The role of different strain backgrounds in bacterial endotoxin-mediated sensitization to neonatal hypoxic-ischemic brain damage.

Genetic background is known to influence the outcome in mouse models of human disease, and previous experimental studies have shown strain variability in the neonatal mouse model of hypoxia-ischemia. To further map out this variability, we compared five commonly used mouse strains: C57BL/6, 129SVJ, BALB/c, CD1 and FVB in a pure hypoxic-ischemic setup and following pre-sensitization with lipopolysaccharide (LPS). Postnatal day 7 pups were subjected to unilateral carotid artery occlusion followed by continuous 30 min 8% oxygen exposure at 36 °C. Twelve hours prior, a third of the pups received a single intraperitoneal LPS (0.6 µg/g) or a saline (vehicle) administration, respectively; a further third underwent hypoxia-ischemia alone without preceding injection. Both C57BL/6 and 129SVJ strains showed minimal response to 30min hypoxia-ischemia alone, BALB/c demonstrated a moderate response, and both CD1 and FVB revealed the highest brain damage. LPS pre-sensitization led to substantial increase in overall brain infarction, microglial and astrocyte response and cell death in four of the five strains, with exception of BALB/c that only showed a significant effect with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Saline administration prior to hypoxia-ischemia resulted in an increase in inflammatory-associated markers, particularly in the astroglial activation of C57BL/6 mice, and in combined microglial activation and neuronal cell loss in FVB mice. Finally, two of the four strongly affected strains--C57BL/6 and CD1--revealed pronounced contralateral astrogliosis with a neuroanatomical localization similar to that observed on the occluded hemisphere. Overall, the current findings demonstrate strain differences in response to hypoxia-ischemia alone, to stress associated with vehicle injection, and to LPS-mediated pre-sensitization, which partially explains the high variability seen in the neonatal mouse models of hypoxia-ischemia. These results can be useful in future studies of fetal/neonatal response to inflammation and reduced oxygen-blood supply.

Efficient gene delivery to the adult and fetal CNS using pseudotyped non-integrating lentiviral vectors.

Non-integrating lentiviral vectors show considerable promise for gene therapy applications as they persist as long-term episomes in non-dividing cells and diminish risks of insertional mutagenesis. In this study, non-integrating lentiviral vectors were evaluated for their use in the adult and fetal central nervous system of rodents. Vectors differentially pseudotyped with vesicular stomatitis virus, rabies and baculoviral envelope proteins allowed targeting of varied cell populations. Efficient gene delivery to discrete areas of the brain and spinal cord was observed following stereotactic administration. Furthermore, after direct in utero administration (E14), sustained and strong expression was observed 4 months into adulthood. Quantification of transduction and viral copy number was comparable when using non-integrating lentivirus and conventional integrating vector. These data support the use of non-integrating lentiviral vectors as an effective alternative to their integrating counterparts in gene therapy applications, and highlight their potential for treatment of inherited and acquired neurological disorders.

Deletion of the mouse RegIIIbeta (Reg2) gene disrupts ciliary neurotrophic factor signaling and delays myelination of mouse cranial motor neurons.

A large number of cytokines and growth factors support the development and subsequent maintenance of postnatal motor neurons. RegIIIbeta, also known as Reg2 in rat and HIP/PAP1 in humans, is a member of a family of growth factors found in many areas of the body and previously shown to play an important role in both the development and regeneration of subsets of motor neurons. It has been suggested that RegIIIbeta expressed by motor neurons is both an obligatory intermediate in the downstream signaling of the leukemia inhibitory factor/ciliary neurotrophic factor (CNTF) family of cytokines, maintaining the integrity of motor neurons during development, as well as a powerful influence on Schwann cell growth during regeneration of the peripheral nerve. Here we report that in mice with a deletion of the RegIIIbeta gene, motor neuron survival was unaffected up to 28 weeks after birth. However, there was no CNTF-mediated rescue of neonatal facial motor neurons after axotomy in KO animals when compared with wild-type. In mice, RegIIIbeta positive motor neurons are concentrated in cranial motor nuclei that are involved in the patterning of swallowing and suckling. We found that suckling was impaired in RegIIIbeta KO mice and correlated this with a significant delay in myelination of the hypoglossal nerve. In summary, we propose that RegIIIbeta has an important role to play in the developmental fine-tuning of neonatal motor behaviors mediating the response to peripherally derived cytokines and growth factors and regulating the myelination of motor axons.

