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.     

Peptides released by ameboid microglia regulate astroglial proliferation

Peptides that stimulate astroglial proliferation are produced in traumatized adult rat brain by 10 d after injury. These same peptides are released by ameboid microglia activated in vitro. Our findings suggest that astroglial scarring is regulated in part by the release of factors from ameboid microglia near the site of brain injury.     

Motility and ramification of human fetal microglia in culture: an investigation using time-lapse video microscopy and image analysis

Microglia are mononuclear phagocytes of the central nervous system and are considered to derive from circulating bone marrow progenitors that colonize the developing human nervous system in the second trimester. They first appear as ameboid forms and progressively differentiate to process-bearing "ramified" forms with maturation. Signals driving this transformation are known to be partly derived from astrocytes. In this investigation we have used cocultures of astrocytes and microglia to demonstrate the relationship between motility and morphology of microglia associated with signals derived from astrocytes. Analysis of progressive cultures using time-lapse video microscopy clearly demonstrates the dynamic nature of microglia. We observe that ameboid microglial cells progressively ramify when cocultured with astrocytes, mirroring the "differentiation" of microglia in situ during development. We further demonstrate that individual cells undergo morphological transformations from "ramified" to "bipolar" to "tripolar" and "ameboid" states in accordance with local environmental cues associated with astrocytes in subconfluent cultures. Remarkably, cells are still capable of migration at velocities of 20-35 microm/h in a fully ramified state overlying confluent astrocytes, as determined by image analysis of motility. This is in keeping with the capacity of microglia for a rapid response to inflammatory cues in the CNS. We also demonstrate selective expression of the chemokines MIP-1alpha and MCP-1 by confluent human fetal astrocytes in cocultures and propose a role for these chemotactic cytokines as regulators of microglial motility and differentiation. The interchangeable morphological continuum of microglia supports the view that these cells represent a single heterogeneous population of resident mononuclear phagocytes capable of marked plasticity.     

Rapid microglial activation induced by traumatic brain injury is independent of blood brain barrier disruption

Following CNS injury, microglia respond and transform into reactive species exhibiting characteristic morphological changes that have been termed "activated" or "ameboid" microglia. In an attempt to establish that microglial reactions induced immediately after injury are caused by intrinsic mechanisms rather than infiltration of blood and its constituents, oxygenized Ringer's solution was perfused into the cerebral circulation of rats so that the circulating blood could be eliminated prior to injury induction. Under artificial respiration, a catheter was inserted from the cardiac apex into the ascending aorta, and oxygenized Ringer's solution was immediately perfused with a pulsatile blood pump, resulting in wash out of the circulating blood from the brain within 1 min. Subsequently, a cortical contusion was induced in the unilateral parietal cortex using a controlled cortical impact (CCI) device. At 5 min following the injury, the brain was fixed by perfusion of fixative through the catheter and removed. Coronal vibratome sections were then processed for CR3 immunohistochemistry to examine the microglial activation. It appeared that microglial activation with both morphological transformation and an increase in CR3 immunoreactivity was induced throughout the hemisphere ipsilateral to the injury side exclusively, even in rats with elimination of circulating blood. The microglial reactions did not differ substantially from those observed in the control rats with extensive BBB disruption. The present results thus provide direct evidence that the microglial activation induced immediately after injury is independent of infiltration of circulating blood induced by concurrent BBB disruption.     

