Extracellular mutant SOD1 induces microglial‐mediated motoneuron injury

Through undefined mechanisms, dominant mutations in (Cu/Zn) superoxide dismutase‐1 (mSOD1) cause the non‐cell‐autonomous death of motoneurons in inherited amyotrophic lateral sclerosis (ALS). Microgliosis at sites of motoneuron injury is a neuropathological hallmark of ALS. Extracellular mutant SOD1 (mSOD1) causes motoneuron injury and triggers microgliosis in spinal cord cultures, but it is unclear whether the injury results from extracellular mSOD1 directly interacting with motoneurons or is mediated through mSOD1‐activated microglia. To dissociate these potential mSOD1‐mediated neurotoxic mechanisms, the effects of extracellular human mSOD1^G93A or mSOD1^G85R were assayed using primary cultures of motoneurons and microglia. The data demonstrate that exogenous mSOD1^G93A did not cause detectable direct killing of motoneurons. In contrast, mSOD1^G93A or mSOD1^G85R did induce the morphological and functional activation of microglia, increasing their release of pro‐inflammatory cytokines and free radicals. Furthermore, only when microglia was co‐cultured with motoneurons did extracellular mSOD1^G93A injure motoneurons. The microglial activation mediated by mSOD1^G93A was attenuated using toll‐like receptors (TLR) 2, TLR4 and CD14 blocking antibodies, or when microglia lacked CD14 expression. These data suggest that extracellular mSOD1^G93A is not directly toxic to motoneurons but requires microglial activation for toxicity, utilizing CD14 and TLR pathways. This link between mSOD1 and innate immunity may offer novel therapeutic targets in ALS. © 2009 Wiley‐Liss, Inc.     

Inflammatory response associated with axonal injury to spinal motoneurons in newborn rats

Axonal injury in peripheral nerve results in massive motoneuron loss during development. The purpose of this study was to examine the response of phagocytic populations (brain macrophages, BMOs, versus microglia) after different types of axonal lesions (distal axotomy or avulsion) in newborn rats. The morphology, spatial location and activation state of these inflammatory cells were observed. Following spinal root avulsion, BMOs were signaled rapidly and specifically to the location of dying motoneurons in the spinal cord. A large number of BMOs were observed around the avulsed motoneurons on the lesioned side of the spinal cord 1 day following the lesion. These BMOs were large, round, and intensely stained by both antibodies against ED1 and OX-42. The number of BMOs decreased by 3 days and disappeared by 5 days after injury. At the same time, reactive microglia appeared in the lesioned area and rapidly reached the peak level by the 5th day following avulsion. These reactive microglia were medium in size with retracted cellular processes and were also intensely stained by both ED1 and OX-42 antibodies. The number and staining intensity of reactive microglia declined sharply by day 7 after the lesion. In contrast, after distal axotomy only microglia but not BMOs were observed in the lesioned area. These microglial cells were small in size with long and fine-branched processes. They were ED1-negative but OX-42-positive.     

Appearance of phagocytic microglia adjacent to motoneurons in spinal cord tissue from a presymptomatic transgenic rat model of amyotrophic lateral sclerosis

Microglial activation occurs early during the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent evidence indicates that the expression of mutant Cu²⁺/Zn²⁺ superoxide dismutase 1 (SOD1) in microglia contributes to the late disease progression of ALS. However, the mechanism by which microglia influence the neurodegenerative process and disease progression in ALS remains unclear. In this study, we revealed that activated microglia aggregated in the lumbar spinal cord of presymptomatic mutant SOD1^H46R transgenic rats, an animal model of familial ALS. The aggregated microglia expressed a marker of proliferating cell, Ki67, and phagocytic marker proteins ED1 and major histocompatibility complex (MHC) class II. The motoneurons near the microglial aggregates showed weak choline acetyltransferase (ChAT) immunoreactivity and contained reduced granular endoplasmic reticulum and altered nucleus electron microscopically. Furthermore, immunopositive signals for tumor necrosis factor‐α (TNFα) and monocyte chemoattractant protein‐1 (MCP‐1) were localized in the aggregated microglia. These results suggest that the activated and aggregated microglia represent phagocytic features in response to early changes in motoneurons and possibly play an important role in ALS disease progression during the presymptomatic stage. © 2010 Wiley‐Liss, Inc.     