The injury response of oligodendrocyte precursor cells is induced by platelets, macrophages and inflammation-associated cytokines.

Oligodendrocyte precursor cells recognized with the NG2 antibody respond rapidly to CNS injuries with hypertrophy and upregulation of the NG2 chondroitin sulfate proteoglycan within 24 h. These cells participate in glial scar formation, remaining around the injury site for several weeks. After injury, reactive oligodendrocyte precursor cells increase their production of several chondroitin sulfate proteoglycans, including NG2: this cell type thus represents a component of the inhibitory environment that prevents regeneration of axons in the injured CNS. This study analyzes factors that activate oligodendrocyte precursor cells. Both microglia and astrocytes become reactive around motor neurons following peripheral nerve lesions. We show that oligodendrocyte precursor cells do not hypertrophy or increase NG2 levels after these lesions. Those lesions that cause an oligodendrocyte precursor cell reaction generally open the blood-brain barrier. We therefore opened the blood-brain barrier with microinjections of vascular endothelial growth factor or lipopolysaccharide to the rat and mouse brain, and examined oligodendrocyte precursor cell reactivity after 24 h. Both treatments led to increases in NG2 and hypertrophy of oligodendrocyte precursor cells. Of directly injected blood components serum and thrombin were without effect, while platelets and macrophages activated oligodendrocyte precursor cells. We tested the effects of a range of injury-related cytokines, of which tumor necrosis factor alpha; interleukin-1; transforming growth factor beta; interferon gamma had effects on oligodendrocyte precursor cells. Oligodendrocyte precursor cell chemokines, and mitogens did not increase NG2 levels.

Major histocompatibility complex (MHC2+) perivascular macrophages in the axotomized facial motor nucleus are regulated by receptors for interferon-gamma (IFNgamma) and tumor necrosis factor (TNF).

The major histocompatibility complex (MHC) glycoproteins, MHC1 and MHC2, play a key role in the presentation of antigen and the development of the immune response. In the current study we examined the regulation of the MHC2 in the mouse brain after facial axotomy. The normal facial motor nucleus showed very few slender and elongated MHC2+ cells. Transection of the facial nerve led to a gradual but strong upregulation in the number of MHC2+ cells, beginning at day 2 and reaching a maximum 14 days after axotomy, correlated with the induction of mRNA for tumor necrosis factor (TNF) alpha, interleukin (IL) 1beta and interferon-gamma (IFNgamma) and a peak in neuronal cell death. In almost all cases, MHC2 immunoreactivity was restricted to perivascular macrophages that colocalized with vascular basement membrane laminin and macrophage IBA1-immunoreactivity, with no immunoreactivity on phagocytic microglia, astrocytes or invading T-cells. Heterologous transplantation and systemic injection of endotoxin or IFNgamma did not affect this perivascular MHC2 immunoreactivity, and transgenic deletion of the IL1 receptor type I, or TNF receptor type 1, also had no effect. However, the deletion of IFNgamma receptor subunit 1 caused a significant increase, and that of TNF receptor type 2 a strong reduction in the number of MHC2+ macrophages, pointing to a counter-regulatory role of IFNgamma and TNFalpha in the immune surveillance of the injured nervous system.

B7.2 on activated and phagocytic microglia in the facial axotomy model: regulation by interleukin-1 receptor type 1, tumor necrosis factor receptors 1 and 2 and endotoxin.

Co-stimulatory factors are involved in different forms of brain pathology and play an important role in the activation of T-cells. In the current study, we explored the regulation of B7.2, a prominent member of the B7 family of costimulatory factors, in the facial motor nucleus (FMN) following facial axotomy and systemic application of lipopolysaccharide (LPS, endotoxin) using light and electron immunohistochemistry and cytokine-receptor-deficient mice. Facial axotomy led to a gradual increase of B7.2 immunoreactivity (IR) on microglial cell surface; similar effects were also observed following application of LPS, but both effects were not additive, suggesting overlapping or saturated signaling pathways. Some B7.2-IR was already present on activated microglia surrounding injured neurons at days 1-4 after injury, but became particularly intense during neuronal cell death, peaking at day 14. Previous studies revealed that these late microglial changes are accompanied by a strong increase in the expression of proinflammatory cytokines such as interleukin-1 beta (IL1beta) tumor necrosis factor-alpha (TNFalpha) and interferon gamma (IFNgamma) [J. Neurosci. 18 (1998a) 5804]. Here, deletion of the receptors for these cytokines-IL1R1, TNFR1 or TNFR2, but not IFNgammaR1-caused a strong and significant reduction in B7.2-IR in reactive microglial cells, compared with their wild type (WT) controls on the same genetic strain background, with a 31% decrease in IL1R1-/- , 39% in TNFR1-/- and 49% in TNFR2-/- mice. These data underscore the significance of IL1beta, TNFalpha and LPS, and their receptors, as potent inflammatory signals that regulate the cellular response in the injured brain as well as the interaction with the rapidly recruited immune system. The broad susceptibility of B7.2 regulation to a wide range of different inflammatory signals also points to its role as a sensor of molecular pathology, and a factor that plays an important accessory role in allowing and shaping the microglia/T-cell interaction in the injured central nervous system.

