Leukocyte recruitment and neuroglial activation during facial nerve regeneration in ICAM-1-deficient mice: effects of breeding strategy

Intercellular adhesion molecule 1 (ICAM-1) is a widely expressed glycoprotein involved in leukocyte extravasation and the interaction of lymphocytes with antigen-presenting cells. We examined these aspects of ICAM-1 function in the central nervous system after axonal injury in wild-type and ICAM-1-deficient mice. ICAM-1 immunoreactivity in the normal mouse facial nucleus was restricted to the vascular endothelium. Transection of the facial nerve led to a fast upregulation of ICAM-1 on activated microglia in the axotomized facial nucleus and the infiltration of ICAM-1-positive lymphocytes. Labeling elsewhere was unchanged. In homozygous ICAM-1 mutant mice, ICAM-1 was absent from endothelial cells and lymphocytes, but low levels of ICAM-1 were detected on cell membranes of reactive microglial cells. Comparison of wild-type animals with homozygously bred, ICAM-1-deficient mice showed a reduction of astrocytic and microglial activation, massive late axonal sprouting, and decreased lymphocyte infiltration. These experiments were repeated in F1 progeny of heterozygous mice on a C57BL/6 background. Neuroglial activation and lymphocyte infiltration in F1 homozygously deficient mice was unaffected compared with wild-type siblings. The invading ICAM-1-deficient lymphocytes also adhered to the ICAM-1-positive phagocytotic microglial cells in the ICAM-1 mutants. No change in the recruitment of macrophages and granulocytes into the crushed facial nerve, and no effect on axonal regeneration occurred. These data argue against the requirement of endothelial ICAM-1 in the recruitment of leukocytes into the crushed peripheral nerve or the axotomized facial motor nucleus and stress the importance of adequately matched controls in studying the effects of gene deletion in experimental animals.     

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.     

Morphological changes and expression of protein kinase CK2 β subunit in the microglia after hypoglossal nerve transection

Following hypoglossal nerve transection, the microglia of the rat hypoglossal nucleus expressed protein kinase CK2 β subunit immunoreactivity. CK2 β immunostaining occurred on the operated side from postoperative day 3; on day 5 we observed strong immunoreactivity and the immunopositive microglial cell processes surrounded the injured neurones. Thereafter, the immunoreactivity decreased gradually and on day 10 the immunopositive cells surrounded only a few injured neurones. Electron microscopic observations on the hypoglossal nucleus revealed microglia-neuronal contact within 3 hours of nerve injury, and by day 3 all the injured neurones were in contact with microglial cells. These observations indicated that microglia-neuronal contact occurred earlier than the CK2 β subunit immunoreactivity. CK2 may not be implicated during the initial migration of the microglia to the injured neurones; however, it may enhance the growth and elongation of the microglial cell processes around the injured neurones.     

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.     

Ultrastructural localization of immunoglobulin G and complement C9 in the brain stem and spinal cord following peripheral nerve injury: an immunoelectron microscopic study

Summary The ultrastructural localization of immunoreactivity for immunoglobulin G (IgG), F(ab′)2 and complement C9 was examined with preembedding immunoelectron microscopy in the hypoglossal nucleus and gracile nucleus as well as in the L4 spinal cord dorsal horn 1 week following hypoglossal or sciatic nerve transection, respectively. Only a few scattered immunoreactive profiles were observed on the unoperated side. On the operated side, IgG and F(ab′)2 immunoreactivity was present in the membranes of all reactive microglial cells observed. In addition, the cell membrane of some hypoglossal motoneurons showed IgG immunoreactivity. Complement C9 immunoreactivity was present in the cytoplasm of all reactive microglial cells examined. In addition, there was diffuse C9 immunoreactivity in motoneuron perikarya ipsilateral to nerve injury as well as in cell membranes in the neuropil, some of which could be identified as neuronal. Our interpretation of these findings is (1) that peripheral nerve injury results in binding of IgG to reactive microglia, as well as to some axotomized neurons, and (2) that C9 is synthesized by reactive microglia in response to axon injury and is also associated with axotomized motoneurons. These findings suggest that IgG and complement C9 are involved in microglia-neuron interactions after peripheral nerve injury.     

