shakings applied to isolate the fraction of OPCs. The second protocol is based on direct cell sorting and uses magnetic microbeads that target the surface antigen of the oligodendrocyte progenitor cell-A2B5. We compared the performance of these methods and analyzed the purity of obtained cultures as well as oligodendrocyte differentiation. Although the yield of OPCs collected with these two methods is similar, both have their advantages and disadvantages. The OPCs obtained with both methods give rise to mature oligodendrocytes within a few days of culture in ITS-supplemented serum-free medium and a 5% O2 atmosphere (mimicking the endogenous oxygen conditions of the nervous tissue). Methods for isolating rat OPCs In the following study we compared methods for isolating neonatal rat oligodendrocyte progenitor cells, for the studies on the in vitro model of neonatal brain injuries. We evaluated the purity of obtained cell cultures and the ability to maturate in physiological normoxia and serum-free culture medium.

The impact of the histone deacetylase inhibitor sodium butyrate on microglial polarization after oxygen and glucose deprivation.

BACKGROUND: Microglia play a major role in the development of brain inflammation after central nervous system injury. On the other hand, microglia also participate in the repair process. The dualistic role of these cells results from the fact that various states of their activation are associated with specific phenotypes. The M1 phenotype is responsible for the production of proinflammatory mediators, whereas the M2 microglia release anti-inflammatory and trophic factors and take part in immunosuppressive and neuroprotective processes. The histone deacetylase inhibitor sodium butyrate (SB) shows anti-inflammatory and neuroprotective effects in some animal models of brain injury. The aim of this study was to examine the effects of sodium butyrate on the proliferation and M1/M2 polarization of primary microglial cells after oxygen and glucose deprivation (OGD) in vitro. METHODS: Primary microglial cultures were prepared from 1-day-old rats, subjected to the OGD procedure and treated with SB (0.1 mM, 1 mM and 10 mM). The effect of OGD and SB on microglial proliferation was assessed by double immunofluorescence, and microglial phenotypes were evaluated by qPCR. RESULTS: The OGD procedure stimulated the proliferation of microglia after 24 h of culturing, and SB treatment reduced the division of these cells. This effect was inversely proportional to the SB concentration. The OGD procedure increased proinflammatory CD86 and IL1ß gene expression and reduced the expression of the anti-inflammatory M2 markers arginase and CD200 in microglia. CONCLUSIONS: SB can change the polarization of microglia after OGD from an unfavourable M1 to a beneficial M2 phenotype. Our results show that SB is a potential immunosuppressive agent that can modulate microglial activation stimulated by ischaemic-like conditions.

Neonatal Rat Glia Cultured in Physiological Normoxia for Modeling Neuropathological Conditions In Vitro.

Cell culture conditions were proven to highly affect crucial biological processes like proliferation, differentiation, intercellular crosstalk, and senescence. Oxygen tension is one of the major factors influencing cell metabolism and thus, modulating cellular response to pathophysiological conditions. In this context, the presented study aimed at the development of a protocol for efficient culture of rat neonatal glial cells (microglia, astrocytes, and oligodendrocytes) in oxygen concentrations relevant to the nervous tissue. The protocol allows for obtaining three major cell populations, which play crucial roles in sustaining tissue homeostasis and are known to be activated in response to a wide spectrum of external stimuli. The cells are cultured in media without supplement addition to avoid potential modulation of cell processes. The application of active biomolecules for coating culturing surfaces might be useful for mirroring physiological cell interactions with extracellular matrix components. The cell fractions can be assembled as cocultures to further evaluate investigated mechanisms, intercellular crosstalk, or cell response to tested pharmacological compounds. Applying additional procedures, like transient oxygen and glucose deprivation, allows to mimic in vitro the selected pathophysiological conditions. The presented culture system for neonatal rat glial cells is a highly useful tool for in vitro modeling selected neuropathological conditions.

Oligodendrocyte Response to Pathophysiological Conditions Triggered by Episode of Perinatal Hypoxia-Ischemia: Role of IGF-1 Secretion by Glial Cells.

Differentiation of oligodendrocyte progenitors towards myelinating cells is influenced by a plethora of exogenous instructive signals. Insulin-like growth factor 1 (IGF-1) is one of the major factors regulating cell survival, proliferation, and maturation. Recently, there is an ever growing recognition concerning the role of autocrine/paracrine IGF-1 signaling in brain development and metabolism. Since oligodendrocyte functioning is altered after the neonatal hypoxic-ischemic (HI) insult, a question arises if the injury exerts any influence on the IGF-1 secreted by neural cells and how possibly the change in IGF-1 concentration affects oligodendrocyte growth. To quantify the secretory activity of neonatal glial cells, the step-wise approach by sequentially using the in vivo, ex vivo, and in vitro models of perinatal asphyxia was applied. A comparison of the results of in vivo and ex vivo studies allowed evaluating the role of autocrine/paracrine IGF-1 signaling. Accordingly, astroglia were indicated to be the main local source of IGF-1 in the developing brain, and the factor secretion was shown to be significantly upregulated during the first 24 h after the hypoxic-ischemic insult. And conversely, the IGF-1 amounts released by oligodendrocytes and microglia significantly decreased. A morphometric examination of oligodendrocyte differentiation by means of the Sholl analysis showed that the treatment with low IGF-1 doses markedly improved the branching of oligodendroglial cell processes and, in this way, promoted their differentiation. The changes in the IGF-1 amounts in the nervous tissue after HI might contribute to the resulting white matter disorders, observed in newborn children who experienced perinatal asphyxia. Pharmacological modulation of IGF-1 secretion by neural cells could be reasonable solution in studies aimed at searching for therapies alleviating the consequences of perinatal asphyxia.

Directed glial differentiation and transdifferentiation for neural tissue regeneration.

Glial cells which are indispensable for the central nervous system development and functioning, are proven to be vulnerable to a harmful influence of pathological cues and tissue misbalance. However, they are also highly sensitive to both in vitro and in vivo modulation of their commitment, differentiation, activity and even the fate-switch by different types of bioactive molecules. Since glial cells (comprising macroglia and microglia) are an abundant and heterogeneous population of neural cells, which are almost uniformly distributed in the brain and the spinal cord parenchyma, they all create a natural endogenous reservoir of cells for potential neurogenerative processes required to be initiated in response to pathophysiological cues present in the local tissue microenvironment. The past decade of intensive investigation on a spontaneous and enforced conversion of glial fate into either alternative glial (for instance from oligodendrocytes to astrocytes) or neuronal phenotypes, has considerably extended our appreciation of glial involvement in restoring the nervous tissue cytoarchitecture and its proper functions. The most effective modulators of reprogramming processes have been identified and tested in a series of pre-clinical experiments. A list of bioactive compounds which are potent in guiding in vivo cell fate conversion and driving cell differentiation includes a selection of transcription factors, microRNAs, small molecules, exosomes, morphogens and trophic factors, which are helpful in boosting the enforced neuro-or gliogenesis and promoting the subsequent cell maturation into desired phenotypes. Herein, an issue of their utility for a directed glial differentiation and transdifferentiation is discussed in the context of elaborating future therapeutic options aimed at restoring the diseased nervous tissue.