Impairment of microglial responses to facial nerve axotomy in cathepsin S-deficient mice

Cathepsin S (CS) is a lysosomal/endosomal cysteine protease especially expressed in cells of a mononuclear lineage including microglia. To better understand the role of CS in microglia, we investigated microglial responses after a facial nerve axotomy in CS-deficient (CS-/-) and wild-type mice. Microglia in both groups accumulated in the facial motor nucleus following axotomy. However, the mean number of microglia in CS-/- mice on the axotomized side was significantly smaller than that in wild-type mice. Microglia were found to adhere to injured motoneurons in wild-type mice, whereas microglia abutted on injured motoneurons without spreading on their surface in CS-/- mice. At the same time, the axotomy-induced down-regulation of tenasin-R, an antiadhesive perineuronal net for microglia, was partially abrogated in CS-/- mice. Primary cultured microglia prepared from CS-/- mice showed that CS deficiency caused significant suppression of migration and transmigration of microglia. In CS-/- mice, impaired recruitments of circulating monocytes and T lymphocytes and reduced expression of the class II major compatibility complex on the axotomized side were observed. Interestingly, cathepsin B, a typical lysosomal cysteine protease, was markedly expressed on the axotomized side in CS-/- but not in wild-type microglia. Finally, we compared axotomy-induced neuronal death in the two groups and found that the percentage of motoneurons that survived in CS-/- mice was significantly smaller than that in wild-type mice. The present study strongly suggests that CS plays a role in the migration and activation of microglia to protect facial motoneurons against axotomy-induced injury.     

Transformation from a neuroprotective to a neurotoxic microglial phenotype in a mouse model of ALS

Neuroinflammation is a prominent pathological feature in the spinal cords of patients with amyotrophic lateral sclerosis (ALS), as well as in transgenic mouse models of inherited ALS, and is characterized by activated microglia. Earlier studies showed that activated microglia play important roles in both motoneuron protection and injury. More recent studies investigating the pathoprogression of disease in ALS mice have demonstrated that the in vivo activation states of microglia, including their anti- versus pro-inflammatory responses, are best characterized as a continuum between two extreme activation states which are represented as a neuroprotective M2 (alternatively-activated) phenotypic state or an injurious/toxic M1 (classically-activated) state; a more complete understanding and determination the temporal transformation of microglia activation states in the ALS disease pathoprogression is therefore warranted. In the current study, we demonstrated a phenotypic and functional transformation of adult ALS mice microglia that overexpress mutant superoxide dismutase (mSOD1). mSOD1 microglia isolated from ALS mice at disease onset expressed higher levels of Ym1, CD163 and BDNF (markers of M2) mRNA and lower levels of Nox2 (a marker of M1) mRNA compared with mSOD1 microglia isolated from ALS mice at end-stage disease. More importantly, when co-cultured with motoneurons, these mSOD1 M2 microglia were neuroprotective and enhanced motoneuron survival than similarly co-cultured mSOD1 M1 microglia; end-stage mSOD1 M1 microglia were toxic to motoneurons. Our study documents that adult microglia isolated from ALS mice at disease onset have an M2 phenotype and protect motoneurons whereas microglia isolated from end-stage disease ALS mice have adopted an M1 phenotype and are neurotoxic supporting the dual phenotypes of microglia and their transformation during disease pathoprogression in these mice. Thus, harnessing the neuroprotective potential of microglia may provide novel avenues for ALS therapies.     

Glutamate transporter GLT-1 is highly expressed in activated microglia following facial nerve axotomy

Glutamate transporters play an important role in the re-uptake of glutamate after its release from glutamatergic synapses. So far five of such transporters subtypes have been cloned from rodent and human brains. The densities of glutamate transporters are recognised to be developmentally regulated, but the role of glutamate transporters in the mechanisms underlying the occurrence of neuronal traumatic injury has not been widely studied. In the present study quantitative Western blotting and immunohistochemical technique were employed to study the expression of GLT-1/EAAT2 in the facial nuclei of adult rats following unilateral facial nerve axotomy. The total content of GLT-1 protein decreased in the ipsilateral axotomised rat facial nucleus. However, activated microglia surrounding motoneurons showed high expression of GLT-1 after facial nerve axotomy. Parallel studies revealed that primary cultured microglial cells also showed GLT-1-immunoreactivity. To our knowledge, this is the first direct demonstration of the expression of GLT-1 protein in activated microglial cells, suggesting a neuroprotective role of microglia against glutamate excitotoxicity following nerve axotomy.     

