Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase

In this study, microglial migration and phagocytosis were examined in mouse organotypic hippocampal slice cultures, which were treated with N-methyl-D-aspartate (NMDA) to selectively injure neuronal cells. Microglial cells were visualized by the expression of enhanced green fluorescent protein. Daily observation revealed microglial accumulation in the pyramidal cell layer, which peaked 5 to 6 days after NMDA treatment. Time-lapse imaging showed that microglia migrated to the pyramidal cell layer from adjacent and/or remote areas. There was no difference in the number of proliferating microglia between control and NMDA-treated slices in both the pyramidal cell layer and stratum radiatum, suggesting that microglial accumulation in the injured areas is mainly due to microglial migration, not to proliferation. Time-lapse imaging also showed that the injured neurons, which were visualized by propidium iodide (PI), disappeared just after being surrounded by microglia. Daily observation revealed that the intensity of PI fluorescence gradually attenuated, and this attenuation was suppressed by pretreatment with clodronate, a microglia toxin. These findings suggest that accumulating microglia phagocytosed injured neurons, and that PI fluorescence could be a useful indicator for microglial phagocytosis. Using this advantage to examine microglial phagocytosis in living slice cultures, we investigated the involvements of mitogen-activated protein (MAP) kinases in microglial accumulation and phagocytosis. p38 MAP kinase inhibitor SB203580, but not MAP kinase/extracellular signal-regulated kinase inhibitor PD98059 or c-Jun N-terminal kinase inhibitor SP600125, suppressed the attenuation of PI fluorescence. On the other hand, microglial accumulation in the injured areas was not inhibited by any of these inhibitors. These data suggest that p38 MAP kinase plays an important role in microglial phagocytosis of injured neurons.     

Intercellular adhesion molecule-1 (ICAM-1) in the mouse facial motor nucleus after axonal injury and during regeneration

Intercellular adhesion molecule 1 (ICAM-1, CD54) is a widely expressed glycoprotein, which plays an important role in leukocyte extravasation and in the interaction of lymphocytes with antigen-presenting cells. In the current study we examined the regulation of ICAM-1 in the mouse facial motor nucleus after facial nerve transection, using immunohistochemistry, confocal laser microscopy and electron microscopy. In the normal facial nucleus ICAM-1 immunoreactivity was restricted to vascular endothelium. Transection of the facial nerve led to a strong and selective upregulation of ICAM-1 on activated microglia. Quantitation of microglial ICAM-1 immunoreactivity revealed a biphasic increase. The first peak 1–2 days post operation paralleling the early stage of microglial activation was followed by a decline at 4–7 days. The second induction of ICAM-1 occured at day 14 accompanying the period of neuronal cell death and microglial phagocytosis of neuronal debris. Immunoelectron microscopy showed strong ICAM-1 reactivity on the cell membrane of activated microglia at day 2. During the second peak (day 14), ICAM-1 was also observed on lymphocytes adhering to phagocytotic microglia forming aggregates around neuronal debris. No immunolabelling was observed on neurons, astrocytes or oligodendroglia. These data suggest the involvement of ICAM-1 in the adhesion of activated microglia, in their phagocytosis of neuronal debris, and also in the interaction with infiltrating lymphocytes following this injury.     

Pinocytosis as a select marker of ramified microglia in vivo and in vitro

Based on previous observations in tissue culture, we investigated pinocytotic activity as a potential cell marker for brain microglia. This functional activity was assessed in three different preparations derived from rat: primary cultures of mixed cerebral cortical cells, tissue slabs of whole cerebrum, and cultures of isolated or enriched microglial cells. Each preparation was incubated with the fluorescent dye lucifer yellow as a soluble tracer and then processed for light microscopy. Under the conditions utilized, ramified microglia specifically exhibited differentially high pinocytotic labeling in all cases; the dye was mainly localized within the cell somata, where it was sequestered in pinocytotic vesicles. In each preparation, the identity of the labeled cell population was confirmed as microglia through immunohistochemical staining with the monoclonal antibody (MAb) OX-42, a specific microglial marker. Therefore, pinocytotic labeling is proposed as a select cell marker for microglia, which may be extremely useful in the identification and study of ramified microglial cells.     

