Microsoft Word - 4AlvisHernando_Microglia_ff 34 Alvis-Miranda et al Microglia: roles and rules in brain traumatic injury Microglia: roles and rules in brain traumatic injury Hernando Raphael Alvis-Miranda, Gabriel Alcala-Cerra1, Luis Rafael Moscote-Salazar1 University of Cartagena, Colombia 1Neurosurgery Abstract Microglia are a special type of supporting cells in the nervous system. between the functions they perform are myelin production, structural support, regulation of extracellular fluid, glial scar formation among others. This cell type for its versatility, is also related to pathological events, where his multiple roles and the release of proinflammatory factors can contribute to understand especially in traumatic brain injury, as secondary injury and the healing process, important aspects the context of brain injury. Key words: microglia, Brain trauma Injury, neurons, neurotrauma Introduction The role of microglia in the neurotrauma has been under continuous debate, researches have been trying to state how microglia-mediated inflammation can contribute to recovery of function after trauma, or fell in secondary injury. Microglia, are resident cells of the brain involved in regulatory processes critical for development, maintenance of the neural environment, injury, and repair (1). Microglia derives from mesodermal precursor cells of hemapoietic lineage that populate the central nervous system (CNS) in early development. Resident microglia cells are turned over slowly, and are replenished throughout adult life by proliferation (2), and represent the resident macrophages of the CNS, accounting for 10% of the adult glial cell population in the normal brain (3). Morphologically distinct from neurons and astrocytes, these cells were classified by Cajal in 1913 as a third element of CNS (4). Del Río Hortega made a further distinction between microglia and other glial cells in 1932. At that time the morphological classification of microglia allowed three kinds of states: ramified, intermediate forms, and ameboid (5). Recent studies have evolved the classification to include a functional connotation like M1 and M2 phenotypes (6). These phenotypes, allows them to actively participate in pathological processes by changing their morphology, expressing various antigens and becoming phagocytic (7). Microglia cells function as sentinels for innate immunity in the CNS continually monitoring and screening the extracellular environment, walling off areas of the CNS from foreign tissue, and removing damaged or dead cells (1), to achieve that, these cells express various sets of pattern recognition receptors (PRRs) in response to pathogen- associated molecular patterns (PAMPs) that include Toll-like receptors (TLRs; e. g: TLR-4) and inflammasomes (8). Very often, microglia cells provide the first line Romanian Neurosurgery (2013) XX 1: 34 - 45 35 defense against invading germs, and interacting with neurons and other glial cells, they can be the first sense of critical changes in the neuronal activity. Although microglial cells are thought to contribute to most pathological conditions, including CNS infections, neuroinflammatory lesions, brain tumors, and neurodegenerative diseases, their exact role in CNS development, homeostasis, and disease remains poorly understood (3). Regarding to neurotrauma, exists a strongly investigated feature, and that is the histological evidence of a prominent microglial activation (9). Currently there are many medical conditions under research because of the microglia activation in their pathological processes (10, 11, 12, 13). This review will explore the current concepts and researches on microglial responses to CNS traumatic injury and also will be discussed the differential role of microglia. General response of microglia to injury For microglial activation no large amount of tissue damage is required; alterations of neural electric transmission or extracellular electrolyte composition and the release of soluble factors normally confined to the intracellular compartment are sufficient to trigger microglia cells activation. In the healthy mature CNS, microglia have a ramified morphology, a small soma with fine cellular processes, this appearance has been associated with microglial “resting state”. When CNS is insulted, either for trauma, infection, ischemia, neurodege- nerative disease o an altered neuronal activity, microglia can evoke rapid and intense changes in their cell shape, gene expression and functional behavior, all these changes conjure the “microglial activation” (14), as will be discussed later more precisely, microglia