ABSTRACT
Tactile agnosia is the inability to recognize objects via haptic exploration, in the absence of an elementary sensory deficit. Traditionally, it has been described as a disturbance in extracting information about the physical properties of objects ("apperceptive agnosia") or in associating object representation with its semantic meaning ("associative agnosia"). However, tactile agnosia is a rare and difficult-to-diagnose condition, due to the frequent co-occurrence of sensorimotor symptoms and the lack of consensus on the terminology and assessment methods. Among tactile agnosia classifications, hyloagnosia (i.e., difficulty in quality discrimination of objects) and morphoagnosia (i.e., difficulty in shape and size recognition) have been proposed to account for the apperceptive level. However, a dissociation between the two has been reported in two cases only. Indeed, very few cases of pure tactile agnosia have been described, mostly associated with vascular damages in somatosensory areas, in pre- and postcentral gyrus, intraparietal sulcus, supramarginal gyrus, and insular cortex. An open question is whether degenerative conditions affecting the same areas could lead to similar impairments. Here, we present a single case of unilateral right-hand tactile agnosia, in the context of corticobasal syndrome (CBS), a rare neurodegenerative disease. The patient, a 55-year-old woman, initially presented with difficulties in tactile object recognition, apraxia for the right hand, and an otherwise intact cognitive profile. At the neuroimaging level, she showed a lesion outcome of a right parietal oligodendroglioma removal and a left frontoparietal atrophy. We performed an experimental evaluation of tactile agnosia, targeting every level of tactile processing, from elementary to higher order tactile recognition processes. We also tested 18 healthy participants as a matched control sample. The patient showed intact tactile sensitivity and mostly intact hylognosis functions. Conversely, she was impaired with the right hand in exploring geometrical and meaningless shapes. The patient's clinical evolution in the following 3 years became consistent with the diagnosis of CBS and unilateral tactile apperceptive agnosia as the primary symptom onset in the absence of a cognitive decline. This is the third case described in the literature manifesting morphoagnosia with almost completely preserved hylognosis abilities and the first description of such dissociation in a case with CBS.
ABSTRACT
AIM: 2-Pentadecyl-2-oxazoline (PEA-OXA), the oxazoline derivative of N-palmitoylethanolamine, exerts anti-inflammatory activity; however, very little is known about the molecular mechanisms underlying this effect. Here, we tested the anti-neuroinflammatory effect of PEA-OXA in primary microglia and we also investigated the possible interaction of the molecule with the Toll-like receptor 4 (TLR4)-myeloid differentiation protein-2 (MD-2) complex. MAIN METHODS: The anti-inflammatory effect of PEA-OXA was analyzed by measuring the expression and release of pro-inflammatory mediators in primary microglia by real-time PCR and ELISA, respectively. The effect of PEA-OXA on the activation of TLR4 signaling was assessed using two stably TLR4-transfected cell lines (i.e., HEK-293 and Ba/F3 cells). Finally, the putative binding mode of PEA-OXA to TLR4-MD-2 was investigated by molecular docking simulations. KEY FINDINGS: Treatment with PEA-OXA resulted in the following effects: (i) it down-regulated gene expression of several pro-inflammatory molecules and the secretion of pro-inflammatory cytokines in LPS stimulated microglia cells; (ii) it did not prevent microglia activation after stimulation with TLR2 ligands; (iii) it prevented TLR4/NF-κB activation triggered by LPS in HEK-Blue™ hTLR4 cells; and (iv) it interfered with the binding of LPS to TLR4-MD-2 complex. Furthermore, molecular docking studies suggested that PEA-OXA could bind MD-2 with a 1:3 (MD-2/PEA-OXA) stoichiometry. CONCLUSION: We show for the first time that the anti-neuroinflammatory effect of PEA-OXA involves its activity against TLR4 signaling, making this molecule a valuable tool for the development of new compounds directed to control neuroinflammation via inhibiting TLR4 signaling.
