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PubMed2006

Role of spinal microglia in rat models of peripheral nerve injury and inflammation.

Mounting evidence supports the hypothesis that spinal microglia modulate the development and maintenance of some chronic pain states. Here we examined the role of spinal microglia following both peripheral inflammatory insult and peripheral nerve injury. We observed significant ipsilateral dorsal horn microglia activation 2 weeks after injury and bilateral activation 50 days following nerve injury as well as 24 h following intraplantar zymosan but not intraplantar complete Freund's adjuvant (CFA). Ipsilateral but not contralateral microglia activation was associated with hind paw mechanical hyperalgesia. Spinal injection of the glial metabolic inactivator fluorocitrate attenuated ipsilateral hyperalgesia and bilateral spinal microglia activation after peripheral nerve injury. Intrathecal fluorocitrate reversed hyperalgesia after intraplantar zymosan and produced no reversal of CFA-induced hyperalgesia. These data suggest a role for spinal glia in the persistence of mechanical hyperalgesia following peripheral nerve injury. However, activation of spinal microglia contralaterally did not correlate to nociception. Furthermore, it would appear that the time course of microglia activation and their contribution to inflammatory pain is dependent on the inflammatory stimulus administered.

Animals↗

PubMed2004

Minocycline delays photoreceptor death in the rds mouse through a microglia-independent mechanism.

PURPOSE: Minocycline, a semi-synthetic tetracycline antibiotic is reported to be neuroprotective in degenerative and ischaemic models of central nervous system disease, via mechanisms involving suppression of both cytotoxic microglial activity and caspase-dependent apoptosis. We have investigated the effect of minocycline treatment on a mouse model of retinitis pigmentosa, an inherited photoreceptor neurodegenerative disorder, and contrasted this with the effect of depleting retinal microglia using liposomal clodronate. METHODS: rds mice were treated intraperitoneally from the second postnatal day (P2) with either daily minocycline until P16, P18, P21, P24 and P27 or alternative day clodronate liposomes until P16. Immunohistochemical and immunofluorescent methods were applied for the detection of microglia (F4/80) and apoptosis (TUNEL and caspase 3 activation). RESULTS: Photoreceptor apoptosis was delayed by minocycline treatment but not, ultimately, prevented. Markedly reduced expression of activated caspase 3 was observed in photoreceptors at the early time point, corresponding with the reduced level of apoptosis. Delayed photoreceptor apoptosis due to minocycline treatment was associated with a 50% reduction in the numbers of microglia at early timepoints. Liposomal clodronate treatment also resulted in a marked reduction in the number of microglia (63% reduction in microglia), but in contrast to minocycline treatment, this had no effect on photoreceptor apoptosis. CONCLUSIONS: Minocycline appears to delay photoreceptor apoptosis through a microglia-independent action. Although microglial cytotoxicity has been implicated during other models of neurodegeneration, microglia are unlikely to play such a role in this model of photoreceptor dystrophy.

Animals↗

PubMed2003

Microglia are activated to become competent antigen presenting and effector cells in the inflammatory environment of the Theiler's virus model of multiple sclerosis.

Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is a well-characterized murine model of the chronic-progressive form of human multiple sclerosis (MS) characterized by the activation of myelin-specific autoreactive CD4 Th1 cells via epitope spreading. To gain an understanding of the potential role of central nervous system (CNS)-resident cells in the presentation of endogenous myelin epitopes, we determined the individual antigen presentation and effector potential of resident microglia vs. infiltrating macrophages in the CNS of mice with ongoing TMEV-IDD by performing functional analysis of these populations separated to high purity by flow cytometric sorting based on their level of CD45 expression. Unlike microglia from nai;ve mice, peptide-pulsed CD45(lo) microglia isolated at the onset of clinical disease were as efficient as CNS-infiltrating CD45(hi) macrophages in activating proliferation and IFN-gamma production by myelin-peptide specific Th1 cells. In contrast, during the chronic stages of TMEV-IDD, CNS-infiltrating macrophages were more highly activated than the resident microglia as reflected both by higher expression of cell surface molecules associated with APC function and enhanced functional ability of spinal cord-infiltrating macrophages to stimulate T cell proliferation in vitro. Interestingly, both microglia and infiltrating macrophages expressed similar profiles of effector molecules such as IL-1, IL-6, IL-12 p40, TNF-alpha, and iNOS. Collectively, this is the first report comparing the antigen-presenting phenotype and function of microglia and infiltrating macrophages in a virus-induced model of CNS demyelination demonstrating that the resident microglia are capable APCs and may play an important role in antigen presentation at the onset of clinical disease and contribute to effector myelin destruction.