Impaired recruitment of neuroprotective microglia and T cells during acute neuronal injury coincides with increased neuronal vulnerability in an amyotrophic lateral sclerosis model

Non-cell-autonomous motor neuronal death is suggested in a mutant Cu/Zn superoxide dismutase 1 (mSOD1)-mediated amyotrophic lateral sclerosis (ALS) model, in which microglia and T cells play significant roles in disease progression. However, it remains unknown whether these cells are toxic or protective. The present study aimed to clarify the developmental age-related alterations of neuronal, glial and T cell responses to acute neuron injury in non-transgenic (N-Tg) mice, and the in vivo effects of mSOD1 on these changes by studying N-Tg and mSOD1-Tg mice subjected to unilateral hypoglossal nerve axotomy at young (8 weeks) and adult (17 weeks) ages. Adult N-Tg mice showed increased neuronal viability on day 21 after axotomy and trends toward increased numbers of recruited microglia on day 3 and T cells on day 7, in the hypoglossal nucleus, compared with young N-Tg mice. Quantitative comparisons between mSOD1-Tg and N-Tg mice at the same ages, on day 3 after axotomy, showed that microglial recruitment was significantly lower in mSOD1-Tg mice than in 17-week-old N-Tg mice (the disease progression stage), but the same difference was not seen in 8-week-old mice (the presymptomatic stage), despite good preservation of hypoglossal neurons. Infiltration of CD3-positive T cells, mostly CD4-positive, on day 7 and the viability rate of hypoglossal neurons on the operated side compared with the contralateral side on day 21 were significantly decreased in mSOD1-Tg mice compared with N-Tg mice aged 17 weeks, but the same difference was not seen in mice aged 8 weeks. On day 3 after axotomy, expression levels of IGF-1 mRNA in the operated hypoglossal nucleus were significantly lower in mSOD1-Tg mice than N-Tg mice at 17 weeks of age. The observation that depressed microglial and T cell responses and expression of neurotrophic factors coincided with reduced neuronal viability in adult mSOD1-Tg mice suggests that diminished neuroprotective functions of mSOD1 microglia and T cells may contribute to exaggerated neuronal death.     

Regulatory T lymphocytes from ALS mice suppress microglia and effector T lymphocytes through different cytokine-mediated mechanisms

Activated microglia and infiltrating lymphocytes are neuropathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motoneuron disease. Although both cell types play pivotal roles in the ALS pathogenic process, the interactions between microglia and lymphocytes, specifically regulatory CD4(+)CD25(High) T lymphocytes (Tregs) and cytotoxic CD4(+)CD25(-) T lymphocytes (Teffs), have not been addressed. When co-cultured with mSOD1 adult microglia, mSOD1 Tregs suppressed the cytotoxic microglial factors NOX2 and iNOS through an IL-4-mediated mechanism, whereas Teffs were only minimally effective; IL-4 inhibitory antibodies blocked the suppressive function of mSOD1 Tregs, and conditioned media from mSOD1 Tregs or the addition of IL-4 reduced microglial NOX2 expression. During the stable disease phase, the total number of Tregs, specifically the numbers of CD4(+)CD25(High)IL-4(+), CD4(+)CD25(High)IL-10(+) and CD4(+)CD25(High)TGF-β(+) Tregs, were increased in ALS mice compared with WT mice; Tregs isolated during this phase reduced Teff proliferation. In contrast, during the rapidly progressing phase, the number of mSOD1 Tregs decreased while the proliferation of mSOD1 Teffs increased. The combination of IL-4, IL-10, and TGF-β was required to inhibit the proliferation of mSOD1 Teffs by mSOD1 Tregs that were isolated during the slow phase, while inhibition of mSOD1 Teffs by mSOD1 Tregs during the rapid phase, as well as WT Teffs, was not dependent on these factors. Thus, mSOD1 Tregs at the slow phase suppressed microglial toxicity and SOD1 Teff proliferation through different mechanisms; microglial activation was suppressed through IL-4 whereas mSOD1 Teffs were suppressed by IL-4, IL-10 and TGF-β. These data suggest that mSOD1 Tregs contribute to the slowly progressing phase in ALS mice and may offer a novel therapeutic option for ALS patients.     