Microglial major histocompatibility complex glycoprotein-1 in the axotomized facial motor nucleus: regulation and role of tumor necrosis factor receptors 1 and 2.

Presentation of antigen is key to the development of the immune response, mediated by association of antigen with major histocompatibility complex glycoproteins abbreviated as MHC1 and MHC2. In the current study, we examined the regulation of MHC1 in the brain after facial axotomy. The normal facial motor nucleus showed no immunoreactivity for MHC1 (MHC1-IR). Transection of the facial nerve led to a strong and selective up-regulation of MHC1-IR on the microglia in the affected nucleus, beginning at day 2 and reaching a maximum 14 days after axotomy, coinciding with a peak influx of the T lymphocytes that express CD8, the lymphocyte coreceptor for MHC1. Specificity of the MHC1 staining was confirmed in beta2-microglobulin-deficient mice, which lack normal cell surface MHC1-IR. MHC1-IR was particularly strong on phagocytic microglia, induced by delayed neuronal cell death, and correlated with the induction of mRNA for tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and interferon-gamma and the influx of T lymphocytes. Mice with severe combined immunodeficiency (scid), lacking T and B cells, showed an increase in the number of MHC1-positive nodules but no significant effect on overall MHC1-IR. Transgenic deletion of the IL1 receptor type I, or the interferon-gamma receptor type 1 subunit, did not affect the microglial MHC1-IR. However, a combined deletion of TNF receptors 1 and 2 (TNFR1&2-KO) led to a decrease in microglial MHC1-IR and to a striking absence of the phagocytic microglial nodules. Deletion of TNFR2 (p75) did not have an effect; deletion of TNFR1 (p55) reduced the diffuse microglial staining for MHC1-IR but did not abolish the MHC1(+) microglial nodules. In summary, neural injury leads to the induction of MHC1-IR on the activated, phagocytic microglia. This induction of MHC1 precedes the interaction with the immune system, at least in the facial motor nucleus model. Finally, the impaired induction of these molecules, up to now, only in the TNFR-deficient mice underscores the central role of TNF in the immune activation of the injured nervous system.

Connective tissue growth factor: a novel marker of layer VII neurons in the rat cerebral cortex.

Connective tissue growth factor (CTGF) belongs to a family of secreted, extracellular matrix-associated proteins that are involved in the regulation of cellular functions such as adhesion, migration, mitogenesis, differentiation and survival. Recent studies have also suggested the up-regulation of CTGF in response to trauma, scar formation and excitotoxicity in the CNS. To further elucidate the localization and regulation of this molecule in the rat brain we performed in situ hybridization experiments and found a very strong and selective expression of CTGF messenger ribonucleic acid (mRNA) on the band of layer VII neurons throughout the adult cerebral cortex. Similarly strong neuronal expression was also present in the dorsal endopiriform nucleus, extending rostrally from the ventrocaudal cortical layer VII, and in the deep layers of the olfactory glomeruli and the accessory olfactory nucleus. Double in situ hybridization confirmed selective CTGF mRNA expression on a subpopulation (approximately 35%) of microtubule-associated protein 2 mRNA-positive neurons in the cortical layer VII and the dorsal endopiriform nucleus. The nucleus of lateral olfactory tract showed moderate signal intensity; other parts of the forebrain, mesencephalon and brain stem only revealed a very weak level of CTGF mRNA expression. Non-neuronal expression was rare, considerably weaker than on cortical layer VII neurons, and normally associated with blood vessels. Developmental analysis of CTGF mRNA expression in embryonic and postnatal mouse also showed a moderately late onset at embryonic day 16-18, and confirmed the presence of CTGF mRNA in cortical layer VII in a second rodent species. Interestingly, injury experiments using direct cerebral trauma or injection of excitotoxic kainic acid into rat brain failed to up-regulate CTGF mRNA after injury and during the ensuing period of neuronal cell death, gliosis and neural scar tissue formation. Altogether, the current data suggest a constitutive role of CTGF, particularly in the adult cerebral cortex. In view of the strong ascending projections of subplate neurons into cortical layer 1, this molecule may be involved in the modulation of synaptic input to apical dendrites of pyramidal neurons.