Animals↗

PubMed2005

Ionizing radiation induces astrocyte gliosis through microglia activation.

The aim of this study was to investigate the role of microglia in radiation-induced astrocyte gliosis. We found that a single dose of 15 Gy radiation to a whole rat brain increased immunostaining of glial fibrillary acidic protein in astrocytes 6 h later, and even more so 24 h later, indicating the initiation of gliosis. While irradiation of cultured rat astrocytes had little effect, irradiation of microglia-astrocyte mixed-cultures displayed altered astrocyte phenotype into more processed, which is another characteristic of gliosis. Experiments using microglia-conditioned media indicated this astrocyte change was due to factors released from irradiated microglia. Irradiation of cultured mouse microglial cells induced a dose-dependent increase in mRNA levels for cyclooxygenase-2 (COX-2), interleukin (IL)-1beta, IL-6, IL-18, tumor necrosis factor-alpha and interferon-gamma-inducible protein-10, which are usually associated with microglia activation. Consistent with these findings, irradiation of microglia activated NF-kappaB, a transcription factor that regulates microglial activation. Addition of prostaglandin E2 (PGE2: a metabolic product of the COX-2 enzyme) to primary cultured rat astrocytes resulted in phenotypic changes similar to those observed in mixed-culture experiments. Therefore, it appears that PGE(2) released from irradiated microglia is a key mediator of irradiation-induced gliosis or astrocyte phenotype change. These data suggest that radiation-induced microglial activation and resultant production of PGE2 seems to be associated with an underlying cause of inflammatory complications associated with radiation therapy for malignant gliomas.

Animals↗

PubMedDate not supplied

Kinin receptors in cultured rat microglia.

Kinins are produced and act at the site of injury and inflammation in various tissues. They are likely to initiate a particular cascade of inflammatory events, which evokes physiological and pathophysiological responses including an increase in blood flow and plasma leakage. In the central nervous system (CNS), kinins are potent stimulators of the production and release of pro-inflammatory mediators represented by prostanoids and cytotoxins. They are known to induce neural tissue damage. Many of the cytotoxins such as cytokines and free radicals and prostanoids are released from glial cells. Among glial cells, astrocytes and oligodendrocytes are known to possess bradykinin (BK) B(2) receptors that phosphoinositide (PI) turnover and raise intracellular Ca(2+) concentration. The presence of bradykinin receptors in microglia has been of great significance. We recently showed that rat primary microglia express kinin receptors. In resting microglia, B(2) receptors but not B(1) receptors are expressed. When the microglia are activated by bradykinin, B(1) receptors are up-regulated, while B(2) receptors are down-regulated. As observed in other glial cells, electrophysiological measurements suggest that B(2) receptors in phosphoinositide turnover and intracellular Ca(2+) concentration in microglia. Release of cytotoxins is likely consequent upon the activation of BK receptors. Our study provides the first evidence that microglia express functional kinin receptors and suggests that microglia play an important role in CNS inflammatory responses.

Animals↗

PubMed2005

Minocycline treatment attenuates microglia activation and non-angiotensin II [125I] CGP42112 binding in brainstem following nodose ganglionectomy.