PK (‘peripheral benzodiazepine’) – binding sites in the CNS indicate early and discrete brain lesions: microautoradiographic detection of [3H]PK 11195 binding to activated microglia

The isoquinoline PK 11195 has been suggested as a marker of glial pathology in the lesioned brain. The aim of the present study is to clarify the precise cellular location of its binding site in the central nervous system. Here, we report that in the facial nucleus after facial nerve axotomy–a lesion causing a retrograde neuronal reaction without nerve cell death while keeping the blood–brain barrier intact–activated microglia are the predominant source of lesion-induced increases of PK 11195 binding. Likewise, increased PK 11195 binding is seen in the gracile nucleus after anterograde neuronal injury following sciatic nerve transection. The peak of PK 11195 binding, using the single isomer R-PK 11195, was observed 4 days after the peripheral nerve lesion, consistent with the well-known time course of microglial activation. Photoemulsion microautoradiography confirmed the restriction of PK 11195 binding to activated microglia. The increase of PK 11195 binding in the facial nucleus seen after selective cell death of facial motoneurons by retrograde suicide transport of toxic ricin, a lesion that is accompanied by the rapid transformation of microglia into phagocytes, was no higher than that seen following axotomy. This suggests that the full transformation of microglia into parenchymal phagocytes is not necessary to reach maximal levels of PK 11195 binding. PK 11195, therefore, is a well-suited marker to detect microglial activation in areas of subtle brain pathology, where neither a disturbance of the blood–brain barrier function nor the presence of macrophages and inflammatory cells indicate an on-going disease process.     

Transmitter- and hormone-activated Ca(2+) responses in adult microglia/brain macrophages in situ recorded after viral transduction of a recombinant Ca(2+) sensor

In vitro studies show that microglia, the resident immune cells of the brain, express neurotransmitter and neuropeptide receptors which are linked to Ca(2+) signaling. Here we describe an approach to obtain Ca(2+) recordings from microglia in situ. We injected a retrovirus encoding a calcium sensor into the cortex of mice 2 days after stimulation of microglial proliferation by a stab wound injury. Microglial cells were identified with tomato lectin in acute slices prepared 3, 6, 21 and 42 days after the injury. The membrane current profile and the ameboid morphology indicated that microglial cells were activated at day 6 while at day 42 they resembled resting microglia. We recorded transient Ca(2+) responses to application of ATP, endothelin-1, substance P, histamine and serotonin. The fluorescence amplitude of ATP was increased only at day 6 compared to other time points, while responses to all other ligands did not vary. Only half of the microglial cells that responded to ATP also responded to endothelin-1, serotonin and histamine. Substance P, in contrast, showed a complete overlap with the ATP responding microglial population at day 6, at day 42 this population was reduced to 55%. Cultured cells were less responsive to these ligands. This study shows that in situ microglia consist of heterogeneous populations with respect to their sensitivity to neuropeptides and -transmitters.     

在LPS诱导的炎症模型中低功率激光照射对小胶质细胞吞噬功能的影响

小胶质细胞的激活在神经退行性疾病的病理发生过程中发挥了重要的作用.一旦被激活,他们便具有类似巨噬细胞的吞噬功能以及释放炎症因子的能力,前者有利于保护中枢神经系统的功能,而后者则会加重神经元的死亡.然而,在神经退行性疾病的发生过程中,脑内的小胶质细胞却不能有效地对死亡细胞甚至Aβ进行吞噬.因此,调控小胶质细胞的吞噬功能被认为是寻求神经保护治疗手段的一个有效策略.在本研究中,我们的实验结果表明了20 J/cm2的LPLI能够增强LPS激活的小胶质细胞的吞噬功能.我们发现LPLI介导的小胶质细胞的吞噬功能增强是一个基于actin聚合的Rac1依赖的过程,持续激活的Rac1（Rac1Q61L）相比野生型Rac1可以诱导更多的actin聚合,而显性负效应的Rac1 （Rac1T17N）却显著抑制了actin的聚合.另外,我们运用一个基于荧光能量共振转移的Raichu-Rac1质粒也进一步证实了在LPLI下Rac1的激活,并且这一激活过程是由PI3K/Akt通路所介导的.我们的研究为控制神经退行性疾病的进程提供了一个可行的的治疗策略.     

Accelerated phagocytosis of amyloid-beta by mouse and human microglia overexpressing the macrophage colony-stimulating factor receptor