Cytotoxic potential of proinflammatory cytokines: combined deletion of TNF receptors TNFR1 and TNFR2 prevents motoneuron cell death after facial axotomy in adult mouse.

Neural injury is known to trigger inflammatory changes, including the synthesis of proinflammatory cytokines such as interleukin-1-beta (IL1beta), tumor necrosis factor-alpha (TNFalpha), and interferon-gamma (IFNgamma) [G. Raivich, L. L. Jones, C. U. A. Kloss, A. Werner, H. Neumann, and G. W. Kreutzberg, 1998, J Neurosci, 18: 5804-5816] that may play a pivotal role in mediating the cellular response in the affected brain tissue. Here we examined the effects of transgenic deletion of receptors for these cytokines on neuronal cell loss in the adult mouse facial motor nucleus after a peripheral, facial nerve cut. Homozygous deletion of IL1 receptor 1 (IL1R1), TNF receptor 1 or 2 (TNFR1 or TNFR2), or IFNgamma receptor 1 (IFNgammaR1) alone had no effect but combined deletion of TNFR1 and TNFR2 caused a striking absence of alphaX beta2 integrin/IBA1-double-labeled, phagocytic microglial nodules in the axotomized facial motor nucleus 14 days after nerve cut. Moreover, this combined deletion also led to an almost complete prevention of cell loss by Day 29. Additional neuronal cell counts at Day 60 revealed a second phase of motoneuron cell disappearance, which did not depend on the presence of TNF receptors. However, there was still the same 22% difference in the total number of motoneurons between the wild-type and TNFR1 & 2-deficient mice, underlining the role of TNF ligands and both TNF receptors in mediating the early phase of neuronal cell loss after traumatic injury.

Systemic LPS injection leads to granulocyte influx into normal and injured brain: effects of ICAM-1 deficiency.

The lipopolysaccharide (LPS) constituents of the gram-negative bacterial wall are among the most potent activators of inflammation. In the current study, we examined the effect of subcutaneous injection of Escherichia coli LPS on leukocyte influx into the normal and injured brain using endogenous peroxidase (EP). Normal brain parenchyma does not contain granulocytes and this does not change after indirect trauma, in facial axotomy. However, systemic injection of 1 mg LPS led to a gradual appearance of EP-positive parenchymal granulocytes within 12 h, with a maximum at 1-4 days after injection. Facial axotomy (day 14) led to a further 50-300% increase in granulocyte number. Of the five mouse strains tested in the current study, four--Balb/C, FVB, C57Bl/6, and C3H/N--showed vigorous granulocyte influx (60-90 cells per 20-microm section in axotomized facial nucleus, 20-40 cells per section on the contralateral side). The influx was an order of magnitude lower in the SJL mice. The peroxidase-positive cells were immunoreactive for neutrophil antigen 7/4 and alpha M beta 2 integrin, were negative for IBA1 (monocytes) and CD3 (T cells), and could be prelabeled by subcutaneous injection with rhodamine B isothiocyanate (RITC), confirming their origin as blood-borne granulocytes. All RITC-positive cells were IBA1 negative. This influx of granulocytes was accompanied by a disruption of the blood-brain barrier to albumin and induction of the cell adhesion molecule ICAM-1 on affected blood vessels. Transgenic deletion of ICAM-1 led to a more than 50% reduction in the number of infiltrating granulocytes compared to litter-matched wild-type controls, in normal brain as well as in axotomized facial motor nucleus. In summary, systemic injection of LPS leads to invasion of granulocytes into the mouse brain and a breakdown of the blood-brain barrier to blood-borne cells and to soluble molecules. Moreover, this mechanism may play a pathogenic role in the etiology of meningitis and in severe bacterial sepsis.