We have previously shown that following unilateral nodose ganglionectomy, [125I] CGP42112 binds to a non-angiotensin II (Ang II) related binding site in rat dorsal motor nucleus of the vagus nerve, ambiguus nucleus and nucleus of the solitary tract. Furthermore, this up-regulated binding site localizes with activated microglia. Given that some tetracyclines may inhibit microglia activation in brain, we examined the effect of minocycline treatment on the binding of [125I] CGP42112 and [3H] PK11195 (an established radioligand for microglia), as well as OX-42 immunoreactivity (an immunomarker for activated microglia), following nodose ganglionectomy. Male Wistar Kyoto rats underwent unilateral nodose ganglionectomy or sham operation and were treated with saline or minocycline (50 mg/kg i.p.) 12 h before surgery and twice daily after surgery (each 50mg/kg i.p.) for 3 days. Subsequent to nodose ganglionectomy, [125I] CGP42112 binding (insensitive to PD123319 or Ang II) was increased approximately two-fold in the ipsilateral nucleus of the solitary tract and was also induced in the ipsilateral dorsal motor nucleus of the vagus nerve and ambiguus nucleus of saline-treated rats. Treatment with minocycline reduced this non-angiotensin II [125I] CGP42112 binding (40-50% reduction) in the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve and ambiguus nucleus. Analogous experiments using [3H] PK11195 also revealed up-regulated binding in the ipsilateral nucleus of the solitary tract ( approximately 205%), dorsal motor nucleus of the vagus nerve (approximately 80%) and ambiguus nucleus (approximately 210%) of saline-treated rats following nodose ganglionectomy, which was reduced by 40-100% with minocycline treatment. Immunoreactivity to OX-42 confirmed an increase in microglia activation and accumulation of macrophages in these brain stem nuclei following nodose ganglionectomy, which was also attenuated following treatment with minocycline. These data demonstrate that non-Ang II [125I] CGP42112 binding following nodose ganglionectomy is attenuated by minocycline treatment. This minocycline-induced effect was associated with reduced activation of microglia and an apparent reduction in the number of macrophages in the abovementioned nuclei. This evidence suggests that a non-Ang II [125I] CGP42112 binding site is located on, or associated with, activated microglia and macrophages, providing a useful tool with which to quantitate the neuroprotective effects of centrally acting anti-inflammatory compounds.

Animals↗

PubMed2006

The intra-arterial injection of microglia protects hippocampal CA1 neurons against global ischemia-induced functional deficits in rats.

In the present study, we have attempted to elucidate the effects of the intra-arterial injection of microglia on the global ischemia-induced functional and morphological deficits of hippocampal CA1 neurons. When PKH26-labeled immortalized microglial cells, GMIR1, were injected into the subclavian artery, these exogenous microglia were found to accumulate in the hippocampus at 24 h after ischemia. In hippocampal slices prepared from medium-injected rats subjected to ischemia 48 h earlier, synaptic dysfunctions including a significant reduction of synaptic responses and a marked reduction of long-term potentiation (LTP) of the CA3-CA1 Schaffer collateral synapses were observed. At this stage, however, neither significant neuronal degeneration nor gliosis was observed in the hippocampus. At 96 h after ischemia, there was a total loss of the synaptic activity and a marked neuronal death in the CA1 subfield. In contrast, the basal synaptic transmission and LTP of the CA3-CA1 synapses were well preserved after ischemia in the slices prepared from the microglia-injected animals. We also found the microglial-conditioned medium (MCM) to significantly increase the frequency of the spontaneous postsynaptic currents of CA1 neurons without affecting the amplitude, thus indicating that MCM increased the provability of the neurotransmitter release. The protective effect of the intra-arterial injected microglia against the ischemia-induced neuronal degeneration in the hippocampus was substantiated by immunohistochemical and immunoblot analyses. Furthermore, the arterial-injected microglia prevented the ischemia-induced decline of the brain-derived neurotrophic factor (BDNF) levels in CA1 neurons. These observations strongly suggest that the arterial-injection of microglia protected CA1 neurons against the ischemia-induced neuronal degeneration. The restoration of the ischemia-induced synaptic deficits and the resultant reduction of the BDNF levels in CA1 neurons, possibly by the release of diffusible factor(s), might thus contribute to the protective effect of the arterial-injection of microglia against ischemia-induced neuronal degeneration.

