Phenotypic characterisation of cell populations in the brains of horses experimentally infected with West Nile virus.

BACKGROUND: West Nile virus (WNV), a mosquito borne member of the Flaviviridae, is one of the most commonly diagnosed agents of viral encephalitis in horses and people worldwide. OBJECTIVES: A cassette of markers for formalin-fixed paraffin-embedded tissue and an archive of tissues from experimental infections in the horse were used to investigate the equine neuroimmune response to WNV meningoencephalomyelitis to phenotype the early response to WNV infection in the horse. STUDY DESIGN: Quantitative analysis using archived tissue from experimentally infected horses. METHODS: The thalamus and hindbrain from 2 groups of 6 horses were compared and consisted of a culture positive tissues from WNV experimentally horses, in the other, normal horses. Formalin-fixed paraffin-embedded tissue from the thalamus and hindbrain were immunolabeled for microglia, astrocytes, B cells, macrophages/neutrophils, CD3+ T cells. Fresh frozen tissues were immunolabeled for CD4+ and CD8+ T lymphocyte cell markers. Cell counts were obtained using a computer software program. Differences, after meeting assumptions of abnormality, were computed using a general linear model with a Tukey test (P<0.05) for pairwise comparisons. RESULTS: In WNV-challenged horses, Iba-1+ microglia, CD3+ T lymphocyte and MAC387+ macrophage staining were significantly increased. The T cell response for the WNV-challenged horses was mixed, composed of CD4+ and CD8+ T lymphocytes. A limited astrocyte response was also observed in WNV-challenged horses, and MAC387+ and B cells were the least abundant cell populations. MAIN LIMITATIONS: The results of this study were limited by a single collection time post-infection. Furthermore, a comprehensive analysis of cellular phenotypes is needed for naturally infected horses. Unfortunately, in clinical horses, there is high variability of sampling in terms of days post-infection and tissue handling. CONCLUSIONS: The data show that WNV-challenged horses recruit a mixed T cell population at the onset of neurologic disease.

In situ characterization and mapping of iron compounds in Alzheimer's disease tissue.

There is a well-established link between iron overload in the brain and pathology associated with neurodegeneration in a variety of disorders such as Alzheimer's (AD), Parkinson's (PD) and Huntington's (HD) diseases [1]. This association was first discovered in AD by Goodman in 1953 [2], where, in addition to abnormally high concentrations of iron in autopsy brain tissue, iron has also been shown to accumulate at sites of brain pathology such as senile plaques [3]. However, since this discovery, progress in understanding the origin, role and nature of iron compounds associated with neurodegeneration has been slow. Here we report, for the first time, the location and characterisation of iron compounds in human AD brain tissue sections. Iron fluorescence was mapped over a frontal-lobe tissue section from an Alzheimer's patient, and anomalous iron concentrations were identified using synchrotron X-ray absorption techniques at 5 mum spatial resolution. Concentrations of ferritin and magnetite, a magnetic iron oxide potentially indicating disrupted brain-iron metabolism, were evident. These results demonstrate a practical means of correlating iron compounds and disease pathology in-situ and have clear implications for disease pathogenesis and potential therapies.

Induction of manganese superoxide dismutase in acute spinal cord injury.

Free radical-mediated mechanisms of cellular damage have been implicated in the early stages of spinal cord injury (SCI). Manganese superoxide dismutase (MnSOD) is a potent scavenger of superoxide radicals and likely serves an important cytoprotective role in preventing cellular damage after SCI. We have evaluated the expression of MnSOD to address its role during the early events of SCI using a well-established rat contusion model. Northern analysis showed a rapid induction of MnSOD mRNA between 2 and 6 h post injury. Observed time-dependent increases in MnSOD message was maximal 6 h post injury over that of MnSOD mRNA levels induced by laminectomy alone. Immunoblot and immunohistochemical analysis demonstrated increased expression of MnSOD protein 24 h after SCI with localization primarily within neurons. Interestingly, laminectomy alone also caused an induction of MnSOD gene and protein expression. To evaluate one potential mechanism of MnSOD induction, we microinjected the naive spinal cord with IL-1beta, which caused a similar fold induction of MnSOD mRNA levels by 6 h as observed with SCI, thus implicating it as a potential inducer of MnSOD during SCI. In summary, these results demonstrate that this potent cytoprotective antioxidant enzyme is rapidly and significantly induced as a consequence of SCI.