Regulation of microglial migration,phagocytosis, and neurite outgrowth by HO‐1/CO signaling

Clearance of infected and apoptotic neuronal corpses during inflammatory conditions is a fundamental process to create a favorable environment for neuronal recovery. Microglia are the resident immune cells and the predominant phagocytic cells of the CNS, showing a multitude of cellular responses upon activation. Here, we investigated in functional assays how the CO generating enzyme heme oxygenase 1 (HO‐1) influences BV‐2 microglial migration, clearance of debris, and neurite outgrowth of human NT2 neurons. Stimulation of HO‐1 activity attenuated microglial migration in a scratch wound assay, and phagocytosis in a cell culture model of acute inflammation comprising lipopolysaccharide (LPS)‐activated microglia and apoptosis‐induced neurons. Application of a CO donor prevented the production of NO during LPS stimulation, and reduced microglial migration and engulfment of neuronal debris. LPS‐activated microglia inhibited neurite elongation of human neurons without requiring direct cell–cell surface contact. The inhibition of neurite outgrowth was totally reversed by application of exogenous CO or increased internal CO production through supply of the substrate hemin to HO. Our results point towards a vital cytoprotective role of HO‐1/CO signaling after microglial activation. In addition, they support a therapeutic potential of CO releasing chemical agents in the treatment of excessive inflammatory conditions in the CNS. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 854–876, 2015     

Nitric oxide regulates antagonistically phagocytic and neurite outgrowth inhibiting capacities of microglia

Traumatic injury or the pathogenesis of some neurological disorders is accompanied by inflammatory cellular mechanisms, mainly resulting from the activation of central nervous system (CNS) resident microglia. Under inflammatory conditions, microglia up‐regulate the inducible isoform of NOS (iNOS), leading to the production of high concentrations of the radical molecule nitric oxide (NO). At the onset of inflammation, high levels of microglial‐derived NO may serve as a cellular defense mechanism helping to clear the damaged tissue and combat infection of the CNS by invading pathogens. However, the excessive overproduction of NO by activated microglia has been suggested to govern the inflammation‐mediated neuronal loss causing eventually complete neurodegeneration. Here, we investigated how NO influences phagocytosis of neuronal debris by BV‐2 microglia, and how neurite outgrowth of human NT2 model neurons is affected by microglial‐derived NO. The presence of NO greatly increased microglial phagocytic capacity in a model of acute inflammation comprising lipopolysaccharide (LPS)‐activated microglia and apoptotic neurons. Chemical manipulations suggested that NO up‐regulates phagocytosis independently of the sGC/cGMP pathway. Using a transwell system, we showed that reactive microglia inhibit neurite outgrowth of human neurons via the generation of large amounts of NO over effective distances in the millimeter range. Application of a NOS blocker prevented the LPS‐induced NO production, totally reversed the inhibitory effect of microglia on neurite outgrowth, but reduced the engulfment of neuronal debris. Our results indicate that a rather simple notion of treating excessive inflammation in the CNS by NO synthesis blocking agents has to consider functionally antagonistic microglial cell responses during pharmaceutic therapy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 566–584, 2016     

Axotomy-dependent urokinase induction in the rat facial nucleus: possible stimulation of microglia by neurons

The phenomenon in which urokinase-type plasminogen activator (uPA) is induced in the axotomized facial nucleus suggests an interaction between injured motoneurons and microglia. We examined the relation of neurons and microglia to the induction of uPA in vitro. The amount of uPA released from a co-culture of neurons and microglia was much greater than the addition of that from each alone, suggesting the occurrence of an interaction between the two. The analysis of conditioned neuronal medium (CNM)-effects on microglia and conditioned microglial medium (CMM)-effects on neurons revealed that microglia enhance uPA release in response to CNM, rather than vice versa. Characterization of the CNM-effect on microglia demonstrated that CNM enhances not only uPA release but also the specific activity of acid phosphatase and 5'-nucleotidase in microglia. The profile of microglial activation caused by CNM was quite different from that caused by lipopolysaccharide (LPS)-activation. These results suggest that a specific soluble constituent(s) derived from neurons activates microglia by a mechanism different from LPS. As a candidate molecule for the microglial activation, brain-derived neurotrophic factor was detected in the CNM. Thus, uPA induction in the axotomized facial nucleus may be explained by a neuronal stimulus leading to uPA induction in microglia.     

Expression of major histocompatibility complex antigens and CR3 complement receptors in activated microglia following an injection of ricin into the sciatic nerve in rats.

