Key Event Title
|Level of Biological Organization|
Key Event Components
|brain inflammation||microglial cell||pathological|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Binding of antagonist to NMDARs can lead to neuroinflammation and neurodegeneration||KeyEvent|
|ionotropic glutamatergic receptors and cognition||KeyEvent|
|Mitochondrial dysfunction and Neurotoxicity||KeyEvent|
|Oxidative stress and Developmental Neurotoxicity||KeyEvent|
|During brain development, adulthood and aging||High|
Key Event Description
Neuroinflammation or brain inflammation differs from peripheral inflammation in that the vascular response and the role of peripheral bone marrow-derived cells are less conspicuous. The most easily detectable feature of neuroinflammation is activation of microglial cells and astrocytes. It is evidenced by changes in shape, increased expression of certain antigens, and accumulation and proliferation of the glial cells in affected regions (Aschner, 1998; Graeber & Streit, 1990; Monnet-Tschudi et al, 2007; Streit et al, 1999; Kraft and Harry, 2011; Claycomb et al., 2013). Upon stimulation by cytokines or inflammogens (e.g. from pathogens or from damaged neurons), both glial cell types activate inflammatory signalling pathways, which result in increased expression and/or release of inflammatory mediators such as cytokines, eicosanoids, and metalloproteinases (Dong & Benveniste, 2001), as well as in the production of reactive oxygen (ROS) and nitrogen species (RNS) (Brown & Bal-Price, 2003). Different types of activation states are possible for microglia and astrocytes, resulting in pro-inflammatory or anti-inflammatory signalling and other cellular functions (such as phagocytosis) (Streit et al., 1999; Nakajima and Kohsaka, 2004).
Therefore, neuroinflammation can have both neuroprotective/neuroreparative and neurodegenerative consequences (Carson et al., 2006 ; Monnet-Tschudi et al, 2007; Aguzzi et al., 2013 ; Glass et al., 2010). Under normal physiological conditions, microglial cells scan the nervous system for neuronal integrity (Nimmerjahn et al, 2005) and for invading pathogens (Aloisi, 2001; Kreutzberg, 1995; Kreutzberg, 1996; Rivest, 2009). They are the first type of cell activated (first line of defence), and can subsequently induce astrocyte activation (Falsig, 2008). Two distinct states of microglial activation have been described (Gordon, 2003; Kigerl et al, 2009; Maresz et al, 2008; Mosser & Edwards, 2008; Perego et al; Ponomarev et al, 2005): The M1 state is classically triggered by interferon-gamma and/or other pro-inflammatory cytokines, and this state is characterized by increased expression of integrin alpha M (Itgam) and CD86, as well as the release of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6), and it is mostly associated with neurodegeneration. The M2 state is triggered by IL-4 and IL-13 (Maresz et al, 2008; Perego et al, 2011; Ponomarev et al, 2007) and induces the expression of mannose receptor 1 (MRC1), arginase1 (Arg 1) and Ym1/2; it is involved in repair processes. The activation of astrocytes by microglia-derived cytokines or TLR agonists resembles the microglial M1 state (Falsig 2006). Although classification of the M1/M2 polarization of microglial cells may be considered as a simplification of authentic microglial reaction states (Ransohoff, 2016), a similar polarization of reactive astrocytes has been descibed recently Liddlelow et al., 2017): Interleukin-1 alpha (IL-1alpha), TNF and subcomponent q (C1q) released by activated microglial cells induce A1-reactive astrocytes, which lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis and induce the death of neurons and oligodendrocytes.
How It Is Measured or Detected
Neuroinflammation, i.e. the activation of glial cells can be measured by quantification of cellular markers (most commonly), or of released mediators (less common). As multiple activation states exist for the two main cell types involved, it is necessary to measure several markers of neuroinflammation:
- Microglial activation can be detected based on the increased numbers of labeled microglia per volume element of brain tissue (due to increase of binding sites, proliferation, and immigration of cells) or on morphological changes. A specific microglial marker, used across different species, is CD11b. Alternatively various specific carbohydrate structures can be stained by lectins (e.g. IB4). Beyond that, various well-established antibodies are available to detect microglia in mouse tissue (F4/80), phagocytic microglia in rat tissue (ED1) or more generally microglia across species (Iba1). Transgenic mice are available with fluorescent proteins under the control of the CD11b promoter to easily quantify microglia without the need for specific stains.