Subject(s)
Inflammation , Lipopolysaccharides , Humans , Lipopolysaccharides/adverse effects , Inflammation/metabolism , Toll-Like Receptor 4/metabolism , Molecular Docking Simulation , Microglia/metabolism , HEK293 Cells , Anti-Inflammatory Agents/pharmacology , NF-kappa B/metabolismABSTRACT
Background: Uncontrolled neuroinflammation and microglia activation lead to cellular and tissue damage contributing to neurodegenerative and neurological disorders. Spirulina (Arthrospira platensis (Nordstedt) Gomont, or Spirulina platensis), a blue-green microalga, which belongs to the class of cyanobacteria, has been studied for its numerous health benefits, which include anti-inflammatory properties, among others. Furthermore, in vivo studies have highlighted neuroprotective effects of Spirulina from neuroinflammatory insults in different brain areas. However, the mechanisms underlying the anti-inflammatory effect of the microalga are not completely understood. In this study we examined the effect of pre- and post-treatment with an acetone extract of Spirulina (E1) in an in vitro model of LPS-induced microglia activation. Methods: The effect of E1 on the release of IL-1ß and TNF-α, expression of iNOS, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1), and the activation of NF-κB was investigated in primary microglia by ELISA, real-time PCR, and immunofluorescence. Results: Pre- and early post-treatment with non-cytotoxic concentrations of E1 down-regulated the release of IL-1ß and TNF-α, and the over-expression of iNOS induced by LPS. E1 also significantly blocked the LPS-induced nuclear translocation of NF-κB p65 subunit, and upregulated gene and protein levels of Nrf2, as well as gene expression of HO-1. Conclusions: These results indicate that the extract of Spirulina can be useful in the control of microglia activation and neuroinflammatory processes. This evidence can support future in vivo studies to test pre- and post-treatment effects of the acetone extract from Spirulina.
ABSTRACT
Remyelination in patients with multiple sclerosis frequently fails, especially in the chronic phase of the disease promoting axonal and neuronal degeneration and progressive disease disability. Drug-based therapies able to promote endogenous remyelination capability of oligodendrocytes are thus emerging as primary approaches to multiple sclerosis. We have recently reported that the co-ultramicronized composite of palmitoylethanolamide and the flavonoid luteolin (PEALut) promotes oligodendrocyte precursor cell (OPC) maturation without affecting proliferation. Since TAM receptor signaling has been reported to be important modulator of oligodendrocyte survival, we here evaluated the eventual involvement of TAM receptors in PEALut-induced OPC maturation. The mRNAs related to TAM receptors -Tyro3, Axl, and Mertk- were all present at day 2 in vitro. However, while Tyro3 gene expression significantly increased upon cell differentiation, Axl and Mertk did not change during the first week in vitro. Tyro3 gene expression developmental pattern resembled that of MBP myelin protein. In OPCs treated with PEALut the developmental increase of Tyro3 mRNA was significantly higher as compared to vehicle while was reduced gene expression related to Axl and Mertk. Rapamycin, an inhibitor of mTOR, prevented oligodendrocyte growth differentiation and myelination. PEALut, administered to the cultures 30 min after rapamycin, prevented the alteration of mRNA basal expression of the TAM receptors as well as the expression of myelin proteins MBP and CNPase. Altogether, data obtained confirm that PEALut promotes oligodendrocyte differentiation as shown by the increase of MBP and CNPase and Tyro3 mRNAs as well as CNPase and Tyro3 immunostainings. The finding that these effects are reduced when OPCs are exposed to rapamycin suggests an involvement of mTOR signaling in PEALut effects.