cells respond by detecting lipopolysaccharide (LPS) (8), beta-amyloid (Aβ) (15), thrombin, IFN-γ, and other proinflammatory cytokines (16). In this late state, microglia suffer a morphological transformation, induction of a wide range of soluble factors, like cytokines, free radicals and nitric oxide, acquire a phagocytic phenotype (17) to clear tissue debris, damaged cells, or germs, and get physically associated with injured neurons. Microglial activation may result in expression of MHC (18) antigens and costimulatory molecules and in the capacity of microglia to act as antigen presenting cells. Microglial activation has key features: first, these cells react to all CNS pathologies, no matter if they are acute or chronic. Second, the response evoked is graded, and can range from very slight morphological changes accompanied by minor immunological activation, to the development of full-blown tissue macrophages with the associated expression of an entire battery of membrane bound and soluble immunologically relevant molecules (19). Because of the “immune privilege” (20) of the CNS, i. e., that the brain parenchyma has to protect itself from the potentially detrimental consequences of an immune reaction, the microglial activation is a highly regulated process. It is important to recognize the fact that microglia in “resting state”, actively scan and monitor the extracellular environment by pinocytosis (21), always ready to transform to a state of “activity”, in which microglia primarily serve for support and protection. So, the transition from a “resting” mode to an executive state thereby 36 Alvis-Miranda et al Microglia: roles and rules in brain traumatic injury represents a shift in activities rather than “activation” phenomenon, then, there are no periods of inactivity. Once microglial cell “activation” occurs, it starts with an emergency like response, such as defense oriented functions, i. e., combat an infection or to limit additional damage after injury. Eventually, the activated population, convert to a repair- oriented response, for tissue reparation. Although much effort has been directed toward characterizing the fundamental properties of an activated microglial cell, surprisingly little is known about the intrinsic molecular mechanisms that program the functional state of these cells in either the healthy or the diseased CNS (22), but these molecular mechanisms can be grouped to produce five stages: proliferation, migration, secretion of soluble products and electrophysiological changes. - Proliferation: Irrespective of the type of CNS lesion, microglial cells show an early proliferative response, usually proliferation indices, i. e., the number of actively proliferating microglial cells divided by the total number of microglial cells, peak around 3–5 days after a lesion (19). This proliferative response rapidly declines subsequently and approaches control levels 7-15 days after injury; but even after the zenith of the proliferation index, the total number of microglial cells can remain elevated, and depending on the type of injury, will still being significantly higher than pre-lesion values around a month after an experimental injury (23, 24, 25). The proliferation of microglial cells is triggered by damage to adjacent neurons, and deafferentation of central neurons by peripheral axotomy is a sufficient stimulus to induce it. A number of different factors have been shown to play important roles in the initiation of microglial proliferation but even actually is an incompletely answered world. In laboratory experiments have been demonstrated the presence of macrophage- colony stimulating factor (M-CSF) as a driver of the cell cycle of microglia, inducing their proliferation (26). Purinergic receptor P2X7 also controls microglial proliferation (27). Others microglial mitogens are the granulocyte/monocyte colony-stimulating factor (GM-CSF), brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, IL-1, IL-5, and chemokines (e. g., fractalkine and its receptor CXC3CL1) (28, 29, 30, 31, 32, 33). In the other hand, the proliferation regulation or inhibition seems to be exerted by the Transforming growth factor beta (TGF-β), mainly produced by activated astrocytes (34). In microglial cells proliferation, also are important the cell-cell interactions, particularly with neurons (35), which have key roles in delivery of regulatory signals, in part through the glycoprotein OX-2 (36). Microglia are equippated with glutamate-, purinergic-, acetylcholine-, adrenergic-, dopamine-, neurokinin-, serotoninergic-, opioid- and gamma-amino-butyric-acid-receptors, implicating that for their function is important the neuronal activity detection, but, microglia neurotransmitter receptor aren’t in a specialized region in close apposition to neurotransmitter release sites. Spatial proximity to synapses can be attained transiently; the long-term association is not likely since microglia cells are highly motile in resting and activated states (21). Neuron-microglia communication relies mostly on release of signaling molecules from extrasynaptic Romanian Neurosurgery (2013) XX 1: 34 - 45 37 axonal varicosities, as well as their diffusion from synapses. - Migration Microglia processes are capable of rapid extension (≈1. 