Microglia and macrophages in the developing CNS.

An understanding of microglial functions during normal CNS development is prerequisite for understanding developmental neurotoxicology. This review provides a brief summary of previous work regarding the origin of microglia and addresses differences and similarities between microglia and brain macrophages. Current concepts and ideas which implicate microglia in diverse developmental processes, such as apoptosis, axon growth, and vasculogenesis are discussed. The study of reactive microgliosis may prove useful in the histopathological analysis of neurotoxicant-induced brain damage during development.

Chemokines and Alzheimer's disease.

In recent years, increasing attention has been focused on chemokines as inflammatory mediators in the CNS. The limited number of studies that have investigated chemokine and chemokine receptor expression in Alzheimer's disease (AD) brain and in cell culture models seem to support a role for inflammation in AD pathogenesis. Here we provide a review of these studies, but in addition, point out the possible role of chemokines as communication molecules between neurons and microglia. Understanding neuron-microglia interactions is essential for understanding AD pathogenesis, and disturbances in chemokine-mediated intercellular communication may contribute toward a generalized impairment of microglial cell function.

Down-regulation of the macrophage lineage through interaction with OX2 (CD200).

OX2 (CD200) is a broadly expressed membrane glycoprotein, shown here to be important for regulation of the macrophage lineage. In mice lacking CD200, macrophage lineage cells, including brain microglia, exhibited an activated phenotype and were more numerous. Upon facial nerve transection, damaged CD200-deficient neurons elicited an accelerated microglial response. Lack of CD200 resulted in a more rapid onset of experimental autoimmune encephalomyelitis (EAE). Outside the brain, disruption of CD200-CD200 receptor interaction precipitated susceptibility to collagen-induced arthritis (CIA) in mice normally resistant to this disease. Thus, in diverse tissues OX2 delivers an inhibitory signal for the macrophage lineage.

Facial motor neuron regeneration induces a unique spatial and temporal pattern of myristoylated alanine-rich C kinase substrate expression.

We have previously shown that the myristoylated alanine-rich C kinase substrate, a primary protein kinase C substrate in brain that binds and cross-links filamentous actin, is enriched in neuronal growth cones and is developmentally regulated in brain. Here we examined myristoylated alanine-rich C kinase substrate expression in the facial motor nucleus during axonal regeneration following facial nerve axotomy or facial nerve resection lesions, which impede regeneration, or following motor neuron degeneration induced by the retrograde neurotoxin ricin. For comparative purposes, the protein kinase C substrates myristoylated alanine-rich C kinase substrate-like protein and growth-associated protein-43 were examined in parallel. Myristoylated alanine-rich C kinase substrate messenger RNA exhibited a robust increase in both neurons and non-neuronal cells in the facial motor nucleus beginning four days after axotomy, peaked at seven days (2.5-fold), and declined back to baseline levels by 40 days. Myristoylated alanine-rich C kinase substrate protein similarly exhibited a twofold elevation in the facial motor nucleus determined four and 14 days post-axotomy. Following nerve resection, myristoylated alanine-rich C kinase substrate messenger RNA levels increased at seven days and returned to baseline levels by 40 days. Unlike myristoylated alanine-rich C kinase substrate messenger RNA, myristoylated alanine-rich C kinase substrate-like messenger RNA levels did not increase in the facial motor nucleus at any time point following nerve axotomy or resection, whereas growth-associated protein-43 messenger RNA exhibited a rapid (one day) and prolonged (40 days) elevation in facial motor nucleus neurons following either nerve axotomy or resection. Ricin-induced degeneration of facial motor neurons elevated myristoylated alanine-rich C kinase substrate and myristoylated alanine-rich C kinase substrate-like messenger RNAs in both microglia (lectin-positive) and astrocytes (glial fibrillary acidic protein-positive).Collectively, these data demonstrate that myristoylated alanine-rich C kinase substrate exhibits a unique expression profile in the facial motor nucleus following facial nerve lesions, and it is proposed that myristoylated alanine-rich C kinase substrate may serve to mediate actin-membrane cytoskeletal plasticity in both neurons and glial cells in response to protein kinaseC-mediated signaling during nerve regeneration and degeneration.

Comparative evaluation of cytokine profiles and reactive gliosis supports a critical role for interleukin-6 in neuron-glia signaling during regeneration.