Animals↗

PubMed2000

Agmatine suppresses nitric oxide production in microglia.

We investigated the effect of agmatine, an arginine metabolite synthesized in the brain, in cultured microglia obtained from neonatal rat cerebral cortex. Agmatine (1-300 microM) did not affect viability of cultured microglia. Activation of microglia by lipopolysaccharide (LPS, 1 microg/ml) caused the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) assessed as the accumulation of nitrite in the culture supernatants. Agmatine had no effect on the expression of iNOS, but significantly suppressed the LPS-induced NO production in a concentration-dependent manner. Agmatine was also effective in suppressing the production of NO induced by a combination of interferon-gamma (500 U/ml) and amyloid beta protein (10 microM). In co-cultures of rat cortical neurons and microglia, LPS caused significant loss of neuron viability. The LPS neurotoxicity was not observed in the absence of microglia, and was completely blocked by the NOS inhibitor diphenyleneiodoium chloride. The neuronal death induced by microglia-derived NO was significantly attenuated by the presence of agmatine. These results suggest that agmatine works to protect neurons by inhibiting the production of NO in microglia.

Agmatine↗

PubMed1999

Neuronal adhesion molecule telencephalin induces rapid cell spreading of microglia.

Telencephalin (TLCN) is a neuronal surface glycoprotein whose expression is restricted to the telencephalon, the most rostral segment of the brain. TLCN binds to lymphocyte function-associated antigen-1 (LFA-1) integrin. In the central nervous system, LFA-1 is selectively and constitutively expressed by microglia, suggesting that TLCN/LFA-1 binding may mediate cell-cell interactions between telencephalic neurons and microglia. In the present study, we investigated the effects of recombinant TLCN protein on the morphology of microglia. TLCN induced an intensive spreading of lamellipodia, causing a rapid change in microglial morphology. In contrast, TLCN induced no significant change in morphology of neuroblastoma and fibroblasts. Furthermore, the TLCN-induced spreading of microglia was accompanied by a clustering of LFA-1 on cell surface membrane. These results provide evidence that TLCN binding to the surface of microglia transduces signals into microglia that mediate or accelerate cell spreading and LFA-1 redistribution, implying that neuronal TLCN may control the state and/or function of microglia in both physiological and pathological conditions.

Animals↗

PubMed2003

Expression and function of bradykinin receptors in microglia.

Expression of bradykinin (BK) receptors and their cellular function were investigated in microglia. Microglial cells were isolated from mixed cultures of cerebrocortical cells from postnatal day 3 Wistar rats. Reverse transcription-PCR (RT-PCR) showed that rat primary microglia express mRNAs for the type 2 bradykinin (B(2)) receptor subtype but not the type 1 (B(1)) receptor subtype under our experimental condition. However, the expression of B(1) receptor was greatly up-regulated after the treatment of microglia with BK for 24 hours. The expression of B(2) receptor in microglia was further confirmed by immunocytochemistry. Membrane currents were measured using whole-cell recording under voltage-clamp conditions. In 14% of patched cells (12/85 cells), BK (100-200 nM) induced an outward current at the holding potential of -20 mV, with oscillations in 2 cases. The BK-induced outward current was transient and desensitized rapidly. TEA inhibited the BK-induced outward current in a dose-dependent manner. These results suggest that microglia express B(2) receptors and presumably increase the intracellular Ca(2+) concentration via inositol trisphosphate with the subsequent activation of Ca(2+)-dependent K(+) channels. Our data provide the first evidence that microglia express functional BK receptors and support the idea that microglia play an important role in CNS inflammatory responses.

Animals↗

PubMed1999

Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain.