Interleukin-6 (IL6) and cellular response to facial nerve injury: effects on lymphocyte recruitment, early microglial activation and axonal outgrowth in IL6-deficient mice.

Nerve injury triggers numerous changes in the injured neurons and surrounding non-neuronal cells. Of particular interest are molecular signals that play a role in the overall orchestration of this multifaceted cellular response. Here we investigated the function of interleukin-6 (IL6), a multifunctional neurotrophin and cytokine rapidly expressed in the injured nervous system, using the facial axotomy model in IL6-deficient mice and wild-type controls. Transgenic deletion of IL6 caused a massive decrease in the recruitment of CD3-positive T-lymphocytes and early microglial activation during the first 4 days after injury in the axotomized facial nucleus. This was accompanied by a more moderate reduction in peripheral regeneration at day 4, lymphocyte recruitment (day 14) and enhanced perikaryal sprouting (day 14). Motoneuron cell death, phagocytosis by microglial cells and recruitment of granulocytes and macrophages into injured peripheral nerve were not affected. In summary, IL6 lead to a variety of effects on the cellular response to neural trauma. However, the particularly strong actions on lymphocytes and microglia suggest that this cytokine plays a central role in the initiation of immune surveillance in the injured central nervous system.

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.

A mouse model of Streptococcus pneumoniae meningitis mimicking several features of human disease.

The course of bacterial titers, meningeal inflammation, behavioral abnormalities, and neuronal damage was studied in a mouse model of Streptococcus pneumoniae meningitis. At 24 h after injection of 10(4) colony-forming units (CFU) S. pneumoniae into the right forebrain, infected mice became severely lethargic. Bacterial titers in cerebrospinal fluid and cerebellum rose to 10(9) CFU/ml, with strong granulocyte invasion into the meninges and neuronal necroses in the neocortex, striatum and hippocampal formation. Meningeal inflammation and neuronal damage in intercellular cell adhesion molecule-1- and macrophage colony-stimulating factor-deficient mice was similar to that in wild-type littermates. Untreated, the infection was fatal. Wild-type mice treated earlier than 24 h after infection with ceftriaxone (2 mg every 12 h for 3 days) survived without apparent behavioral abnormalities. Delay of treatment beyond 30 h led to the death of more than 50% of the infected mice. This mouse model is suitable for therapeutic studies and for the investigation of inflammation in knockout mice. The neuronal damage resembles morphological abnormalities observed in humans.

Alpha4 integrin is expressed during peripheral nerve regeneration and enhances neurite outgrowth.

We have shown previously that repair in the peripheral nervous system is associated with a reversion to an embryonic pattern of alternative splicing of the extracellular matrix molecule fibronectin. One of the consequent changes is a relative increase in the number of fibronectins expressing the binding site for alpha4 integrins. Here we show that alpha4 integrins are expressed on dorsal root ganglion neuron cell bodies and growth cones in the sciatic nerve during regeneration and that the interaction of alpha4 integrin with alternatively spliced isoforms of recombinant fibronectins containing the alpha4 binding site enhances neurite outgrowth in dorsal root ganglion neurons. The pheochromocytoma (PC12) neuronal cell line, which normally extends neurites poorly on fibronectin, does so efficiently when alpha4 is expressed in the cells. Experiments using chimeric integrins expressed in PC12 cells show that the alpha4 cytoplasmic domain is necessary and sufficient for this enhanced neurite outgrowth. In both dorsal root ganglion neurons and PC12 cells the alpha4 cytoplasmic domain is tightly linked to the intracellular adapter protein paxillin. These experiments suggest an important role for alpha4 integrin and paxillin in peripheral nerve regeneration and show how alternative splicing of fibronectin may provide a mechanism to enhance repair after injury.

Microglia and the early phase of immune surveillance in the axotomized facial motor nucleus: impaired microglial activation and lymphocyte recruitment but no effect on neuronal survival or axonal regeneration in macrophage-colony stimulating factor-deficient mice.