Using reverse transcription polymerase chain reaction (RT-PCR), we have studied the temporal expression of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNAs in three axotomy paradigms with distinct functional outcomes. Axotomy of adult rat facial motoneurons results in neuronal regeneration, axotomy of neonatal facial motoneurons results in neuronal apoptosis, and axotomy of rubrospinal neurons results in neuronal atrophy. Our RT-PCR findings show that a significant and sustained upregulation of IL-6 mRNA is associated uniquely with the regeneration of adult facial motoneurons. Histochemical studies using IL-6 immunohistochemistry show intense IL-6 immunoreactivity in axotomized adult facial motoneurons. Assessment of reactive glial changes with astroglial and microglial markers reveals that the reactive gliosis following adult facial nerve axotomy is more intense than that observed in either of the other two paradigms. Exposure of cultured microglial cells to IL-6 stimulates microglial proliferation in a dose-dependent manner. Cultured microglia also show expression of IL-6 receptor mRNA, as determined by RT-PCR. Our findings support the idea that reactive gliosis is required for neuron regeneration to occur, and more specifically, they suggest that neuron-derived IL-6 serves as a signalling molecule that induces microglial proliferation during motoneuron regeneration.

Interleukin-2 gene deletion produces a robust reduction in susceptibility to experimental autoimmune encephalomyelitis in C57BL/6 mice.

Dysregulation of interleukin-2 (IL-2), the prototypical T cell growth factor and immunoregulatory cytokine, may modify self-tolerance and predisposition to autoimmunity. The available literature suggested that IL-2 could be hypothesized to either propagate or inhibit the development autoimmune demyelinating disorders of the central nervous system such as multiple sclerosis. Thus, the present study sought to test these competing hypotheses by examining whether disrupting one or both IL-2 gene alleles would render mice more or less vulnerable to experimental autoimmune encephalomyelitis (EAE). Myelin oligodendrocyte glycoprotein was used to induce EAE in C57BL/6-IL-2(-/-) knockout, C57BL/6-IL-2(+/-) heterozygote and C57BL/6-IL-2(+/+) wild-type mice. All of the wild-type and heterozygote mice developed signs of EAE compared with only 23% of the IL-2 knockout mice. Histopathological examination of lumbar spinal cord sections confirmed that subpial perivascular inflammatory infiltrates found in wild-type and heterozygote mice were absent in the unaffected IL-2 knockout mice. These data demonstrate that vulnerability to EAE is markedly reduced in C57BL/6 mice lacking IL-2, and suggest that this cytokine may play a critical role in autoimmune processes of the central nervous system.

The induction of EAE is only partially dependent on TNF receptor signaling but requires the IL-1 type I receptor.

Experimental autoimmune encephalomyelitis develops in mice immunized with CNS antigens. To elucidate the role that specific proinflammatory cytokines play in the induction of this process we examined the development of EAE in mice with targeted disruptions of the TNF p55 or p75 or the IL-1 p80 receptors. EAE developed in mice with either one or both TNF receptors deleted although the onset of disease in mice with the p55 receptor deleted was delayed. However, mice with a deletion of the IL-1 p80 receptor failed to develop any inflammatory lesions in the CNS or evidence of clinical EAE. Thus we conclude that TNF or its receptors contribute to, but are not necessary for, the induction of EAE while the IL-1 p80 receptor is absolutely required. The p55 TNF receptor plays a role in determining the onset of disease and its severity.

Microglial response to brain injury: a brief synopsis.

In addition to astrocytes and oligodendrocytes, microglia represent the third major population of glial cells within the central nervous system (CNS). Microglia are distributed ubiquitously throughout the brain and spinal cord, and one of their main functions is to monitor and sustain neuronal health. Microglial cells are quite sensitive to even minor disturbances in CNS homeostasis, and they become readily activated during most neuropathologic conditions, including peripheral nerve injury, trauma and stroke, inflammatory disease, and neurotoxicant-induced neuronal injury. During activation, microglia display conspicuous functional plasticity, which involves changes in cell morphology, cell number, cell surface receptor expression, and production of growth factors and cytokines. The many changes occurring in activated cells reflect the altered functional states of microglia that are induced by signals arising from injured neurons. Thus, neuronal-microglial signaling plays a fundamental role in understanding how the CNS responds to injury. Reactive microgliosis should be viewed as a cellular effort to initiate ameliorative and reparative measures in the injured brain.