ABSTRACT
BACKGROUND: Persistent and/or recurrent inflammatory processes are the main factor leading to multiple sclerosis (MS) lesions. The composite ultramicronized palmitoylethanolamide, an endogenous N-acylethanolamine, combined with the flavonoid luteolin, PEALut, have been found to exert neuroprotective activities in experimental models of spinal and brain injury and Alzheimer disease, as well as a clinical improvement in human stroke patients. Furthermore, PEALut enhances the expression of different myelin proteins in oligodendrocyte progenitor cells suggesting that this composite might have protective effects in MS experimental models. METHODS: The mouse model of experimental autoimmune encephalomyelitis (EAE) based on active immunization with a fragment of myelin oligodendrocyte glycoprotein (MOG35-55) was used. The daily assessment of clinical score and the expression of serum amyloid A (SAA1), proinflammatory cytokines TNF-α, IL-1ß, IFN-γ, and NLRP3 inflammasome, as well as TLR2, Fpr2, CD137, CD3-γ, and TCR-ζ chain, heterodimers that form T cell surface glycoprotein (TCR), and cannabinoid receptors CB1, CB2, and MBP, were evaluated in the brainstem and cerebellum at different postimmunization days (PIDs). RESULTS: Vehicle-MOG35-55-immunized (MOG35-55) mice developed ascending paralysis which peaked several days later and persisted until the end of the experiment. PEALut, given intraperitoneally daily starting on day 11 post-immunization, dose-dependently improved clinical score over the range 0.1-5 mg/kg. The mRNA expression of SAA1, TNF-α, IL-1ß, IFN-γ, and NLRP3 were significantly increased in MOG35-55 mice at 14 PID. In MOG35-55 mice treated with 5 mg /kg PEALut, the increase of SAA1, TNF- α, IL-1ß, and IFN-γ transcripts at 14 PID was statistically downregulated as compared to vehicle-MOG35-55 mice (p < 0.05). The expression of TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 receptors showed a significant upregulation in vehicle-MOG35-55 mice at 14 PID. Instead, CB1 and MBP transcripts have not changed in expression at any time. In MOG/PEALut-treated mice, TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 mRNAs were significantly downregulated as compared to vehicle MOG35-55 mice. CONCLUSIONS: The present results demonstrate that the intraperitoneal administration of the composite PEALut significantly reduces the development of clinical signs in the MOG35-55 model of EAE. The dose-dependent improvement of clinical score induced by PEALut was associated with a reduction in transcript expression of the acute-phase protein SAA1, TNF-α, IL-1ß, IFN-γ, and NLRP3 proinflammatory proteins and TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 receptors.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental/pathology , Ethanolamines/pharmacology , Luteolin/pharmacology , Neuroprotective Agents/pharmacology , Palmitic Acids/pharmacology , Amides , Animals , Biomarkers/analysis , Cytokines/drug effects , Cytokines/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Inflammation/immunology , Inflammation/pathology , Mice , Mice, Inbred C57BLABSTRACT
[This corrects the article DOI: 10.3389/fncel.2018.00072.].
ABSTRACT
Epigenetics is the study of changes in gene expression which may be triggered by both genetic and environmental factors, and independent from changes to the underlying DNA sequence-a change in phenotype without a change in genotype-which in turn affects how cells read genes. Epigenetic changes represent a regular and natural occurrence but can be influenced also by factors such as age, environment, and disease state. Epigenetic modifications can manifest themselves not only as the manner in which cells terminally differentiate, but can have also deleterious effects, resulting in diseases such as cancer. At least three systems including DNA methylation, histone modification, and non-coding RNA (ncRNA)-associated gene silencing are thought to initiate and sustain epigenetic change. For example, in Alzheimer's disease (AD), both genetic and non-genetic factors contribute to disease etiopathology. While over 250 gene mutations have been related to familial AD, less than 5% of AD cases are explained by known disease genes. More than likely, non-genetic factors, probably triggered by environmental factors, are causative factors of late-onset AD. AD is associated with dysregulation of DNA methylation, histone modifications, and ncRNAs. Among the classes of ncRNA, microRNAs (miRNAs) have a well-established regulatory relevance. MicroRNAs are highly expressed in CNS neurons, where they play a major role in neuron differentiation, synaptogenesis, and plasticity. MicroRNAs impact higher cognitive functions, as their functional impairment is involved in the etiology of neurological diseases, including AD. Alterations in the miRNA network contribute to AD disease processes, e.g., in the regulation of amyloid peptides, tau, lipid metabolism, and neuroinflammation. MicroRNAs, both as biomarkers for AD and therapeutic targets, are in the early stages of exploration. In addition, emerging data suggest that altered transcription of long ncRNAs, endogenous, ncRNAs longer than 200 nucleotides, may be involved in an elevated risk for AD.