Microglia, the resident CNS macrophages, represent about 10% of the adult brain cell population. Although described a long time ago, their origin and developmental lineage is still debated. While del Rio-Hortega suggested that microglia originate from meningeal macrophages penetrating the brain during embryonic development, many authors claim that brain parenchymal microglia derive from circulating blood monocytes originating from bone marrow. We have previously reported that the late embryonic and adult mouse brain parenchyma contains potential microglial progenitors [F. Alliot, E. Lecain, B. Grima, B. Pessac, Microglial progenitors with a high proliferative capacity in the embryonic and the adult mouse brain, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 1541-1545]. We now report that they can be detected in the brain rudiment from embryonic day 8, after their appearance in the yolk sac and that their number increases until late gestation. We also show that microglia appear during embryonic development and that their number increases steadily during the first two postnatal weeks, when about 95% of microglia are born. Finally, the main finding of this study is that microglia is the result of in situ proliferation, as shown by the high proportion of parenchymal microglial cells that express PCNA, a marker of cell multiplication, in embryonic and postnatal brain. Taken together, our data support the hypothesis that terminally differentiated brain parenchymal microglia are derived from cells originating from the yolk sac whose progeny actively proliferates in situ during development.

Animals↗

PubMed2004

Protein kinase C alpha requirement in the activation of p38 mitogen-activated protein kinase, which is linked to the induction of tumor necrosis factor alpha in lipopolysaccharide-stimulated microglia.

Activated microglia have been suggested to produce a cytotoxic cytokine, tumor necrosis factor alpha (TNF alpha), in many pathological brains. Thus, determining the molecular mechanism of this induction and suppression has been the focus of a great deal of research. Using lipopolysaccharide (LPS) as an experimental inducer of TNF alpha, we investigated the regulatory mechanism by which TNFalpha is induced or suppressed in microglia. We found that LPS-induced TNF alpha is suppressed by pretreatment with the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580. Similar suppression was achieved by pretreatment with specific protein kinase C (PKC) inhibitors, Gö6976, myristoylated pseudosubstrate (20-28), and bisindolylmaleimide. These results suggest that PKC alpha activity as well as p38MAPK activity is associated with TNF alpha induction in LPS-stimulated microglia. The requirement of PKC alpha in LPS-dependent TNFalpha induction was verified in PKC alpha-downregulated microglia which could be induced by phorbol-12-myristate-13-acetate pretreatment. Simultaneously, PKC alpha was found to be requisite for the activation of p38MAPK in LPS-stimulated microglia. In addition, the PKC alpha levels in the LPS-stimulated microglia were observed to decrease in response to the p38MAPK inhibitor, indicating that the PKC alpha levels are regulated by the p38MAPK activity. We therefore concluded that PKC alpha and p38MAPK are interactively linked to the signaling cascade inducing TNFalpha in LPS-stimulated microglia, and that in this cascade, PKC alpha is requisite for the activation of p38MAPK, leading to the induction of TNF alpha.

Animals↗

PubMed2000

Beta-chemokines and human immunodeficiency virus type-1 proteins evoke intracellular calcium increases in human microglia.

Activation of beta-chemokine receptors, co-receptors for human immunodeficiency virus type-1 (HIV-1), stimulates movement and secretion in microglia, possibly through a Ca(2+)-dependent mechanism. We studied chemokine activation of Ca(2+) signaling processes in microglia. Human fetal microglia were grown in primary culture and chemokine-induced increases in intracellular calcium concentration ([Ca(2+)](i)) were measured in single cells using indo-1-based microfluorimetry. Application of 50 ng/ml regulated on activation, normal T expressed and secreted (RANTES; 120 s) evoked responses in 26% of the microglia (187/719 cells). [Ca(2+)](i) increased from a basal level of 66+/-6 nM to peak at 268+/-23 nM (n=187). Chemokine-evoked responses rapidly desensitized as indicated by the rapid return to basal [Ca(2+)](i) levels in the maintained presence of RANTES. The removal of extracellular Ca(2+) or stimulation in the presence of Ni(2+) (2mM) or La(3+) (100 microM) blocked the RANTES-elicited [Ca(2+)](i) increase. The L-type calcium channel antagonist nimodipine (10 microM) inhibited the RANTES-mediated increase in [Ca(2+)](i) by 80+/-16%. Thus, the RANTES-evoked calcium transient appears to result from Ca(2+) influx with little if any release from intracellular stores. Application of gp120(clade) (E) and gp120(CM235) (50 ng/ml) neither mimicked nor antagonized the RANTES-evoked response. Application of 50 ng/ml eotaxin (120 s) evoked an increase in [Ca(2+)](i) in 13% of the human microglia in culture (61/469 cells). The HIV-1 regulatory protein Tat (50 ng/ml) increased the [Ca(2+)](i) in a subset of eotaxin-responsive cells (16/30). The L-type calcium channel antagonist nimodipine (3 microM) inhibited eotaxin- and Tat-mediated increases in [Ca(2+)](i) by 88+/-6% and 93+/-6%, respectively. Thus, activation of CCR3 appears to evoke Ca(2+) influx through L-type Ca(2+) channels.These results indicate that beta-chemokines, RANTES and eotaxin, activate a nimodipine sensitive Ca(2+) influx pathway in human fetal microglia. HIV-1 Tat protein mimicked chemokine-mediated Ca(2+) signaling and may modulate the migratory and secretory responses of microglia.