Microglia surrounding A beta plaques in Alzheimer's disease and in the APPV717F transgenic mouse model of Alzheimer's disease have enhanced immunoreactivity for the macrophage colony-stimulating factor receptor (M-CSFR), encoded by the proto-oncogene c-fms. Increased expression of M-CSFR on cultured microglia results in proliferation and release of pro-inflammatory cytokines and expression of inducible nitric-oxide synthase. We transfected mouse BV-2 and human SV-A3 microglia to overexpress M-CSFR and examined microglial phagocytosis of fluorescein-conjugated A beta. Flow cytometry and laser confocal microscopy showed accelerated phagocytosis of A beta in mouse and human microglia because of M-CSFR overexpression that was time- and concentration-dependent. In contrast, microglial uptake of 1-microm diameter polystyrene microspheres was not enhanced by M-CSFR overexpression. Microglial uptake of A beta was blocked by cytochalasin D, which inhibits phagocytosis. M-CSFR overexpression increased the mRNA for macrophage scavenger receptor A, and fucoidan blocking of macrophage scavenger receptors inhibited uptake of A beta. M-CSFR antibody blocking experiments demonstrated that increased A beta uptake depended on the interaction of the M-CSFR with its ligand. These results suggest that overexpression of M-CSFR in APPV717F mice may prime microglia for phagocytosis of A beta after immunization.     

A dual role of lipocalin 2 in the apoptosis and deramification of activated microglia

Activated microglia are thought to undergo apoptosis as a self-regulatory mechanism. To better understand molecular mechanisms of the microglial apoptosis, apoptosis-resistant variants of microglial cells were selected and characterized. The expression of lipocalin 2 (lcn2) was significantly down-regulated in the microglial cells that were resistant to NO-induced apoptosis. lcn2 expression was increased by inflammatory stimuli in microglia. The stable expression of lcn2 as well as the addition of rLCN2 protein augmented the sensitivity of microglia to the NO-induced apoptosis, while knockdown of lcn2 expression using short hairpin RNA attenuated the cell death. Microglial cells with increased lcn2 expression were more sensitive to other cytotoxic agents as well. Thus, inflammatory activation of microglia may lead to up-regulation of lcn2 expression, which sensitizes microglia to the self-regulatory apoptosis. Additionally, the stable expression of lcn2 in BV-2 microglia cells induced a morphological change of the cells into the round shape with a loss of processes. Treatment of primary microglia cultures with the rLCN2 protein also induced the deramification of microglia. The deramification of microglia was closely related with the apoptosis-prone phenotype, because other deramification-inducing agents such as cAMP-elevating agent forskolin, ATP, and calcium ionophore also rendered microglia more sensitive to cell death. Taken together, our results suggest that activated microglia may secrete LCN2 protein, which act in an autocrine manner to sensitize microglia to the self-regulatory apoptosis and to endow microglia with an amoeboid form, a canonical morphology of activated microglia in vivo.     

Phagocytosis of Microglia in the Central Nervous System Diseases

Microglia, the resident macrophages of the central nervous system, rapidly activate in nearly all kinds of neurological diseases. These activated microglia become highly motile, secreting inflammatory cytokines, migrating to the lesion area, and phagocytosing cell debris or damaged neurons. During the past decades, the secretory property and chemotaxis of microglia have been well-studied, while relatively less attention has been paid to microglial phagocytosis. So far there is no obvious concordance with whether it is beneficial or detrimental in tissue repair. This review focuses on phagocytic phenotype of microglia in neurological diseases such as Alzheimer’s disease, multiple sclerosis, Parkinson’s disease, traumatic brain injury, ischemic and other brain diseases. Microglial morphological characteristics, involved receptors and signaling pathways, distribution variation along with time and space changes, and environmental factors that affecting phagocytic function in each disease are reviewed. Moreover, a comparison of contributions between macrophages from peripheral circulation and the resident microglia to these pathogenic processes will also be discussed.     

Population control of microglia: does apoptosisplay a role?

Brain lesions, even of the most subtle type, are accompanied by the activation of microglia, the main immune cells of the brain. Microglial cells dramatically increase in number through proliferation and adhere to the injured neurons, where they displace the synaptic input. After proliferation, microglia gradually migrate into the nearby parenchyma and appear to decrease in number. Here we examined the possible involvement of apoptosis in the regulation of the microglial cell number using Terminal transferase mediated d-UTP Nick End-Labelling (TUNEL). In vitro, cell death is a common phenomenon in microglial cell cultures, and is enhanced by the withdrawal of the mitogen, granulocyte-macrophage colony stimulating factor. In vivo, application of the TUNEL-reaction revealed TUNEL-positive microglia beginning at day 4, with a peak 7 days after transection of the facial nerve. Surprisingly, TUNEL-labelling in vivo was localized on the outer side of the nuclear membrane and in the microglial cytoplasm, with very little staining within the nucleus itself. These TUNEL-labelled cells also lacked other classic morphological signs of apoptosis, like membrane blebbing, chromatin condensation and apop-totic bodies. These data suggest that the regulation of post-mitotic microglia is not mediated by the classic pathway of apoptosis.     