The ventral horn motor neurons in the lower lumbar cord underwent rapid degeneration following an injection of Ricinus communis agglutinin-60 (RCA) into the sciatic nerve. The cell death which was most drastic between the fifth and seventh post-injection day elicited a significant increase in the number of microglia. The activated microglia were scattered throughout the neuropil but the dramatic feature was their close association with the somata of the degenerating neurons. Often several microglial cells were seen surrounding the soma of a degenerating neuron. Immunocytochemical study showed that both the interstitial as well as the perineuronal activated microglia were labelled with the monoclonal antibodies OX-18 and OX-42 for the detection of MHCI encoded antigen and type three complement receptors, respectively. Intense immunoreactivity was observed especially in the perineuronal microglia with OX-18. Electron microscopic study confirmed the identification of the activated microglia. Although the activated microglia closely apposed the neuronal soma, there was no sign of a direct endocytosis. The cytoplasm of the activated microglia, however, contained massive lipofuscin bodies in longer survival animals. Electron microscopic immunocytochemical study showed that the immunoreactivity of the activated microglia was localized along their plasma membrane facing the neuronal soma. Since the microglia cells on the contralateral side of the ventral horn were not marked by the antibodies used, it was postulated that the vigorous expression of MHCI antigen and CR3 receptors on the activated microglia was induced by the neuronal degeneration resulting from the application of the toxin ricin.     

Regulation of Microglial Phagocytosis by RhoA/ROCK-Inhibiting Drugs

Inflammation within the central nervous system (CNS) is a major component of many neurodegenerative diseases. The underlying mechanisms of neuronal loss are not fully understood, but the activation of CNS resident phagocytic microglia seems to be a significant element contributing to neurodegeneration. At the onset of inflammation, high levels of microglial phagocytosis may serve as an essential prerequisite for creating a favorable environment for neuronal regeneration. However, the excessive and long-lasting activation of microglia and the augmented engulfment of neurons have been suggested to eventually govern widespread neurodegeneration. Here, we investigated in a functional assay of acute inflammation how the small GTPase RhoA and its main target the Rho kinase (ROCK) influence microglial phagocytosis of neuronal debris. Using BV-2 microglia and human NT2 model neurons, we demonstrate that the pain reliever Ibuprofen decreases RhoA activation and microglial phagocytosis of neuronal cell fragments. Inhibition of the downstream effector ROCK with the small-molecule agents Y-27632 and Fasudil reduces the engulfment of neuronal debris and attenuates the production of the inflammatory mediator nitric oxide during stimulation with lipopolysaccharide. Our results support a therapeutic potential for RhoA/ROCK-inhibiting agents as an effective treatment of excessive inflammation and the resulting progression of microglia-mediated neurodegeneration in the CNS.     

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.     

Neuronal regulation by which microglia enhance the production of neurotrophic factors for GABAergic, catecholaminergic, and cholinergic neurons

A phenomenon-in which microglia are activated in axotomized rat facial nucleus suggests that a certain neuronal stimulus triggers the activation of microglia. However, how the microglial characteristics are regulated by this neuronal stimulus has not previously been determined. In this study, therefore, the regulation of microglial properties by neurons was characterized in vitro from a neurotrophic perspective. To evaluate the neurotrophic effects of microglia stimulated with neurons, the effects of conditioned medium (CM) of microglia stimulated with neuronal CM (NCM) were assessed in neuronal cultures. The amounts of tyrosine hydroxylase (TH) in neuronal culture exposed to CM of microglia stimulated with NCM was much more than those in neurons exposed to CM of control microglia, suggesting that neuronal stimulus enhances the production of neurotrophic factors for catecholaminergic neurons in microglia. Therefore, the neurotrophic effects of CM of microglia stimulated with NCM were analyzed in detail. The immunocytochemical and biochemical experiments revealed that the CM of microglia stimulated with NCM enhances the survival/maturation of GABAergic and catecholaminergic neurons. The levels of choline acetyltransferase specific to cholinergic neurons also significantly increased in response to stimulation with the same microglial CM. These results allowed us to investigate the production of neurotrophic factors in the CM of microglia stimulated with NCM. The results indicated that NCM induces nerve growth factor (NGF), and enhances neurotrophin-4/5 (NT-4/5), transforming growth factor beta1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), interleukin-3 (IL-3), and IL-10 in microglia. The promoted neurotrophic effects of CM of microglia stimulated with NCM were significantly abrogated by deprivation of neurotrophic factors by means of an immunoprecipitation method. Taken together, neuronal stimulus was found to activate microglia to produce more neurotrophic factors as above, thereby changing microglia into more neurotrophic cells.     