Acquired Immunodeficiency Syndrome↗

PubMed2000

Microglia in organotypic hippocampal slice culture and effects of hypoxia: ultrastructure and lipocortin-1 immunoreactivity.

Lipocortin-1 immunocytochemistry was used to study the various cell forms of microglia that appear during organotypic hippocampal tissue culture, as well as in the in vitro toxic hypoxia model. Antibodies against lipocortin-1 identified activated and phagocytic cells that were abundant in a slice after the plating of a culture: cells of the intermediate form at the later time-points of culturing, resting ramified microglia beginning from the seventh day of culturing, as well as activated and phagocytic cells that appeared in the slice after experimental toxic hypoxia induced by potassium cyanide treatment. Lipocortin-1-positive microglia cell forms corresponded well to the description of the microglia in vivo, and the morphology of microglia corresponded to the circumstances under which these cells were observed in slice cultures. Electron microscopic studies have demonstrated, for the first time, that microglia in organotypic slice culture preserve morphological features typical of different microglial forms in vivo, as well as specific contacts and interactions with the other neural tissue elements. After experimental toxic hypoxia, rapid changes in microglial ultrastructure and localization were observed, reminiscent of in vivo models of ischaemia. In conclusion, observations of microglial morphology and behaviour allow us to suggest that microglia in the organotypic culture preserve their essential characteristic features and properties, thus providing an important model system for studying the structure and function of these cells.

Animals↗

PubMed1998

Lipopolysaccharide induction of MARCKS-related protein and cytokine secretion are differentially impaired in microglia from LPS-nonresponsive (C3H/HeJ) mice.

Many events involved in activation of microglia and leukocytes by lipopolysaccharide (LPS) are mediated by protein kinase C (PKC), and we have recently demonstrated that a major PKC substrate, MARCKS-related protein (MRP), is selectively induced by LPS in murine microglia. In microglia from LPS-nonresponsive (C3H/HeJ) mice, induction of MRP and secretion of CSF-1 required much higher LPS concentrations (> or = 100 ng/ml) than in normal (C3H/OuJ) microglia (< or = 10 ng/ml). By contrast, TNF alpha production was not significantly increased in C3H/HeJ microglia even at 1 microgram LPS/ml. Microglia expressed PKC isoforms alpha, beta, delta, and zeta (but not gamma and epsilon); PKC isoform levels were similar in both normal and C3H/HeJ microglia and no significant change in response to LPS was noted. Our results indicate that LPS alters PKC substrate (rather than kinase) expression, and that the Lpsd mutation in C3H/HeJ mice differentially affects regulation of several gene products implicated in microglial function.

Animals↗

PubMed2025

Lymphoid gene expression supports neuroprotective microglia function.