Secondary lymphoid tissue chemokine (CCL21) activates CXCR3 to trigger a Cl- current and chemotaxis in murine microglia

Microglial cells represent the major immunocompetent element of the CNS and are activated by any type of brain injury or disease. A candidate for signaling neuronal injury to microglial cells is the CC chemokine ligand CCL21, given that damaged neurons express CCL21. Investigating microglia in acute slices and in culture, we demonstrate that a local application of CCL21 for 30 s triggered a Cl(-) conductance with lasted for tens of minutes. This response was sensitive to the Cl(-) channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 4-acetamide-4'-isothiocyanatostilbene, 2,2'-disulfonic acid. Moreover, CCL21 triggered a chemotaxis response, which was sensitive to Cl(-) channel blockers. In microglial cells cultured from CCR7 knockout mice, CCL21 produced the same type of Cl(-) current as well as a chemotaxis response. In contrast, in microglial cells from CXCR3 knockout mice, CCL21 triggered neither a Cl(-) conductance nor a chemotaxis response after CCL21 application. We conclude that the CCL21-induced Cl(-) current is a prerequisite for the chemotaxis response mediated by the activation of CXCR3 but not CCR7 receptors, indicating that in brain CCL21 acts via a different receptor system than in lymphoid organs.     

miR‐124 Contributes to the functional maturity of microglia

During early development of the central nervous system (CNS), a subset of yolk‐sac derived myeloid cells populate the brain and provide the seed for the microglial cell population, which will self‐renew throughout life. As development progresses, individual microglial cells transition from a phagocytic amoeboid state through a transitional morphing phase into the sessile, ramified, and normally nonphagocytic microglia observed in the adult CNS under healthy conditions. The molecular drivers of this tissue‐specific maturation profile are not known. However, a survey of tissue resident macrophages identified miR‐124 to be expressed in microglia. In this study, we used transgenic zebrafish to overexpress miR‐124 in the mpeg1 expressing yolk‐sac‐derived myeloid cells that seed the microglia. In addition, a systemic sponge designed to neutralize the effects of miR‐124 was used to assess microglial development in a miR‐124 loss‐of‐function environment. Following the induction of miR‐124 overexpression, microglial motility and phagocytosis of apoptotic cells were significantly reduced. miR‐124 overexpression in microglia resulted in the accumulation of residual apoptotic cell bodies in the optic tectum, which could not be achieved by miR‐124 overexpression in differentiated neurons. Conversely, expression of the miR‐124 sponge caused an increase in the motility of microglia and transiently rescued motility and phagocytosis functions when activated simultaneously with miR‐124 overexpression. This study provides in vivo evidence that miR‐124 activity has a key role in the development of functionally mature microglia. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 507–518, 2016     

Phagocytosis is regulated by nitric oxide in murine microglia.

Nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) in activated microglia and has been shown to participate in host defense mechanisms. However, the role of NO produced by constitutive nitric oxide synthase (cNOS) in microglia is poorly understood. In this report, NO was found to regulate phagocytosis in murine BV-2 microglial cells as quantified by flow cytometry. Addition of NO-generating compounds caused impaired phagocytosis as compared to untreated microglia. The addition of nitric oxide synthase (NOS) inhibitors to microglial cells resulted in potentiation of phagocytosis, suggesting that constitutive NO was participating in the regulation of phagocytosis. The inverse correlation between NO production and phagocytosis was also observed when Alzheimer's beta-amyloid peptide was added. With beta-amyloid treatment, constitutive NO production decreased while phagocytosis increased. Cell extracts prepared from untreated microglia were found to contain both neuronal and endothelial NOS isoforms, but not the inducible form. The correlation of spontaneous NO production with attenuated phagocytosis suggests that constitutive NOS enzymes participate in microglial regulation.     

Role of Ciliary Neurotrophic Factor in Microglial Phagocytosis

Microglia, CNS-resident macrophages, serve as scavengers to remove cellular debris and facilitate tissue remodeling in the developing and injured CNS. Little is known as what and how microenvironmental factors mediate the phagocytotic ability of microglia. Our previous study has indicated that treatment with glial cell line-derived neurotrophic factor (GDNF) increased the phagocytotic activity of primary rat microglia possibly through the upregulation of α5 integrin. In the present study, ciliary neurotrophic factor (CNTF), which has been reported to be produced by glia, was shown to have stimulatory effect on the phagocytosis of primary rat microglia and mouse microglial cell line BV2. Ca²⁺ imaging analysis and the application of intracellular calcium chelator BAPTA-AM revealed that CNTF-induced increase in microglial phagocytosis was mediated by a calcium signaling pathway. Furthermore, treatment with CNTF led to an increase in the expression of αv integrin, which has been reported to be involved in the phagocytosis of the apoptotic cells. In summary, we have provided evidence that CNTF can increase microglial phagocytosis through a calcium-mediated pathway. Our results also suggest that the upregulation of αv integrin by CNTF could be involved in the increased phagocytotic activity of microglia. Special issue article in honor of Dr. George DeVries.     