UDP Facilitates Microglial Phagocytosis Through P2Y6 Receptors

Microglia engage in the clearance of dead cells or dangerous debris. When neighboring cells are injured, the cells release or leak ATP into extracellular space and microglia rapidly move toward or extend a process to the nucleotides as chemotaxis through P2Y12 receptors. In the meanwhile, microglia express the metabotropic P2Y6 receptors, the activation of which by uridine 5’-diphosphate (UDP) triggers microglial phagocytosis in a concentration-dependent fashion. UDP/UTP was leaked when hippocampal neurons were damaged by kainic acid in vivo and in vitro. Systemic administration of kainic acid in rats resulted in neuronal cell death in the hippocampal CA1 and CA3 regions, where increases in mRNA for P2Y6 receptors in activated microglia. Thus, the P2Y6 receptor is upregulated when neurons are damaged, and would function as a sensor for phagocytosis by sensing diffusible UDP signals.     

Minocycline decreases in vitro microglial motility, beta1-integrin, and Kv1.3 channel expression

Minocycline is a semisynthetic, tetracycline derivative that exerts anti-inflammatory and neuroprotective effects unrelated to its anti-microbial action. We have previously shown that minocycline prevented peripheral nerve injury-induced mechanical allodynia. Minocycline's mechanisms of action as a neuroprotective and anti-allodynic agent are unknown. In response to injury, microglia become activated, proliferate, and migrate. Resting microglia express voltage-dependent inward K⁺ currents and blocking Kv1.3 channels has been shown to inhibit microglial-mediated neuronal death. We investigated the effect of minocycline on the expression of Kv channels, cell motility, and β-integrin expression using primary rat cortical microglia, transwell assays, and by flow cytometry. Minocycline significantly reduced microglial migration to cellular debris, astrocyte-conditioned medium, ADP, and algesic mediators and significantly reduced the expression of CD29 (β₁-integrin) but not CD18 (β₂-integrin). Minocycline reduced the effect of extracellular potassium and later decreased microglial Kv1.3 expression. In summary, we uncovered a novel effect of minocycline that demonstrates this agent decreases microglial β₁-integrin expression, which leads to inhibition of motility. We propose an in vivo model whereby reduced microglial trafficking to injured neurons following nerve injury decreases the release of proinflammatory mediators into the synaptic milieu, preventing neuronal sensitization, the pathological correlate to chronic pain.     

Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury

Peripheral nerve injury can induce spinal microglial/astrocyte activation. Substances released by activated glial cells excite spinal nociceptive neurons. Pharmacological disruption of glial activation or antagonism of substances released by activated glia prevent or reverse pain hypersensitivity. It is not known, however, what causes spinal cord glia to shift from a resting to an activated state. In an attempt to understand the potential role of monocyte chemoattractant protein-1 (MCP-1) in triggering spinal glial activation and its contribution to the development of neuropathic pain, we investigated the effect of peripheral nerve injury on MCP-1 expression in dorsal root ganglia (DRG) and the spinal cord, and established its temporal relationship with activation of spinal microglia and astrocytes. We observed that MCP-1 was induced by chronic constriction of the sciatic nerve in DRG sensory neurons, spinal cord motor neurons and in the superficial dorsal horn, ipsilateral to the injury. Neuronal MCP-1 induction was followed by surrounding microglial activation. After peaking at day 7 after injury, MCP-1 levels began to decline rapidly and had returned to baseline by day 150. In contrast, microglial activation peaked by day 14 and declined afterwards to reach a lower, yet significantly raised level beyond day 22 and remained increased until the end of the test period. Astrocyte activation became detectable later, progressed more slowly and also remained increased until the end of the test period, in parallel with a decreased nociceptive threshold. Our results suggest that neuronal MCP-1 may serve as a trigger for spinal microglial activation, which participates in the initiation of neuropathic pain. Delayed, sustained astrocyte activation may participate with microglia in the persistent phase of pain hypersensitivity.     

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.     