Microglia, the innate immune cells of the brain, play a defining role in the progression of Alzheimer's disease (AD)1. The microglial response to amyloid plaques in AD can range from neuroprotective to neurotoxic2. Here we show that the protective function of microglia is governed by the transcription factor PU.1, which becomes downregulated following microglial contact with plaques. Lowering PU.1 expression in microglia reduces the severity of amyloid disease pathology in mice and is linked to the expression of immunoregulatory lymphoid receptor proteins, particularly CD28, a surface receptor that is critical for T cell activation3,4. Microglia-specific deficiency in CD28, which is expressed by a small subset of plaque-associated PU.1low microglia, promotes a broad inflammatory microglial state that is associated with increased amyloid plaque load. Our findings indicate that PU.1low CD28-expressing microglia may operate as suppressive microglia that mitigate the progression of AD by reducing the severity of neuroinflammation. This role of CD28 and potentially other lymphoid co-stimulatory and co-inhibitory receptor proteins in governing microglial responses in AD points to possible immunotherapy approaches for treating the disease by promoting protective microglial functions.

Microglia↗

PubMed2003

Selective gene expression in brain microglia mediated via adeno-associated virus type 2 and type 5 vectors.

Microglia represent a crucial cell population in the central nervous system, participating in the regulation and surveillance of physiological processes as well as playing key roles in the etiologies of several major brain disorders. The ability to target gene transfer vehicles selectively to microglia would provide a powerful new approach to investigations of mechanisms regulating brain pathologies, as well as enable the development of novel therapeutic strategies. In this study, we evaluate the feasibility of specifically and efficiently targeting microglia relative to other brain cells, using vectors based on two different serotypes of adeno-associated virus (AAV) carrying cell-type-specific transcriptional elements to regulate gene expression. Among a set of promoter choices examined, an element derived from the gene for the murine macrophage marker F4/80 was the most discriminating for microglia. Gene expression from vectors controlled by this element was highly selective for microglia, both in vitro and in vivo. To our knowledge, this is the first demonstration of selective expression of transferred genes in microglia using AAV-derived vectors, as well as the first utilization of recombinant AAV-5 vectors in any macrophage lineage. These results provide strong encouragement for the application of these vectors and this approach for delivering therapeutic and other genes selectively to microglia.

Animals↗

PubMed1999

Prostaglandin E receptor subtypes in cultured rat microglia and their role in reducing lipopolysaccharide-induced interleukin-1beta production.

Prostaglandins (PGs) are potent modulators of brain function under normal and pathological conditions. The diverse effects of PGs are due to the various actions of specific receptor subtypes for these prostanoids. Recent work has shown that PGE2, while generally considered a proinflammatory molecule, reduces microglial activation and thus has an antiinflammatory effect on these cells. To gain further insight to the mechanisms by which PGE2 influences the activation of microglia, we investigated PGE receptor subtype, i.e., EP1, EP2, EP3, and EP4, expression and function in cultured rat microglia. RT-PCR showed the presence of the EP1 and EP2 but not EP3 and EP4 receptor subtypes. Sequencing confirmed their identity with previously published receptor subtypes. PGE2 and the EP1 agonist 17-phenyl trinor PGE2 but not the EP3 agonist sulprostone elicited reversible intracellular [Ca2+] increases in microglia as measured by fura-2. PGE2 and the EP2/EP4-specific agonists 11-deoxy-PGE1 and 19-hydroxy-PGE2 but not the EP4-selective agonist 1-hydroxy-PGE1 induced dose-dependent production of cyclic AMP (cAMP). Interleukin (IL)-1beta production, a marker of activated microglia, was also measured following lipopolysaccharide exposure in the presence or absence of the receptor subtype agonists. PGE2 and the EP2 agonists reduced IL-1beta production. IL-1beta production was unchanged by EP1, EP3, and EP4 agonists. The adenylyl cyclase activator forskolin and the cAMP analogue dibutyryl cAMP also reduced IL-1beta production. Thus, the inhibitory effects of PGE2 on microglia are mediated by the EP2 receptor subtype, and the signaling mechanism of this effect is likely via cAMP. These results show that the effects of PGE2 on microglia are receptor subtype-specific. Furthermore, they suggest that specific and selective manipulation of the effects of PGs on microglia and, as a result, brain function may be possible.

Adenylyl Cyclases↗