Prion protein expression differences in microglia and astroglia influence scrapie-induced neurodegeneration in the retina and brain of transgenic mice

Activated microglia and astroglia are known to be involved in a variety of neurodegenerative diseases, including prion diseases. In the present experiments, we studied activation of astroglia and microglia after intraocular scrapie infection in transgenic mice expressing prion protein (PrP) in multiple cell types (tg7 mice) or in neurons only (tgNSE mice). In this model, scrapie infection and protease-resistant PrP deposition occurs in the retinas of both strains of mice, but retinal degeneration is observed only in tg7 mice. Our results showed that the retinas of tg7 and tgNSE mice both had astroglial activation with increased chemokine expression during the course of infection. However, only tg7 retinas exhibited strong microglial activation compared to tgNSE retinas, which showed little microglial activation by biochemical or morphological criteria. Therefore, microglial PrP expression might be required for scrapie-induced retinal microglial activation and damage. Furthermore, microglial activation preceded retinal neurodegeneration in tg7 mice, suggesting that activated microglia might contribute to the degenerative process, rather than being a response to the damage. Surprisingly, brain differed from retina in that an altered profile of microglial activation markers was upregulated, and the profiles in the two mouse strains were indistinguishable. Microglial activation in the brain was associated with severe brain vacuolation and neurodegeneration, leading to death. Thus, retinal and brain microglia appeared to differ in their requirements for activation, suggesting that different activation pathways occur in the two tissues.     

Aminoguanidine inhibits caspase-3 and calpain activation without affecting microglial activation following neonatal transient cerebral ischemia

Microglial cells, the resident macrophages of the CNS, can be both beneficial and detrimental to the brain. These cells play a central role as mediators of neuroinflammation associated with many neurodegenerative states, including cerebral ischemia. Because microglial cells are both a major source of inducible nitric oxide synthase (iNOS)/nitric oxide (NO) production locally in the injured brain and are activated by NO-mediated injury, we tested whether iNOS inhibition reduces microglial activation and ischemic injury in a neonatal focal ischemia-reperfusion model. Post-natal day 7 rats were subjected to a 2 h transient middle cerebral artery (MCA) occlusion. Pups with confirmed injury on diffusion-weighted magnetic resonance imaging (MRI) during occlusion were administered 300 mg/kg/dose aminoguanidine (AG) or vehicle at 0, 4 and 18 h after reperfusion, and animals were killed at 24 or 72 h post-reperfusion. The effect of AG on microglial activation as judged by the acquisition of ED1 immunoreactivity and proliferation of ED1-positive cells, on activation of cell death pathways and on injury volume, was determined. The study shows that while AG attenuates caspase 3 and calpain activation in the injured tissue, treatment does not affect the rapidly occurring activation and proliferation of microglia following transient MCA occlusion in the immature rat, or reduce injury size.     

The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1β (IL-1β), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

This study shows that inflammatory responses of microglial cells are markedly influenced by the ion transporter, NKCC1; blockade or genetic deletion of microglial NKCC1 has broad cell-autonomous effects, leading to changes in morphology, membrane conductance, process recruitment after injury, and cytokine production, with worsened neurological outcome after stroke.     

Microglia proliferation is regulated by hydrogen peroxide from NADPH oxidase

Microglia are resident brain macrophages that become activated and proliferate following brain damage or stimulation by immune mediators, such as IL-1beta or TNF-alpha. We investigated the mechanisms by which microglial proliferation is regulated in primary cultures of rat glia. We found that basal proliferation of microglia was stimulated by proinflammatory cytokines IL-1beta or TNF-alpha, and this proliferation was completely inhibited by catalase, implicating hydrogen peroxide as a mediator of proliferation. In addition, inhibitors of NADPH oxidase (diphenylene iodonium or apocynin) also prevented microglia proliferation, suggesting that this may be the source of hydrogen peroxide. IL-1beta and TNF-alpha rapidly stimulated the rate of hydrogen peroxide produced by isolated microglia, and this was inhibited by diphenylene iodonium, implying that the cytokines were acting directly on microglia to stimulate the NADPH oxidase. Low concentrations of PMA or arachidonic acid (known activators of NADPH oxidase) or xanthine/xanthine oxidase or glucose oxidase (generating hydrogen peroxide) also increased microglia proliferation and this was blocked by catalase, showing that NADPH oxidase activation or hydrogen peroxide was sufficient to stimulate microglia proliferation. In contrast to microglia, the proliferation of astrocytes was unaffected by the presence of catalase. In conclusion, these findings indicate that microglial proliferation in response to IL-1beta or TNF-alpha is mediated by hydrogen peroxide from NADPH oxidase.     