Microglia and microglia-derived brain macrophages in culture: generation from axotomized rat facial nuclei, identification and characterization in vitro

In order to study microglial cells and microglia-derived brain macrophages in vitro, a method has been developed which allows the transfer of mitotic microglial cells from adult rat brain into tissue culture. The studies were performed on facial motor nuclei which were explanted after axotomy of the facial nerve. Outgrowing cells were identified and characterized by (i) morphological criteria using light and electron microscopy, (ii) in vivo [3H]thymidine labeling combined with subsequent in vitro autoradiography, (iii) immunocytochemistry for vimentin, GFAP, Fc and complement receptors, MHC antigens, laminin, fibronectin, factor VIII related- and 04 antigen as well as lectin histochemistry, and (iv) functional in vitro tests. In addition, a microglial cell line was established from proliferating cells. The results indicate that perineuronal microglia rather than astrocytes, perivascular cells, oligodendrocytes or endothelial cells may become phagocytic after having been activated by axotomy in situ.     

Phagocytic microglia during delayed neuronal loss in the facial nucleus of the rat: Time course of the neuronofugal migration of brain macrophages

The injection of Fluoro-Gold (FG) into the whisker pad of rats yields a stable fluorescent labeling of the motoneurons in the lateral facial subnucleus. Following resection of 8–10 mm of the facial nerve, the microglia phagocytose the FG-preloaded neurons and assume the label. Employing this vital labeling of microglia in situ we studied the fate of same after completion of phagocytic activity. Starting at 56 days post resection (DPR) the FG-labeled microglia spread out from the lateral facial subdivision and invaded the entire facial nucleus. The quantitative analysis of this redistribution of the fluorescent marker revealed a prolonged increase in the number of labeled microglia strictly proportional to the delayed loss of neurons. The differentiation between microglia and shrunken neurons was performed with the new method of immunoquenching: the staining of vibratome sections with anti-rat neuron-specific enolase (NSE) combined with an ABC-HRP kit and DAB as detector totally extinguished (quenched) all fluorescence from the pre-labeled facial motoneurons. The fluorescent microglia were additionally stained with GSA I-B₄ and OX–42, which should completely quench all fluorescence in the section. However, a few small round cells, always closely opposed to neuronal perikarya, still fluoresced. These NSE-negative, GSA I-B₄ and OX–42 negative, but fluorescent cells may represent a new, immunologically uncharacterized microglial cell type, that participates in neuronophagia. (c) 1995 Wiley-Liss, Inc.     

Regulation of thrombospondin in the regenerating mouse facial motor nucleus

Thrombospondin (TSP) is a multifunctional extracellular matrix protein that plays a role in neuronal migration and axonal outgrowth in the developing central nervous system. In the current study we have examined the localization and regulation of TSP immunoreactivity (TSP-IR) during neuronal regeneration in the axotomized facial motor nucleus using Western blotting and light and electron microscopy. Transection of the facial nerve led to a gradual increase in TSP-IR in the regenerating motoneurons, peaking 4–7 days after injury (DAI). In addition to regenerating neurons, axotomy also caused a rapid upregulation of TSP-IR on activated microglia throughout the facial nucleus, with a maximum of 2–3 DAI, and a second increase at 14–21 DAI on microglial aggregates surrounding degenerating motoneurons and in neuronophagic microglia. In summary, injury leads to the induction of thrombospondin on axotomized neurons and activated microglia, peaking at the times of maximal posttraumatic microglial proliferation and during neuronal phagocytosis. Since thrombospondin is a multimodal extracellular matrix protein with a variety of cell attachment sites, thrombospondin might serve to link microglia and injured neurons, followed by microglial proliferation and removal of the neuronal debris. © 1996 Wiley-Liss, Inc.     

Origin and distribution of bone marrow-derived cells in the central nervous system in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is associated with increased numbers of microglia within the central nervous system (CNS). However, it is unknown whether the microgliosis results from proliferation of CNS resident microglia, or recruitment of bone marrow (BM)-derived microglial precursors. Here we assess the distribution and number of BM-derived cells in spinal cord using transplantation of green fluorescent protein (GFP)-labeled BM cells into myelo-ablated mice over-expressing human mutant superoxide dismutase 1 (mSOD), a murine model of ALS. Transplantation of GFP+ BM did not affect the rate of disease progression in mSOD mice. Mean numbers of microglia and GFP+ cells in spinal cords of control mice were not significantly different from those in asymptomatic mSOD mice and showed no change with animal age. The number of GFP+ cells and microglia (F4/80+ and CD11b+ cells) within the spinal cord of mSOD mice increased compared to age-matched controls at a time when mSOD mice exhibited disease symptoms, continuing up to disease end-stage. Although we observed an increase in the number of GFP+ cells in spinal cords of mSOD mice with disease symptoms, mean numbers of GFP+ F4/80+ cells comprised less than 20% of all F4/80+ cells and did not increase with disease progression. Furthermore, the relative rates of proliferation in CD45+GFP- and CD45+GFP+ cells were comparable. Thus, we demonstrate that the microgliosis present in spinal cord tissue of mSOD mice is primarily due to an expansion of resident microglia and not to the recruitment of microglial precursors from the circulation.     