25 mm/min) towards sites of acute CNS damage (37). The main candidate for a chemoattractant sensed by microglia is adenosine triphosphate [ATP; involves G(i/o) proteins and the mitogen- activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)-dependent pathway] (38) through microglial P2Y12 receptors (39) and an outward potassium current (40). Neuropeptides such as angiotensin II, bradykinin (controlled by a G(i/o)-protein- independent pathway), endothelin, galanin [induces microglial migration in a similar way to bradykinin, but induces intracellular Ca(2+) mobilization by inositol-3, 4, 5- trisphosphate (InsP(3))-dependent Ca(2+) release from the intracellular store] and neurotensin are also chemoattractants for microglia. The fact that there are different intracellular signaling cascades indicates that chemoattractants control distinct microglial functions. Nitric oxide (NO) also is an additional guidance cue involved in microglia migration. - Secretion of soluble factors These are derived mainly from the activated microglia, and consist of cytokines, proteases, free radicals and growth factors. Proinflammatory cytokines, which are closely involved in various diseases (including trauma, ischemia, Alzheimer's disease, epilepsy, and others), play important roles in the facilitation of activated microglia for astrocytic activation (34). Among the most important cytokines is IL-1 (41), showing neurotoxic actions, and stimulating astrocytes and other microglial cells in paracrine and autocrine way. Its receptor is also founded both in neurons and glial cells. IL-1 induces expression of other proinflammatory mediators like IL-6 (42), and TNF-α (43), thus this interleukin seems to play a regulating role in the concert of cytokines, and binding results in activation of a series of intracellular adaptor molecules, mitogen activated protein (MAP) kinases and the transcription factor, nuclear factor kappa B (NF-kB), leading to changes in gene expression. The first cells to express IL-1 are microglia, but other brain cells (astrocytes, invading immune cells, vascular cells and probably neurons) can also produce IL-1 (41). Once the microenvironment of the CNS becomes activated, local cells also produce proinflammatory cytokines, chemokines and upregulate inmunomodulatory surface markers. These changes in turn decrease the stringency of the blood-brain barrier, allowing entry of other soluble factors and peripheral immune cells, including macrophages, natural killer cells and lymphocytes. Proteases are also very important after CNS injury, because they mediate tissue damage and invasion of blood-derived immunocompetent cells. A factor recognized as a potential neurotoxic microglial secretion product is tissue- plasminogen activator (t-PA); it aggravates neuronal injury after ischemia (44). One key mediators of cytotoxicity in the CNS secreted by microglia is NO (45), this molecule displays a wide array of biological activities, ranging from cytotoxicity to neuroprotective effects and neurotransmission (46). NO acts directly as neurotoxic via excitotoxic mechanisms (47). Little is known about cellular mechanisms regulating reactive oxygen species (ROS) production by activated microglia under 38 Alvis-Miranda et al Microglia: roles and rules in brain traumatic injury pathological conditions, but, in microglia can produce ROS in different ways, e. g., NADPH oxidase, mitochondria or xanthine oxidase (48). Phagocytic and cytotoxic functions of microglia are also triggered during CNS injury. Upon activation, microglia upregulate opsonic receptors including complement receptors (CR1, CR3, CR4) and Fc gamma receptors (I, II, III), which enhance phagocytic activity by binding to complement components and immunoglobulin fragments respectively (36). - Electrophysiological changes In the ion channels relays key functions of microglial physiology, for example, deramification of microglial cells in culture was inhibited