Microglia: Proliferation and activation driven by the P2X7 receptor

Microglial activation is associated with the pathogenesis and progression of conditions such as Alzheimer's disease (AD), Parkinsons’ disease, prion disease, multiple sclerosis, and ischemic and traumatic brain injury. The molecular mechanism of microglial activation is largely unknown. The expression of the purinergic, P2X7 receptor (P2X7R), is known to be enhanced in many brain pathologies where presence of activated microglia is a concurrent feature. This review focuses on the links between P2X7R expression and microglial activation and proliferation. The P2X7R is identified as a key player in the process of microgliosis, where by driving microglial activation, it can potentially lead to a deleterious cycle of neuroinflammation and neurodegeneration.     

Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits

The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R). Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1(op)) gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure.     

Microglial replacement in the aged brain restricts neuroinflammation following intracerebral hemorrhage

Aged microglia display augmented inflammatory activity after neural injury. Although aging is a risk factor for poor outcome after brain insults, the precise impact of aging-related alterations in microglia on neural injury remains poorly understood. Microglia can be eliminated via pharmacological inhibition of the colony–stimulating factor 1 receptor (CSF1R). Upon withdrawal of CSF1R inhibitors, microglia rapidly repopulate the entire brain, leading to replacement of the microglial compartment. In this study, we investigated the impact of microglial replacement in the aged brain on neural injury using a mouse model of intracerebral hemorrhage (ICH) induced by collagenase injection. We found that replacement of microglia in the aged brain reduced neurological deficits and brain edema after ICH. Microglial replacement-induced attenuation of ICH injury was accompanied with alleviated blood-brain barrier disruption and leukocyte infiltration. Notably, newly repopulated microglia had reduced expression of IL-1β, TNF-α and CD86, and upregulation of CD206 in response to ICH. Our findings suggest that replacement of microglia in the aged brain restricts neuroinflammation and brain injury following ICH.Subject terms: Neuroimmunology, Cognitive ageing     

Brain peptides and glial growth. I. Glia-promoting factors as regulators of gliogenesis in the developing and injured central nervous system

Glia-promoting factors (GPFs) are peptides of the central nervous system which accelerate the growth of specific glial populations in vitro. Although these factors were first discovered in the goldfish visual system (Giulian, D., Y. Tomozawa, H. Hindman, and R. Allen, 1985, Proc. Natl. Acad. Sci. USA., 83:4287-4290), we now report similar peptides are found in mammalian brain. The cerebral cortex of rat contains oligodendroglia-stimulating peptides, GPF1 (15 kD) and GPF3 (6 kD), as well as astroglia-stimulating peptides, GPF2 (9 kD) and GPF4 (3 kD). The concentrations of specific GPFs increase in brain during periods of gliogenesis. For example, GPF1 and GPF3 are found in postnatal rat brain during a peak of oligondendroglial growth while GPF2 and GPF4 are first detected at a time of astroglial proliferation in the embryo. Stab wound injury to the cerebral cortices of rats stimulates astroglial proliferation and induces marked elevations in levels of GPF2 and GPF4. Our findings suggest that two distinct classes of GPFs, those acting upon oligodendroglia and those acting upon astroglia, help to regulate cell growth in the developing and injured central nervous system.     