Characterization of the chicken GCAP gene array and analyses of GCAP1, GCAP2, and GC1 gene expression in normal and rd chicken pineal.

PURPOSE: This study had three objectives: (1) to characterize the structures of the chicken GCAP1 and GCAP2 genes; (2) to determine if GCAP1, GCAP2, and GC1 genes are expressed in chicken pineal gland; (3) if GC1 is expressed in chicken pineal, to determine if the GC1 null mutation carried by the retinal degeneration (rd) chicken is associated with degenerative changes within the pineal glands of these animals. METHODS: GCAP1 and GCAP2 gene structures were determined by analyses of chicken cosmid and cDNA clones. The putative transcription start points for these genes were determined using 5'-RACE. GCAP1, GCAP2 and GC1 transcripts were analyzed using Northern blot and RT-PCR. Routine light microscopy was used to examine pineal morphology. RESULTS: Chicken GCAP1 and GCAP2 genes are arranged in a tail-to-tail array. Each protein is encoded by 4 exons that are interrupted by 3 introns of variable length, the positions of which are identical within each gene. The putative transcription start points for GCAP1 and GCAP2 are 314 and 243 bases upstream of the translation start codons of these genes, respectively. As in retina, GCAP1, GCAP2 and GC1 genes are expressed in the chicken pineal. Although the GC1 null mutation is present in both the retina and pineal of the rd chicken, only the retina appears to undergo degeneration. CONCLUSIONS: The identical arrangement of chicken, human, and mouse GCAP1/2 genes suggests that these genes originated from an ancient gene duplication/inversion event that occurred during evolution prior to vertebrate diversification. The expression of GC1, GCAP1, and GCAP2 in chicken pineal is consistent with the hypothesis that chicken pineal contains a functional phototransduction cascade. The absence of cellular degeneration in the rd pineal gland suggests that GC1 is not critical for pineal cell survival.

Glial cells in neurotoxicity development.

Neuroglial cells of the central nervous system include the astrocytes, oligodendrocytes, and microglia. Their counterparts in the peripheral nervous system are the Schwann cells. The term neuroglia comes from an erroneous concept originally coined by Virchow (1850), in which he envisioned the neurons to be embedded in a layer of connective tissue. The term, or its shortened form--glia, has persisted as the preferred generic term for these cells. A reciprocal relationship exists between neurons and glia, and this association is vital for mutual differentiation, development, and functioning of these cell types. Therefore, perturbations in glial cell function, as well as glial metabolism of chemicals to active intermediates, can lead to neuronal dysfunction. The purpose of this review is to explore neuroglial sites of neurotoxicant actions, discuss potential mechanisms of glial-induced or glial-mediated central nervous system and peripheral nervous system damage, and review the role of glial cells in neurotoxicity development.

Reactive microgliosis.

Damage to the central nervous system (CNS) elicits the activation of both astrocytes and microglia. This review is focused on the principal features that characterize the activation of microglia after CNS injury. It provides a critical discussion of concepts regarding microglial biology that include the relationship between microglia and macrophages, as well as the role of microglia as immunocompetent cells of the CNS. Mechanistic and functional aspects of microgliosis are discussed primarily in the context of microglial neuronal interactions. The controversial issue of whether reactive microgliosis is a beneficial or a harmful process is addressed, and a resolution of this dilemma is offered by suggesting different interpretations of the term 'activated microglia' depending on its usage during in vivo or in vitro experimentation.

Cytokine transcripts expressed by microglia in vitro are not expressed by ameboid microglia of the developing rat central nervous system.

Because of morphological similarities between ameboid microglia in the developing central nervous system (CNS), brain macrophages in the injured CNS, and cultured microglia in vitro, it is thought that these cell types are functionally equivalent. To investigate the validity of this assumption, we have compared mRNA levels of interleukin-1alpha and -1beta (IL-1alpha and IL-1beta), tumor necrosis factor-alpha and -beta (TNF-alpha and TNF-beta), transforming growth factor-beta1 (TGF-beta1), and macrophage colony-stimulating factor (M-CSF) in the postnatal day 4 (P4) supraventricular corpus callosum (SVCC) with those in unstimulated cultured microglia. Control tissues included spleen, cortex, hippocampus, and cerebellum. Our analyses have shown that while IL-1alpha, IL-1beta, TNF-alpha, TNF-beta, and TGF-beta1 transcripts are abundantly expressed by cultured microglia, they are very low to virtually undetectable in the SVCC. These data strongly suggest that ameboid microglia, which are concentrated in the SVCC, are unlikely to be a significant source of these cytokines. Our study, which shows clear differences in the functional status of cultured microglia vs. ameboid microglia in vivo, stresses the importance of using caution when interpreting in vitro findings in terms of the in vivo functions of microglia.

Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia.

A recently identified chemokine, fractalkine, is a member of the chemokine gene family, which consists principally of secreted, proinflammatory molecules. Fractalkine is distinguished structurally by the presence of a CX3C motif as well as transmembrane spanning and mucin-like domains and shows atypical constitutive expression in a number of nonhematopoietic tissues, including brain. We undertook an extensive characterization of this chemokine and its receptor CX3CR1 in the brain to gain insights into use of chemokine-dependent systems in the central nervous system. Expression of fractalkine in rat brain was found to be widespread and localized principally to neurons. Recombinant rat CX3CR1, as expressed in Chinese hamster ovary cells, specifically bound fractalkine and signaled in the presence of either membrane-anchored or soluble forms of fractalkine protein. Fractalkine stimulated chemotaxis and elevated intracellular calcium levels of microglia; these responses were blocked by anti-CX3CR1 antibodies. After facial motor nerve axotomy, dramatic changes in the levels of CX3CR1 and fractalkine in the facial nucleus were evident. These included increases in the number and perineuronal location of CX3CR1-expressing microglia, decreased levels of motor neuron-expressed fractalkine mRNA, and an alteration in the forms of fractalkine protein expressed. These data describe mechanisms of cellular communication between neurons and microglia, involving fractalkine and CX3CR1, which occur in both normal and pathological states of the central nervous system.

Chemokine receptor expression in cultured glia and rat experimental allergic encephalomyelitis.

Chemokines are a group of pro-inflammatory peptides that mediate leukocyte migration and activation. Several members of the chemokine family have been shown to be synthesized by cells of the central nervous system (CNS). To begin to address the role of chemokine receptors in CNS physiology, we identified, by molecular cloning techniques, the rat orthologs of the chemokine receptors, CCR2, CCR3, CCR5, and CXCR4. CCR2 and CCR5 expression was detected in rat spleen, lung, kidney, thymus and macrophages; CCR5 mRNA was also detected in rat brain. Primary cultures of rat microglia expressed CCR5 mRNA that was regulated by IFN-gamma, while both cultured astrocytes and microglia were found to contain mRNA for CXCR4 and CX3CR1. Induction of experimental allergic encephalomyelitis (EAE) in the rat was accompanied by increased levels of CCR2, CCR5, CXCR4, and CX3CR1 mRNAs in the lumbar spinal cords of animals displaying clinical signs of the disease. These data identify the rat orthologs of chemokine receptors and demonstrate that brain, spinal cord, and cultured glial cells express chemokine receptors that can be regulated both in vitro and in vivo.

IFN-tau suppresses both the autoreactive humoral and cellular immune responses and induces stable remission in mice with chronic experimental allergic encephalomyelitis.

We have previously shown that interferon-tau (IFN-tau) pretreatment inhibits the development of both acute and chronic mouse experimental allergic encephalomyelitis (EAE), an animal model for the human demyelinating disease multiple sclerosis (MS). IFN-tau is a type I IFN that has pregnancy recognition hormone activity in ruminants. Here we show that IFN-tau induced remission in SJL/J mice that had ongoing chronic active EAE disease and protected mice against secondary relapses. IFN-tau treatment reversed lymphocyte infiltration and microglial activation in the central nervous system. Mice that were treated with IFN-tau had lower levels of anti-MBP antibodies than untreated mice in both chronic and acute forms of EAE. MBP induced proliferation in B cells from EAE mice, but treatment with IFN-tau either in vivo or in vitro blocked activation. Furthermore, IFN-tau inhibited MBP activation of T cells from EAE mice. Thus, IFN-tau inhibits the humoral arm as well as the cellular arm of the autoimmune disease EAE. The data presented here show that IFN-tau inhibits both B cell and T cell responses in EAE as well as active, chronic EAE, and this may help explain the effectiveness of type I IFNs in treatment of MS.