Fractalkine attenuates excito-neurotoxicity via microglial clearance of damaged neurons and antioxidant enzyme heme oxygenase-1 expression

Glutamate-induced excito-neurotoxicity likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases. Microglial clearance of dying neurons and associated debris is essential to maintain healthy neural networks in the central nervous system. In fact, the functions of microglia are regulated by various signaling molecules that are produced as neurons degenerate. Here, we show that the soluble CX3C chemokine fractalkine (sFKN), which is secreted from neurons that have been damaged by glutamate, promotes microglial phagocytosis of neuronal debris through release of milk fat globule-EGF factor 8, a mediator of apoptotic cell clearance. In addition, sFKN induces the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) in microglia in the absence of neurotoxic molecule production, including NO, TNF, and glutamate. sFKN treatment of primary neuron-microglia co-cultures significantly attenuated glutamate-induced neuronal cell death. Using several specific MAPK inhibitors, we found that sFKN-induced heme oxygenase-1 expression was primarily mediated by activation of JNK and nuclear factor erythroid 2-related factor 2. These results suggest that sFKN secreted from glutamate-damaged neurons provides both phagocytotic and neuroprotective signals.     

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.     

Role of sodium/hydrogen exchanger isoform 1 in microglial activation and proinflammatory responses in ischemic brains

Our recent study reveals that Na?/H? exchanger isoform 1 (NHE-1) mediates H? extrusion during "respiratory bursting", which is important for microglial activation. In the present study, we further investigated whether NHE-1 plays a role in proinflammatory activation of microglia in vivo using a mouse model of transient focal cerebral ischemia and reperfusion (I/R). Activated microglial cells were identified by their expression of two microglial marker proteins (CD11b and Iba1) as well as by their transformation from a "ramified" to an "amoeboid" morphology. An immediate increase in activated microglial numbers was detected in the ipsilateral ischemic core area of NHE-1?/? brains at 1 hour (h) I/1 h R, which gradually decreased during 6-24 h I/R. This was followed by a sharp rise in microglial activation in the peri-infarct area and an increase in proinflammatory cytokine formation at 3 day after I/R. Interestingly, HOE 642 (a potent NHE-1 inhibitor) -treated or NHE-1 heterozygous (NHE-1?/?) mice exhibited less microglia activation, less NADPH oxidase activation, or a reduced proinflammatory response at 3-7 day after I/R. Blocking NHE-1 activity also significantly decreased microglial phagocytosis in vitro. In contrast, astrogliosis formation in the peri-infarct area was not affected by NHE-1 inhibition. Taken together, our results demonstrate that NHE-1 protein was abundantly expressed in activated microglia and astrocytes. NHE-1 inhibition reduced microglial proinflammatory activation following ischemia.     

Glial cell responses, complement and apolipoprotein J expression following axon injury in the neonatal rat

Immature motoneurons are highly susceptible to degeneration following axon injury. The response of perineuronal glia to axon injury may significantly influence neuronal survival and axon regeneration. We have examined the central reactions to neonatal facial nerve transection with emphasis on the expression of complement component C3 (C3) and the multifunctional apolipoprotein J (ApoJ). Axotomy was performed on one-day-old rats. Animals were perfused from eight hours to two weeks after the lesion. The astroglial marker, glial fibrillary acidic protein (GFAP) was increased from one day and the microglial marker OX-42 from two days after injury. ApoJ immunoreactivity was increased in axotomized neuronal perikarya and astroglial cells from one day postaxotomy, but no C3 immunoreactive profiles were found at any postoperative survival time. Cell proliferation as judged by bromodeoxyuridine labeling and immunoreactivity for the cyclin Ki-67 antigen (antibody MIB5) occurred only at two days after injury. Double immunostaining revealed that the vast majority of proliferating cells were microglia, although occasional cells double labeled astrocytes were found as well. Our results indicate that the non-neuronal response in neonatal animals differ from that of adult ones as follows: 1) microglia transform rapidly into phagocytes in parallel with the degeneration of axotomized neurons, 2) despite the presence of neuronal degeneration, no expression of C3 was found, and the upregulation of the expression of the complement C3 receptor (CR3) is delayed, 3) ApoJ is strongly upregulated in perineuronal astrocytes as well as in the axotomized motoneurons. The marked upregulation of ApoJ in both instances suggests a general role of this protein in the neuronal response to axotomy.