Microglial reaction in focal cerebral ischaemia induced by intra-carotid homologous clot injection

This study examined the microglial reaction in a simulated thrombo-embolus ischaemia in rats given an intracarotid injection of a suspension of homologous blood clot. All rats including the controls receiving vehicle injection were perfused at 5 hours, and 1, 3 and 7 days post-operation. The brains were removed and processed for immunohistochemistry using a panel of monoclonal antibodies: OX-42, OX-18 and OX-6 for labeling of microglia. In rats given saline injection OX-42 immunoreactive microglial cells were observed to be distributed quite evenly throughout the whole brain. When injection of clot suspension was given, microglial cells responded vigorously, particularly in the ipsilateral hippocampus. Microglial reaction was also detected in the ipsilateral cerebral cortex, caudate as well as septal nuclei. The majority of the detected reactive microglial cells were hypertrophied showing thick or stout processes. Some rod-like and amoeboid microglia were also observed. Rarely did the reactive microglia express OX-6 immunoreactivity. All microglial cells were unreactive for OX-18. The actual mechanisms leading to the microglial activation as well as functions of reactive microglia in focal cerebral ischaemia remain speculative. In the absence of direct evidence, it could only be suggested that they may act as sensor cells for detection of subtle alterations in the microenvironment, probably in response to focal ischaemia and/or leakage of serum-derived factors induced by thrombo-embolus stroke.     

Inhibition of granulocyte-macrophage colony-stimulating factor signaling and microglial proliferation by anti-CD45RO: role of Hck tyrosine kinase and phosphatidylinositol 3-kinase/Akt

Increasing evidence suggests that CD45, a transmembrane protein tyrosine phosphatase, is an important modulator of macrophage activation. Microglia, resident brain macrophages, express CD45 and proliferate under pathologic conditions. In this study, we examined the role of CD45 in modulating GM-CSF-induced proliferation and signal transduction in primary human microglial cultures. Soluble, but not immobilized anti-CD45RO induced tyrosine phosphatase activity and inhibited GM-CSF-induced microglial proliferation. Microglial proliferation was also inhibited by PP2 (Src inhibitor), LY294002 (PI3K inhibitor), and U0126 (MEK inhibitor). GM-CSF induced phosphorylation of Jak2, Stat5, Hck (the myeloid-restricted Src kinase), Akt, Stat3, and Erk MAPKs in microglia. Of these, anti-CD45RO inhibited phosphorylation of Hck and Akt, and PP2 inhibited phosphorylation of Hck and Akt. In a macrophage cell line stably overexpressing wild-type or kinase-inactive Hck, GM-CSF increased proliferation of the control (empty vector) and wild-type but not kinase-inactive cells, and this was inhibited by anti-CD45RO. Together, these results demonstrate that, in macrophages, Hck tyrosine kinase is activated by GM-CSF, and that Hck plays a pivotal role in cell proliferation and survival by activating the PI3K/Akt pathway. Ab-mediated activation of macrophage and microglial CD45 tyrosine phosphatase may have therapeutic implications for CNS inflammatory diseases.     

Microglial motility in the rat facial nucleus following peripheral axotomy

Microglial motility was studied in living mammalian brain tissue using infrared gradient contrast microscopy in combination with video contrast enhancement and time lapse video recording. The infrared gradient contrast allows the visualization of living cells up to a depth of 60 μm in brain slices, in regions where cell bodies remain largely uninjured by the tissue preparation and are visible in their natural environment. In contrast to other techniques, including confocal microscopy, this procedure does not require any staining or labeling of cell membranes and thus guarantees the investigation of tissue which has not been altered, apart from during preparation. Microglial cells are activated and increase in number in the facial nucleus following peripheral axotomy. Thus we established the preparation of longitudinal rat brainstem slices containing the axotomized facial nucleus as a source of activated microglial cells. During prolonged video time lapse recordings, two different types of microglial cell motility could be observed. Microglial cells which had accumulated at the surface of the slice remained stationary but showed activity of the cell soma, developing pseudopods of different shape and size which undulated and which were used for phagocytosis of cell debris. Microglial phagocytosis of bacteria could be documented for the first time in situ. In contrast, ameboid microglia which did not display pseudopods but showed migratory capacity, could be observed exclusively in the depth of the tissue. Some of these cells maintained a close contact to neurons and appeared to move along their dendrites, a finding that may be relevant to the role of microglia in “synaptic stripping”, the displacement of synapses following axotomy. This approach provides a valuable opportunity to investigate the interactions between activated microglial cells and the surrounding cellular and extracellular structures in the absence of staining or labeling, thus opening a wide field for the analysis of the cellular mechanisms involved in numerous pathologies of the CNS.     