Subject(s)
Alzheimer Disease/genetics , Epigenesis, Genetic/physiology , Alzheimer Disease/metabolism , DNA Methylation , HumansABSTRACT
BACKGROUND: Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves production of acute-phase proteins, including serum amyloid A (SAA). Interleukin-1ß (IL-1ß), a master regulator of neuroinflammation produced by activated inflammatory cells of the myeloid lineage, in particular microglia, plays a key role in the pathogenesis of acute and chronic diseases of the peripheral nervous system and CNS. IL-1ß release is promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with toll-like receptor (TLR) ligands. METHODS: Purified (> 99%) microglia cultured from neonatal rat cortex and cerebellum were first primed with the putative TLR4/TLR2 agonist SAA (recombinant human Apo-SAA) or the established TLR4 agonist lipopolysaccharide (LPS) followed by addition of ATP. Expression of genes for the NLRP3 inflammasome, IL-1ß, tumor necrosis factor-α (TNF-α), and SAA1 was measured by quantitative real-time polymerase chain reaction (q-PCR). Intracellular and extracellular amounts of IL-1ß were determined by ELISA. RESULTS: Apo-SAA stimulated, in a time-dependent manner, the expression of NLRP3, IL-1ß, and TNF-α in cortical microglia, and produced a concentration-dependent increase in the intracellular content of IL-1ß in these cells. A 2-h 'priming' of the microglia with Apo-SAA followed by addition of ATP for 1 h, resulting in a robust release of IL-1ß into the culture medium, with a concomitant reduction in its intracellular content. The selective P2X7R antagonist A740003 blocked ATP-dependent release of IL-1ß. Microglia prepared from rat cerebellum displayed similar behaviors. As with LPS, Apo-SAA upregulated SAA1 and TLR2 mRNA, and downregulated that of TLR4. LPS was less efficacious than Apo-SAA, perhaps reflecting an action of the latter at TLR4 and TLR2. The TLR4 antagonist CLI-095 fully blocked the action of LPS, but only partially that of Apo-SAA. Although the TLR2 antagonist CU-CPT22 was inactive against Apo-SAA, it also failed to block the TLR2 agonist Pam3CSK4. CONCLUSIONS: Microglia are central to the inflammatory process and a major source of IL-1ß when activated. P2X7R-triggered IL-1ß maturation and export is thus likely to represent an important contributor to this cytokine pool. Given that SAA is detected in Alzheimer disease and multiple sclerosis brain, together with IL-1ß-immunopositive microglia, these findings propose a link between P2X7R, SAA, and IL-1ß in CNS pathophysiology.
Subject(s)
Adenosine Triphosphate/pharmacology , Interleukin-1beta/metabolism , Microglia/drug effects , Serum Amyloid A Protein/pharmacology , Animals , Animals, Newborn , Brain/cytology , Cells, Cultured , Interleukin-1beta/genetics , Lipopolysaccharides/pharmacology , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , RNA, Messenger/metabolism , Rats , Time FactorsABSTRACT
Inflammation is a complex biological response fundamental to how the body deals with injury and infection to eliminate the initial cause of cell injury and effect repair. Unlike a normally beneficial acute inflammatory response, chronic inflammation can lead to tissue damage and ultimately its destruction, and often results from an inappropriate immune response. Inflammation in the nervous system ("neuroinflammation"), especially when prolonged, can be particularly injurious. While inflammation per se may not cause disease, it contributes importantly to disease pathogenesis across both the peripheral (neuropathic pain, fibromyalgia) and central [e.g., Alzheimer disease, Parkinson disease, multiple sclerosis, motor neuron disease, ischemia and traumatic brain injury, depression, and autism spectrum disorder] nervous systems. The existence of extensive lines of communication between the nervous system and immune system represents a fundamental principle underlying neuroinflammation. Immune cell-derived inflammatory molecules are critical for regulation of host responses to inflammation. Although these mediators can originate from various non-neuronal cells, important sources in the above neuropathologies appear to be microglia and mast cells, together with astrocytes and possibly also oligodendrocytes. Understanding neuroinflammation also requires an appreciation that non-neuronal cell-cell interactions, between both glia and mast cells and glia themselves, are an integral part of the inflammation process. Within this context the mast cell occupies a key niche in orchestrating the inflammatory process, from initiation to prolongation. This review will describe the current state of knowledge concerning the biology of neuroinflammation, emphasizing mast cell-glia and glia-glia interactions, then conclude with a consideration of how a cell's endogenous mechanisms might be leveraged to provide a therapeutic strategy to target neuroinflammation.