by blockade of nonselective cation channels, this suggest that cation influx through such channels increases the intracellular osmolarity with a subsequent osmotic entry of water and cell swelling (49), and the ramification, or the adquisition of a amoeboid shape, is likely o be associated with stretch-activated Cl- channels (50). Also has been observed that voltage- and/or Ca2+-activated K+ channels regulate microglial proliferation (51), migration (52, 53), cytokine secretion (54, 55, 56), production of free radicals (57, 58, 59). Strikingly, the intracellular conversion of pre-IL-1b to biologically active IL-1b is dependent for the decrease the cytoplasmic K+ concentration (60) and blockade of delayed outward rectifier K+ channels resulted in an inhibition of LPL- stimulated microglial IL-1- release (61). Chloride channels have been found to be involved in microglial proliferation (62, 63), production of ROS (64, 65), iNOS expression and nitric oxide production (66), migration (52), phagocytosis (67) and ramification (68). During NADPH oxidase mediated respiratory burst of microglia are important the proton channels for charge compensation (69). Calcium has been evidenced to participate in transmission of brain-damage signals to microglia through long-range waves, this type of waves is induced by glutamate and is independent of ATP (70). What happens after activation is an even less known fact than the microglial activation. Microglial activation when no longer needed, finishes being an unremarkable phenomenon, and not even much noticed, but, may be some residual effects (71). When being challenged again, the experienced microglia could behave differently, and this could explain some chronically related problems like age- related dysfunction or a neurodegenerative process (72, 17). However this behavior could be a protective effect, because the affected cells have been prepared by the former activation to respond quickly and efficiently in a second injury. Microglia: dual behavior? Exists the concept of secondary neuronal damage, i. e., microglia-mediated inflammation fell in secondary neuronal injury, and is in part built over the base that immunocompetent cells can aggravate neuronal demise, but whether microglial cells have a dual role, supportive or detrimental in neurotrauma and in others CNS acute or chronic pathologies has been the key question of almost a century. Has been stated that microglial behavior depends on several factors: the kind of stress and damage signals, the duration/timing of an impact, the microenvironment, the interaction with other cell types and, interestingly, even the age of an organism (73). There are a Romanian Neurosurgery (2013) XX 1: 34 - 45 39 contrast between the potentially neurotoxic substances and the growth factors secreted by microglia, but it is impossible to predict the kind of microglia behavior (benefit or harm to surviving neurons) that will be induced by the lesion in an acute and chronic way. Macrophages and microglia can be classified into at least two subsets with distinct molecular phenotypes and effector functions depending on the activation pathway. The “activated” proinflammatory M1 macrophages, activated by LPS and by the proinflammatory cytokine IFN-γ, express CD86 and CD16/32 and produce high amounts of oxidative metabolites (NO and superoxide), proteases and proinflammatory cytokines. They play a central role in host defense against pathogens and tumor cells, and they can also damage healthy cells such as neurons and glial cells. In contrast, M2 macrophages are ‘alternatively activated’, anti-inflammatory macrophages induced by IL-4 and IL-13, and they express CD206 and arginase 1. The latter downregulate inflammation and promote tissue remodeling/repair and angiogenesis (73, 6). As previously mentioned, microglia can both produce and respond to cytokines, these can be either neuroprotective [e. g. IL-10, tumor growth factor (TGF)-β, TNF-α] and/or neurotoxic (e. g. IL-1, TNF-α, IFN-γ) (74). At high levels TNF-α is neurotoxic, but at low levels it can be neuroprotective (75). In models that simulate chronic systemic inflammation has been shown that microglia in young mice protect dopaminergic neurons against LPS, but in the old mice, microglia promote the death cell (76). Beneficial responses Almost all studies for evaluation of the role of microglia in pathological circumstances have been performed using in vitro cultured microglia, whereby there are limitations because those only shows one cell phenotype simulating a physiological or pathological condition, also, these models lacks of