- The most frequently used astrocyte marker is GFAP (99% of all studies) (Eng et al., 2000). This protein is highly specific for astrocytes in the brain, and antibodies are available for immunocytochemical detection. In neuroinflammatory brain regions, the stain becomes more prominent, due to an upregulation of the protein, a shape change/proliferation of the cells, and/or better accessibility of the antibody. Various histological quantification approaches can be used. Occasionally, alternative astrocytic markers, such as vimentin of the S100beta protein, have been used for staining of astrocytes (Struzynska et al., 2007). Antibodies for complement component 3 (C3), the most characteristic and highly upregulated marker of A1 neurotoxic reactive astrocytes are commercially available.
- All immunocytochemical methods can also be applied to cell culture models.
- In patients, microglial accumulation can be monitored by PET imaging, using [11C]-PK 11195 as a microglial marker (Banati et al., 2002).
- Activation of glial cells can be assessed in tissue or cell culture models also by quantification of sets of activation markers. This can for instance be done by PCR quantification of inflammatory factors, by measurement of the respective mediators, e.g. by ELISA-related immuno-quantification. Such markers include:
- Pro- and anti-inflammatory cytokine expression (IL-1β; TNF-α, Il-6, IL-4); or expression of immunostimmulatory proteins (e.g. MHC-II)
- Itgam, CD86 expression as markers of M1 microglial phenotype
- Arg1, MRC1, as markers of M2 microglial phenotype
(for descriptions of techniques, see also Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014)
Regulatory example using the KE:Measurement of glial fibrillary acidic protein (GFAP) in brain tissue, whose increase is a marker of astrocyte reactivity, is required by the US EPA in rodent toxicity studies for fuel additives (40 CFR 79.67), but is optional for other toxicant evaluations.
Domain of Applicability
Neuroinflammation is observed in human, monkey, rat, mouse, and zebrafish, in association with neurodegeneration or following toxicant exposure. Some references (non-exhaustive list) are given below for illustration:
In human: Vennetti et al., 2006
In monkey (Macaca fascicularis): Charleston et al., 1994, 1996
Iin rat: Little et al., 2012; Zurich et al., 2002; Eskes et al., 2002
In mouse: Liu et al., 2012
In zebrafish: Xu et al., 2014.
Aschner M (1998) Immune and inflammatory responses in the CNS: modulation by astrocytes. ToxicolLett 103: 283-287
Banati, R. B. (2002). "Visualising microglial activation in vivo." Glia 40: 206-217.
Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355
Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM. 1996. Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. NeuroToxicology 17: 127-138.
Charleston JS, Bolender RP, Mottet NK, Body RL, Vahter ME, Burbacher TM. 1994. Increases in the number of reactive glia in the visual cortex of Macaca fascicularis following subclinical long-term methyl mercury exposure. ToxicolApplPharmacol 129: 196-206.
Dong Y, Benveniste EN (2001) Immune Function of Astrocytes. Glia 36: 180-190
Eng LF, Ghirnikar RS, Lee YL (2000) Glial Fibrillary Acidic Protein: GFAP-Thirty-One Years (1969-2000). NeurochemRes 25: 1439-1451
Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.
Falsig J, Latta M, Leist M. Defined inflammatory states in astrocyte cultures correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004 Jan;88(1):181-93.
Falsig J, Pörzgen P, Lund S, Schrattenholz A, Leist M. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006 Feb;96(3):893-907.
Falsig J, van Beek J, Hermann C, Leist M. Molecular basis for detection of invading pathogens in the brain. J Neurosci Res. 2008 May 15;86(7):1434-47.
Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010). Mechanisms underlying inflammation in neurodegeneration. Cell. 2010 Mar 19;140(6):918-34.
Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3: 23-35
Graeber MB, Streit WJ (1990) Microglia: immune network in the CNS. Brain Pathol 1: 2-5
Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 29: 13435-13444
Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17-42
Kreutzberg GW (1995) Microglia, the first line of defence in brain pathologies. Arzneimttelforsch 45: 357-360
Kreutzberg GW (1996) Microglia : a sensor for pathological events in the CNS. Trends Neurosci 19: 312-318
Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, et al. 2017. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541(7638): 481-487.
Little AR, Miller DB, Li S, Kashon ML, O'Callaghan JP. 2012. Trimethyltin-induced neurotoxicity: gene expression pathway analysis, q-RT-PCR and immunoblotting reveal early effects associated with hippocampal damage and gliosis. Neurotoxicol Teratol 34(1): 72-82.
Liu Y, Hu J, Wu J, Zhu C, Hui Y, Han Y, et al. 2012. alpha7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation. J Neuroinflammation 9: 98.
Lund S, Christensen KV, Hedtjärn M, Mortensen AL, Hagberg H, Falsig J, Hasseldam H, Schrattenholz A, Pörzgen P, Leist M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J Neuroimmunol. 2006 Nov;180(1-2):71-87.
Maresz K, Ponomarev ED, Barteneva N, Tan Y, Mann MK, Dittel BN (2008) IL-13 induces the expression of the alternative activation marker Ym1 in a subset of testicular macrophages. J Reprod Immunol 78: 140-148
Monnet-Tschudi F, Zurich MG, Honegger P (2007) Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures. Hum Exp Toxicol 26: 339-346
Monnet-Tschudi, F., A. Defaux, et al. (2011). "Methods to assess neuroinflammation." Curr Protoc Toxicol Chapter 12: Unit12 19.
Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8: 958-969
Nakajima K, Kohsaka S. 2004. Microglia: Neuroprotective and neurotrophic cells in the central nervous system. Current Drug Targets-Cardiovasc & Haematol Disorders 4: 65-84.
Perego C, Fumagalli S, De Simoni MG (2011) Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice. J Neuroinflammation 8: 174
Ponomarev ED, Maresz K, Tan Y, Dittel BN (2007) CNS-derived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells. J Neurosci 27: 10714-10721
Ponomarev ED, Shriver LP, Maresz K, Dittel BN (2005) Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J Neurosci Res 81: 374-389
Ransohoff RM. 2016. A polarizing question: do M1 and M2 microglia exist? Nat Neurosci 19(8): 987-991.
Sandstrom von Tobel, J., D. Zoia, et al. (2014). "Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures." Toxicol Lett. DOI : 10.1016/j.toxlet.2014.02.001
Struzynska L, Dabrowska-Bouta B, Koza K, Sulkowski G (2007) Inflammation-Like Glial Response in Lead-Exposed Immature Rat Brain. Toxicol Sc 95:156-162
von Tobel, J. S., P. Antinori, et al. (2014). "Repeated exposure to Ochratoxin A generates a neuroinflammatory response, characterized by neurodegenerative M1 microglial phenotype." Neurotoxicology 44C: 61-70.
Venneti S, Lopresti BJ, Wiley CA. 2006. The peripheral benzodiazepine receptor (Translocator protein 18kDa) in microglia: from pathology to imaging. Prog Neurobiol 80(6): 308-322.
Xu DP, Zhang K, Zhang ZJ, Sun YW, Guo BJ, Wang YQ, et al. 2014. A novel tetramethylpyrazine bis-nitrone (TN-2) protects against 6-hydroxyldopamine-induced neurotoxicity via modulation of the NF-kappaB and the PKCalpha/PI3-K/Akt pathways. Neurochem Int 78: 76-85.
Zurich M-G, Eskes C, Honegger P, Bérode M, Monnet-Tschudi F. 2002. Maturation-dependent neurotoxicity of lead aceate in vitro: Implication of glial reactions. J Neurosc Res 70: 108-116.