ABSTRACT
Several studies suggest that curcumin and related compounds possess antioxidant and anti-inflammatory properties including modulation of lipopolysaccharide- (LPS-) mediated signalling in macrophage cell models. We here investigated the effects of curcumin and the two structurally unrelated analogues GG6 and GG9 in primary human blood-derived macrophages as well as the signalling pathways involved. Macrophages differentiated from peripheral blood monocytes for 7 days were activated with LPS or selective Toll-like receptor agonists for 24 h. The effects of test compounds on cytokine production and immunophenotypes evaluated as CD80+/CCR2+ and CD206+/CD163+ subsets were examined by ELISA and flow cytometry. Signalling pathways were probed by Western blot. Curcumin (2.5-10 µM) failed to suppress LPS-induced inflammatory responses. While GG6 reduced LPS-induced IκB-α degradation and showed a trend towards reduced interleukin-1ß release, GG9 prevented the increase in proinflammatory CD80+ macrophage subset, downregulation of the anti-inflammatory CD206+/CD163+ subset, increase in p38 phosphorylation, and increase in cell-bound and secreted interleukin-1ß stimulated by LPS, at least in part through signalling pathways not involving Toll-like receptor 4 and nuclear factor-κB. Thus, the curcumin analogue GG9 attenuated the LPS-induced inflammatory response in human blood-derived macrophages and may therefore represent an attractive chemical template for macrophage pharmacological targeting.
Subject(s)
Curcumin/analogs & derivatives , Lipopolysaccharides/pharmacology , Blotting, Western , Cells, Cultured , Curcumin/chemistry , Curcumin/pharmacology , Diarylheptanoids , Humans , Immunophenotyping , Interleukin-1beta/metabolism , Macrophage Activation/drug effects , Macrophages/drug effects , Macrophages/metabolism , NF-kappa B/metabolism , Signal Transduction/drug effectsABSTRACT
Oligodendrocytes, the myelin-producing cells of the central nervous system (CNS), have limited capability to bring about repair in chronic CNS neuroinflammatory demyelinating disorders such as multiple sclerosis (MS). MS lesions are characterized by a compromised pool of undifferentiated oligodendrocyte progenitor cells (OPCs) unable to mature into myelin-producing oligodendrocytes. An attractive strategy may be to replace lost OLs and/or promote their maturation. N-palmitoylethanolamine (PEA) is an endogenous fatty acid amide signaling molecule with anti-inflammatory and neuroprotective actions. Recent studies show a co-ultramicronized composite of PEA and the flavonoid luteolin (co-ultraPEALut) to be more efficacious than PEA in improving outcome in CNS injury models. Here, we examined the effects of co-ultraPEALut on development of OPCs from newborn rat cortex cultured under conditions favoring either differentiation (Sato medium) or proliferation (fibroblast growth factor-2 and platelet-derived growth factor (PDGF)-AA-supplemented serum-free medium ("SFM")). OPCs in SFM displayed high expression of PDGF receptor alpha gene and the proliferation marker Ki-67. In Sato medium, in contrast, OPCs showed rapid decreases in PDGF receptor alpha and Ki-67 expression with a concomitant rise in myelin basic protein (MBP) expression. In these conditions, co-ultraPEALut (10 µM) enhanced OPC morphological complexity and expression of MBP and the transcription factor TCF7l2. Surprisingly, co-ultraPEALut also up-regulated MBP mRNA expression in OPCs in SFM. MBP expression in all cases was sensitive to inhibition of mammalian target of rapamycin. Within the context of strategies to promote endogenous remyelination in MS which focus on enhancing long-term survival of OPCs and stimulating their differentiation into remyelinating oligodendrocytes, co-ultraPEALut may represent a novel pharmacological approach.