Activation of microglia is among the first cellular changes in the injured CNS. However, little is known about their specific contribution to secondary damage or repair processes in neighboring neurons and nonneuronal cells or to the immune surveillance of the damaged tissue. Animal models with defective microglial response such as osteopetrosis provide an approach to explore these effects. Osteopetrosis (op) is an autosomal recessive mutation with a complete deficiency of the macrophage-colony stimulating factor (MCSF; CSF-1), an important mitogen for brain microglia. In the current study we examined the effects of this MCSF deficiency on the microglial reaction and the overall cellular response to nerve injury in the mouse axotomized facial motor nucleus. In the brain, MCSF receptor immunoreactivity was found only on microglia and was strongly up-regulated following injury. MCSF deficiency led to a failure of microglia to show a normal increase in early activation markers (thrombospondin, MCSF receptor, alpha M beta 2- and alpha 5 beta 1-integrins), to spread on the surface of axotomized motoneurons, and to proliferate after injury. Early recruitment of CD3(+) T-lymphocytes to the facial nucleus 24 hours after injury was reduced by 60%. In contrast, the neuronal and astrocyte response was not affected. There was a normal increase in the neuropeptides calcitonin gene-related peptide and galanin, neuronal c-JUN, and NADPH-diaphorase and a decrease in choline acetyltransferase and acetylcholinesterase. Astrocyte glial fibrillary acidic protein immunoreactivity also showed a normal increase. There was a normal influx of macrophages and granulocytes into the injured facial nerve. Synaptic stripping, neuronal survival, and speed of axonal regeneration were also not affected. The current results show a strong, selective effect of MCSF on the early activation of microglia and, indirectly, on lymphocyte recruitment. This early phase of microglial activation appears not to be involved in the process of repair following peripheral nerve injury. However, it is important in the initiation of inflammatory changes in the brain and in the interaction with the immune system.

In vitro model of microglial deramification: ramified microglia transform into amoeboid phagocytes following addition of brain cell membranes to microglia-astrocyte cocultures.

Changes in the morphology of ramified microglia are a common feature in brain pathology and culminate in the appearance of small, rounded, microglia-derived phagocytes in the presence of neural debris. Here, we explored the effect of adding brain cell membranes on the morphology of alphaMbeta2-integrin (CD11b/CD18, CR3) positive microglia cultured on a confluent astrocyte substrate as an in vitro model of deramification. Addition of brain membranes led to a loss of microglial ramification, with full transformation to small, rounded, macrophages at 20-40 microg/ml. Time course studies showed a rapid response, with first effects at 1-3 hours, and full transformation at 24-48 hours. Removal of cell membranes and exchange of the culture medium led to a similarly rapid process of reramification. Comparison of cell membranes from different tissues at 20 microg/ml showed strong transforming effect for the brain, more moderate for kidney and liver, and very weak for spleen and skeletal muscle. Fluorescent labeling of brain membranes revealed uptake by almost all rounded macrophages, by a subpopulation of glial fibrillary acidic protein (GFAP)-positive astrocytes, but not by ramified microglia. Phagocytosis of inert fluorobeads did not lead to a transformation into macrophages but their phagocytosis was inhibited by brain membranes, pointing to a saturable uptake mechanism. In summary, addition of brain cell membranes and their phagocytosis leads to a rapid and reversible loss of ramification. The differences in transforming activity from different tissues and the absence of effect from phagocytosed fluorobeads suggest, however, the need for a second stimulus following the phagocytosis of cell debris.

The role of macrophages in demyelinating peripheral nervous system of mice heterozygously deficient in p0.

Mice heterozygously deficient in the p0 gene (P0(+/-)) are animal models for some forms of inherited neuropathies. They display a progressive demyelinating phenotype in motor nerves, accompanied by mild infiltration of lymphocytes and increase in macrophages. We have shown previously that the T lymphocytes are instrumental in the demyelination process. This study addresses the functional role of the macrophage in this monogenic myelin disorder. In motor nerves of P0(+/)- mice, the number of macrophages in demyelinated peripheral nerves was increased by a factor of five when compared with motor nerves of wild-type mice. Immunoelectron microscopy, using a specific marker for mouse macrophages, displayed macrophages not only in the endoneurium of the myelin mutants, but also within endoneurial tubes, suggesting an active role in demyelination. To elucidate the roles of the macrophages, we crossbred the myelin mutants with a spontaneous mouse mutant deficient in macrophage colony-stimulating factor (M-CSF), hence displaying impaired macrophage activation. In the P0-deficient double mutants also deficient in M-CSF, the numbers of macrophages were not elevated in the demyelinating motor nerves and demyelination was less severe. These findings demonstrate an active role of macrophages during pathogenesis of inherited demyelination with putative impact on future treatment strategies.

Effect of lipopolysaccharide on the morphology and integrin immunoreactivity of ramified microglia in the mouse brain and in cell culture.