Microglial Depletion Causes Region‐Specific Changes to Developmental Neuronal Cell Death in the Mouse Brain

Developmental neuronal cell death has been characterized as a cell autonomous “suicide” program, but recent findings suggest that microglia play an active role in determining the survival of developing neurons. Results have been contradictory, however, with some studies concluding that microglia promote cell death, while others report that microglia are neuroprotective. Here, we depleted microglia throughout the newborn mouse brain using intracerebroventricular injections of clodronate liposomes, and examined effects on naturally occurring cell death across multiple brain areas. Microglial density varied significantly by brain region, and clodronate liposome treatment at birth reduced the number of microglia in all regions examined. The effect of microglia reduction on cell death, however, varied by region: the number of dying cells was reduced in the medial septum and medial amygdala in clodronate treated animals, but was increased in the oriens layer of the hippocampus, and unchanged in several other brain regions. In most brain regions, the average size of microglia was greater in microglia‐depleted than in control animals, suggesting that the remaining microglia compensate to some extent for a reduction in microglial number. The hippocampal oriens was exceptional in this regard, in that microglial size was reduced following treatment with clodronate. Microglia produce cytokines which mediate many of their effects, and we found higher expression of inflammatory cytokines in the hippocampus than in the septum, independent of clodronate treatment. Thus, microglial depletion has opposite effects on cell death in different brain regions of the newborn brain, which may be related to regional heterogeneity in microglia.     

Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease

Microglia are the principal immune cells of the brain. In Alzheimer disease, these brain mononuclear phagocytes are recruited from the blood and accumulate in senile plaques. However, the role of microglia in Alzheimer disease has not been resolved. Microglia may be neuroprotective by phagocytosing amyloid-beta (Abeta), but their activation and the secretion of neurotoxins may also cause neurodegeneration. Ccr2 is a chemokine receptor expressed on microglia, which mediates the accumulation of mononuclear phagocytes at sites of inflammation. Here we show that Ccr2 deficiency accelerates early disease progression and markedly impairs microglial accumulation in a transgenic mouse model of Alzheimer disease (Tg2576). Alzheimer disease mice deficient in Ccr2 accumulated Abeta earlier and died prematurely, in a manner that correlated with Ccr2 gene dosage, indicating that absence of early microglial accumulation leads to decreased Abeta clearance and increased mortality. Thus, Ccr2-dependent microglial accumulation plays a protective role in the early stages of Alzheimer disease by promoting Abeta clearance.     

Cytokines Reduce Toxic Effects of Ethanol on Oligodendroglia

To characterize immunomodulatory mechanisms that affect oligodendroglia (OL) and white matter following ethanol exposure during early CNS development, we investigated the direct effects of ethanol and cytokines on glia. Mixed glial cultures from newborn rat brain were exposed to 6.5–130 mM ethanol for 1–3 days. OL were sensitive to ethanol, with death ranging from 32 to 88% with increasing time and ethanol concentrations. Little cell death occurred in astroglia or microglia. Mixtures of cytokines representative of those produced by pro-inflammatory Th1 and monocyte/macrophage (M/M) cells as well as those produced by anti-inflammatory Th2 cells were all protective. Three of the cytokines in the Th1 mixture, IL-2, TNF-α and IFN-γ, were protective individually, although no single cytokine was as effective as the mixture. The protective effects of the Th1 mixture and of IL-2 were reversed by inhibition of both MAP kinase and PI-3 kinase signaling pathways. We conclude that cytokines can act either directly on OL or indirectly through effects on astroglia or microglia to protect OL from ethanol toxicity.     

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.