Subject(s)
Cell Differentiation/drug effects , Ethanolamines/pharmacology , Luteolin/pharmacology , Oligodendrocyte Precursor Cells/drug effects , Oligodendroglia/drug effects , Animals , Animals, Newborn , Cattle , Cell Differentiation/physiology , Cells, Cultured , Drug Combinations , Humans , Oligodendrocyte Precursor Cells/physiology , Oligodendroglia/physiology , RatsABSTRACT
Neurons cultured from rodent central nervous system tissues represent important tools in the study of neurodegenerative disease mechanisms and neuroregenerative processes, including the survival- and axon growth-promoting properties of neurotrophic factors. This chapter presents a detailed protocol for the preparation of rat and mouse cortical, hippocampal, and striatal neuron cell cultures, using either embryonic or postnatal tissue with enzymatic digestion.
Subject(s)
Cell Culture Techniques/methods , Nerve Growth Factors/metabolism , Neurons/cytology , Animals , Animals, Newborn , Cells, Cultured , Cerebral Cortex/cytology , Corpus Striatum/cytology , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Hippocampus/cytology , Mice , Neurons/metabolism , Rats , RodentiaABSTRACT
The protocol described in this chapter covers the preparation and culture of enriched populations of microglia, astrocytes, and oligodendrocytes from the cortex and spinal cord of neonatal rat and mouse. The procedure is based on enzymatic digestion of the tissue, followed by the culture of a mixed glial cell population which is then utilized as the starting point for the isolation, via differential attachment, of the different cell types.
Subject(s)
Astrocytes/cytology , Cell Culture Techniques/methods , Microglia/cytology , Oligodendroglia/cytology , Animals , Animals, Newborn , Cell Separation , Cells, Cultured , Cerebellum/cytology , Cerebral Cortex/cytology , Mice , Rats , Rodentia , Spinal Cord/cytologyABSTRACT
Glial cell activation plays an important role in the pathogenesis of various neurodegenerative disorders. This article presents a protocol for the preparation of cultures consisting of rat embryonic cortical neurons grown in the presence of cortical microglia, in which the glia are present in physical contact with the neurons or separated by a semipermeable membrane barrier. An example of how such systems can be used to evaluate potential neuroprotective agents will also be described.
Subject(s)
Cerebral Cortex/embryology , Coculture Techniques/methods , Neuroglia/cytology , Neurons/cytology , Animals , Cells, Cultured , Cerebral Cortex/cytology , Cerebral Cortex/drug effects , Models, Biological , Neuroglia/drug effects , Neurons/drug effects , Neuroprotective Agents/pharmacology , RatsABSTRACT
Dopaminergic neuronal cell degeneration is the principal characteristic feature of the neuropathology of Parkinson disease. Cultures of mesencephalic neurons are widely used as a source of dopaminergic neurons for the study of mechanisms implicated in dopaminergic cell death and for the evaluation of potential dopaminergic neuroprotective agents, including neurotrophic factors. This chapter presents a detailed protocol for the preparation of rat mesencephalic cell cultures and their application to evaluating the effect of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium and the neuroprotective action of brain-derived neurotrophic factor.
Subject(s)
Cell Culture Techniques/methods , Dopaminergic Neurons/cytology , Mesencephalon/cytology , Neuroprotective Agents/pharmacology , Neurotoxins/pharmacology , 1-Methyl-4-phenylpyridinium/pharmacology , Animals , Brain-Derived Neurotrophic Factor/pharmacology , Cell Count , Cells, Cultured , Dopaminergic Neurons/drug effects , Mesencephalon/drug effects , RatsABSTRACT
Glial cell activation plays an important role in the pathogenesis of various neurodegenerative disorders as well as in chronic and neuropathic pain. This chapter describes a model which allows one to assess the individual and combined contributions of astrocytes and microglia in response to a pro-inflammatory stimulus, with emphasis on ionotropic purinergic receptors.
Subject(s)
Astrocytes/cytology , Coculture Techniques/methods , Inflammation/metabolism , Microglia/cytology , Animals , Animals, Newborn , Astrocytes/metabolism , Cells, Cultured , Female , Male , Microglia/metabolism , Models, Biological , Rats , Receptors, Purinergic/metabolismABSTRACT
The protocol presented in this chapter covers the application of rat cortical oligodendrocyte progenitor cells cultured under conditions of differentiation for the evaluation of agents with potential trophic activity, that is, which are capable of promoting maturation under such conditions. As an example we have chosen a co-ultramicronized N-palmitoylethanolamine/luteolin composite, a formulation described in the literature as possessing anti-inflammatory, neuroprotective, and neuroregenerative actions.