Microglial cells form the first line of defense in brain infection. They are related to monocytes and macrophages and can be readily activated by cell wall components of bacteria such as lipopolysaccharides (LPS). In the present study, we explored the effect of this endotoxin in mouse on the morphology of microglia and their immunoreactivity for the integrin family of cell adhesion molecules in vitro and in vivo. Subcutaneous injection of LPS led to a dose-dependent activation of alpha M beta 2-positive microglia, with a saturating effect at 1 microg LPS in the blood-brain barrier deficient area postrema, at 10 microg in the directly adjacent tissue, and at 100 microg throughout the brainstem and cerebellum. Morphologically, this activation was characterized by the swelling of the microglial cell body, a thickening of the proximal processes, and a reduction in distal ramification. Microglial immunoreactivity for the integrins alpha 4 beta 1, alpha 5 beta 1, alpha 6 beta 1, and alpha M beta 2 was strongly increased. In vitro, ramified microglia were obtained using a coculture on top of a confluent astrocyte monolayer. Two days exposure to LPS resulted in a morphological activation of the cultured cells with an increase of the integrin immunoreactivity for alpha 5 (5.7-fold), alpha 4 (3.1-fold), beta 1 (2.3-fold), and alpha M (1.5-fold), and a decrease in the alpha 6-staining intensity by 39%. Even a sublethal dose of LPS (3 mg in vivo and 500 microg/ml in vitro, respectively) did not induce the phagocyte-associated integrin alpha X beta 2 (CD11c/CD18, p150,95) and did not lead to a morphological transformation of the ramified microglia into phagocytes.

Regulation of the cell adhesion molecule CD44 after nerve transection and direct trauma to the mouse brain.

CD44 is a cell surface glycoprotein involved in cell adhesion during neurite outgrowth, leukocyte homing, and tumor metastasis. In the current study, we examined the regulation of this molecule 4 days after neural trauma in different forms of central and peripheral injury. Transection of the hypoglossal, vagus, or sciatic nerve led to the appearance of CD44-immunoreactivity (CD44-IR) on the surface of the affected motoneurons, their dendrites, and their axons. Fimbria fornix transection led to CD44-IR on a subpopulation of cholinergic neurons in the ipsi- and contralateral medial septum and diagonal band of Broca and colocalized with galanin-IR. Central projections of axotomized sensory neurons to the spinal cord (substantia gelatinosa, Clarke's column) also showed an increase in CD44-IR, which was abolished by spinal root transection. Nonneuronal CD44-IR was mainly restricted to sites of direct injury. In the crushed sciatic nerve, CD44-IR was found on the demyelinating Schwann cells and on infiltrating monocytes and granulocytes. Direct parasagittal transection of the cerebral cortex led to CD44-IR on resident astrocytes and on leukocytes entering the injured forebrain tissue. CD44-IR also increased on reactive retinal astrocytes and microglia after the optic nerve crush. Additional time points in the retina and hypoglossal nucleus (days 1, 2, and 14) and cerebral cortex (day 2) injury models also showed the same cell type pattern for the CD44-IR. Finally, polymerase chain reaction analysis confirmed the posttraumatic expression of CD44 mRNA and detected only the standard haematopoietic CD44 splice isoform both in direct and indirect brain injury models. Overall, the current study shows the widespread, graded appearance of CD44-IR on neurons and on nonneuronal cells, depending on the form of neural injury. Here, the ability of CD44 to bind to a variety of extracellular matrix and cell adhesion proteins and its common presence in different forms of brain pathology could suggest an important role for this cell surface glycoprotein in the neuronal, glial, and leukocyte response to trauma and in the repair of the damaged nervous system.

Impaired axonal regeneration in alpha7 integrin-deficient mice.

The interplay between growing axons and the extracellular substrate is pivotal for directing axonal outgrowth during development and regeneration. Here we show an important role for the neuronal cell adhesion molecule alpha7beta1 integrin during peripheral nerve regeneration. Axotomy led to a strong increase of this integrin on regenerating motor and sensory neurons, but not on the normally nonregenerating CNS neurons. alpha7 and beta1 subunits were present on the axons and their growth cones in the regenerating facial nerve. Transgenic deletion of the alpha7 subunit caused a significant reduction of axonal elongation. The associated delay in the reinnervation of the whiskerpad, a peripheral target of the facial motor neurons, points to an important role for this integrin in the successful execution of axonal regeneration.