NTPDase1 activity attenuates microglial phagocytosis

Purinergic signaling plays a major role in the regulation of phagocytosis in microglia. Interplay between P2 and P1 receptor activation is controlled by a cascade of extracellular enzymes which dephosphorylate purines resulting in the formation of adenosine. The ATP- and ADP-degrading capacity of cultured microglia depends on the expression of ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) and is several times higher when compared to astrocytes which lack this enzyme. In brain slices, deletion of CD39 resulted in a 50 % decrease of ADP-degrading ability, while the degradation of ATP was decreased to about 75 % of the values measured in wild-type brain tissue. Microglia in acute slices from cd39^?/? animals had increased constitutive phagocytic activity which could not be further enhanced by ATP in contrast to control animals. Pharmacological blockage of P2 receptors decreased the constitutive phagocytic activity to a similar base level in wild-type and cd39^?/? microglia. Activation of P1 receptors by non-hydrolysable adenosine analog significantly decreased phagocytic activity. Deletion of CD73, an enzyme expressed by microglia which converts AMP to adenosine did not affect phagocytic activity. Taken together, these data show that CD39 plays a prominent role in controlling ATP levels and thereby microglial phagocytosis.     

Effects of the pig renal epithelial cell line LLC-PK1 and its conditioned medium on the phenotype of porcine microglia in vitro

Microglia are dispersed throughout the central nervous system. Under physiological circumstances they display a 'ramified' resting phenotype. In different neuropathologies microglia reversibly transform into the activated form, an amoeboid phagocyte with a broad spectrum of immune effector functions. In this study, a coculture of porcine microglia and the pig renal epithelial cell line LLC-PK1 was used to investigate microglial cell biology. The morphology of the cocultures was elucidated as well as the functionality of the microglia cells by proliferation, superoxide and phagocytosis assays. Our results demonstrate that direct intercellular contact between the two cell types was necessary for microglia to acquire a ramified morphology. Moreover, the conditioned medium of the renal cells promoted proliferation of microglia, inhibited giant cell formation and stimulated microglia to retain their capability to generate superoxide and to perform phagocytosis. In conclusion, we have constructed a cell culture system showing differentiation of microglia in vitro and keeping them in optimal conditions.     

Microglial activation in different models of peripheral nerve injury of the rat

Pain and pain modulation has been viewed as being mediated entirely by neurons. However, new research implicates spinal cord glia as key players in the creation and maintenance of pathological pain. Sciatic nerve lesions are one of the most commonly studied pain-related injuries. In our study we aimed to characterize changes in microglial activation in the rat spinal cord after axotomy and chronic constriction injury of the sciatic nerve and to evaluate this activation in regard to pain behavior in injured and control groups of rats. Microglial activation was observed at ipsilateral side of lumbar spinal cord in all experimental groups. There were slight differences in the level and extent of microglial activation between nerve injury models used, however, differences were clear between nerve-injured and sham animals in accordance with different level of pain behavior in these groups. It is known that activated microglia release various chemical mediators that can excite pain-responsive neurons. Robust microglial activation observed in present study could therefore contribute to pathological pain states observed following nerve injury.     

Microglial activation and recruitment,but not proliferation,suffice to mediate neurodegeneration

Microglial activation occurs during excitotoxin-induced neurodegeneration. We have reported that microglia can exhibit neurotoxic behaviors after injection of excitotoxins into the hippocampus. It is not known, however, whether microglial proliferation, which is part of the activation response, is required for neurodegeneration to be observed, or whether activation of the pre-existing resident microglia suffices. Using osteopetrotic (op/op) mice, in which injury-induced microglial proliferation does not take place, we demonstrate that only the microglia initially residing in the CNS are adequate to promote neurodegeneration. Our data suggest that there is a threshold at which a maximal microglial contribution to neurotoxicity is observed. This threshold appears to be sufficiently low, such that activation of just 40% of the microglia present in wild-type mice serves to trigger neurodegeneration. Furthermore, since the decrease in microglial numbers coincides with a decrease in tissue plasminogen activator's activity, we suggest that tissue plasminogen activator can be used as a marker for microglial proliferation.