Subject(s)
Cell Culture Techniques/methods , Nerve Regeneration/drug effects , Neuroprotective Agents/pharmacology , Oligodendrocyte Precursor Cells/cytology , Amides , Animals , Cell Differentiation , Cells, Cultured , Ethanolamines/pharmacology , Luteolin/pharmacology , Myelin Sheath/drug effects , Myelin Sheath/physiology , Palmitic Acids/pharmacology , RatsABSTRACT
Glial cell activation, in particular microglia, plays an important role in the pathogenesis of various neurodegenerative disorders as well as in chronic and neuropathic pain. This chapter compares two established cell enumeration assays, namely, the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and the protein-binding sulforhodamine B assay for microglia as a function of culture condition and activation state. The pros and cons of each are then described.
Subject(s)
Cell Count/methods , Lipopolysaccharides/pharmacology , Microglia/cytology , Animals , Animals, Newborn , Calorimetry , Cells, Cultured , Mice , Microglia/drug effects , Microglia/metabolism , Rats , Rhodamines/metabolism , Tetrazolium Salts/metabolism , Thiazoles/metabolismABSTRACT
The perspective of neuroinflammation as an epiphenomenon following neuron damage is being replaced by the awareness of glia and their importance in neural functions and disorders. Systemic inflammation generates signals that communicate with the brain and leads to changes in metabolism and behavior, with microglia assuming a pro-inflammatory phenotype. Identification of potential peripheral-to-central cellular links is thus a critical step in designing effective therapeutics. Mast cells may fulfill such a role. These resident immune cells are found close to and within peripheral nerves and in brain parenchyma/meninges, where they exercise a key role in orchestrating the inflammatory process from initiation through chronic activation. Mast cells and glia engage in crosstalk that contributes to accelerate disease progression; such interactions become exaggerated with aging and increased cell sensitivity to stress. Emerging evidence for oligodendrocytes, independent of myelin and support of axonal integrity, points to their having strong immune functions, innate immune receptor expression, and production/response to chemokines and cytokines that modulate immune responses in the central nervous system while engaging in crosstalk with microglia and astrocytes. In this review, we summarize the findings related to our understanding of the biology and cellular signaling mechanisms of neuroinflammation, with emphasis on mast cell-glia interactions.
Subject(s)
Cell Communication/physiology , Encephalitis/pathology , Mast Cells/pathology , Neuroglia/pathology , Animals , Cytokines/metabolism , HumansABSTRACT
Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves hepatic production of acute-phase proteins, including serum amyloid A (SAA). Extrahepatically, SAA immunoreactivity is found in axonal myelin sheaths of cortex in Alzheimer's disease and multiple sclerosis (MS), although its cellular origin is unclear. We examined the responses of cultured rat cortical astrocytes, microglia and oligodendrocyte precursor cells (OPCs) to master pro-inflammatory cytokine tumour necrosis factor (TNF)-α and lipopolysaccaride (LPS). TNF-α time-dependently increased Saa1 (but not Saa3) mRNA expression in purified microglia, enriched astrocytes, and OPCs (as did LPS for microglia and astrocytes). Astrocytes depleted of microglia were markedly less responsive to TNF-α and LPS, even after re-addition of microglia. Microglia and enriched astrocytes showed complementary Saa1 expression profiles following TNF-α or LPS challenge, being higher in microglia with TNF-α and higher in astrocytes with LPS. Recombinant human apo-SAA stimulated production of both inflammatory mediators and its own mRNA in microglia and enriched, but not microglia-depleted astrocytes. Co-ultramicronized palmitoylethanolamide/luteolin, an established anti-inflammatory/ neuroprotective agent, reduced Saa1 expression in OPCs subjected to TNF-α treatment. These last data, together with past findings suggest that co-ultramicronized palmitoylethanolamide/luteolin may be a novel approach in the treatment of inflammatory demyelinating disorders like MS.