the microenvironmental interactions with other cells like neurons and astrocytes. Thanks to the phagocytic function, microglia can enter damaged brain regions and remove toxic detritus, invading pathogens and cell debris. In case of stimulation of TLR by pathogens is induced a proinflammatory reaction but, in cases of brain injury, the recognizing of phosphatidylserine of the cell membrane of damaged neurons induces an anti- inflammatory reaction (74). Also in brain injury, microglial shows other beneficial activities like promoting protection to neurons (77), reestablishment of the neural environment (78), and has been evidenced that insufficient removal of myelin by microglia impairs the recruitment of oligodendrocyte precursor cells and induces an arrest of oligodendrocyte differentiation (78). Some cytokines secrete by microglia have an important role in protection, IL-6 can act on astrocytes and induce brain tissue repair (79), the IL-10 is an inhibitory cytokine of microglial apoptosis (80) and the TGF-β also can be neuroprotective (81, 82, 83) and it is elevated after injury (84, 85). These pleiotropic cytokines have central roles in vascular remodeling, immune suppression, immune homeostasis and repair after injury (86, 87, 88, 89). Furthermore, both TGF-β and IL-10 inhibit macrophage and microglia activation by downregulating the expression of molecules associated with antigen presentation and production of proinflammatory cytokines, chemokines, and nitric and oxygen free radicals (90, 91, 40 Alvis-Miranda et al Microglia: roles and rules in brain traumatic injury 92). Microglia promote neuroplasticity and axonal regeneration, in addition to the monitoring and pruning of synapses (93, 94). Microglia can modify neural networks in multiple ways: promoting neuronal cell death, influencing neural circuitry by neuroplastic reorganization or synaptic stripping (95). Activated microglia also enhance angiogenesis (96, 97, 98). Has been suggested that T cells, once activated (by encountering their relevant antigen- presenting cells), control the local innate response by activating resident microglia in a well-controlled way (99). They can also express neuroprotective proteins such as neurotrophins and glutamate transporters (100, 101). The latter transporters might help to reduce glutamate toxicity via uptake of glutamate. Detrimental responses Microglia have been implicated as a contributor to neuron damage, in models of excitotoxic cell death, phagocytic microglia contribute to neuronal degeneration and cell loss. Microglia can release glutamate, this neurotransmitter is well known to trigger excitotoxic neurodegeneration and cell death of astrocytes and oligodendrocytes (102, 103, 104, 105). The exact mechanism leading to microglial overactivation is still not fully understood, but glial-neuronal crosstalk seems to be central (106), but because of activated microglia can release cytotoxic factors such as NO and ROS and secrete pro-inflammatory cytokines that can potentially damage neurons, oligodendrocytes or extracellular matrix structures directly or indirectly, i. e., activating astrocytes trough IL-1-mediated neurotoxicity (107, 34, 108) have been considered as a “double-edged sword”, and inhibition of its functions have been beneficial in some circumstances (109, 110, 19, 111). Evidence is available to show the presence of activated microglia and their ability to induce cell death in the immature white matter, both in oligodendrocytes precursors and in astrocytes (112, 113). The ROS (NO, hydrogen peroxide, superoxide) can kill invading microbes but also can induce neural damage and reactive gliosis (74). The intracellular ROS derived from NADPH oxidase play special roles in neurodegeneration for his dual functions (114, 115). The ROS released extracellularly can be directly toxic for neurons, especially dopaminergic ones, and when intracellular ROS concentrations raise, can alter the signaling cascade leading to microglia activation (116, 117, 118, 119). Microglia have been related to the progressive nature of neurodegenerative diseases through their capacity to be chronically activated by neuron death and fuelling, a self-renewing cycle of microglial activation followed by further neuron damage (reactive microgliosis). This self- propelling cycle in microglial cells is possibly mediated by intracellular ROS and NO when they become activated by LPS (120). The activation of the prostaglandin E2 receptor of the prostaglandin E2 pathway can lead to microglia-induced paracrine neurotoxicity (121). 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