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.     

Retrograde response of the rat facial motor nucleus to muscle inflammation elicited by phytohaemagglutinin

To investigate whether motoneurons react to signals deriving from target inflammation, we studied the facial motor nucleus after injections of phytohaemagglutinin in the snout of adult rats. This plant lectin is a tool widely used to induce proliferation and activation of T lymphocytes, and we observed marked lymphocyte infiltration in the injected facial muscles. Retrograde labelling of motoneurons was not detected after peripheral injections of fluorochrome-conjugated phytohaemagglutinin. Nitric oxide synthase, revealed by NADPH-diaphorase histochemistry, OX-42-immunoreactive microglia, and expression of the cell death repressor gene bcl-2, investigated with nonradioactive in situ hybridization and immunohistochemistry, were evaluated in the facial nucleus. Daily phytohaemagglutinin injections for 4 days, mimicking repeated muscle exposure to inflammatory stimuli, resulted after 2-day survival in NADPH-diaphorase induction in motoneurons and marked activation of the surrounding microglia. Quantitative image analysis of NADPH-diaphorase staining, and OX-42 immunoreactivity and microglial cell counts indicated highly significant increases with respect to saline-injected control cases. The occurrence of a neuroprotective retrograde response was evaluated monitoring bcl-2 expression. Following single phytohaemagglutinin administration, bcl-2 mRNA was significantly upregulated at 6 h in facial motoneurons and returned to basal levels at 24 h. Bcl-2 immunoreactivity was markedly upregulated at 24 h and was still significantly higher than in controls at 7 days, when concomitant NADPH-diaphorase induction in motoneurons and microglia activation was also observed. No degenerative features were observed in motoneurons after phytohaemagglutinin injections at the examined time-points. The data point out that local muscle inflammation retrogradely elicits gene activation in motoneurons and their microenvironment.     

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.     

Endogenous T lymphocytes and microglial reactivity in the axotomized facial motor nucleus of mice: effect of genetic background and the RAG2 gene

Following facial nerve axotomy in mice, peripheral T cells home to the injured facial motor nucleus (FMN) where they may influence the glial response. Interactions between T cells and microglia, which proliferate in response to axotomy, appear to confer neuroprotection to injured motoneurons. The primary objective of this study was to determine whether T lymphocytes could influence the microglial reaction to motoneuron injury. These experiments tested the hypotheses that (1) C57BL/6 (B6) and 129 mice, inbred strains which have high and low levels of astroglial reactivity in the axotomized FMN, respectively, would also exhibit high and low levels of T cell infiltration, and (2) that these differences would correspond with levels of microglial reactivity and neuronal regeneration. Thus, we compared the response to facial nerve axotomy in B6, 129, and immunodeficient RAG2 knockout (RAG2 KO) mice on these two backgrounds at 14 day post-axotomy for differences in levels of 1) CD3+ T cell infiltration; (2) major histocompatibility complex II (MHC2) expression by microglia; (3) perineuronal microglial phagocytic clusters, an indirect measure of neuronal death; and (4) overall microglial activity as assessed by CD11b expression. To examine the inheritance pattern of the abovementioned neuroimmune measures, we also made assessments in B6x129 F1 generation mice. B6 and 129 mice displayed high and low levels of T cell infiltration to the affected FMN and low and high MHC2 expression, respectively. Levels of microglial activity did not differ between the two strains. In immunodeficient RAG2 KO mice on both backgrounds, the number of MHC2+ microglia did not differ from their immunologically normal background controls. Moreover, deletion of either the RAG2 or RAG1 genes in B6 mice was not associated with increased neuronal death at day 14 post-axotomy, as we had previously found in B6 mice with the severe combined immunodeficiency (SCID) mutation. Contrary to our hypothesis, the paucity of T cells in the affected FMN of the 129 mice was associated with less neuronal death when compared to B6 mice, which showed a robust T cell response. Moreover, the data suggest that parameters of the central and peripheral immune responses to axotomy are independently regulated. Assessments in B6x129 F1 generation mice revealed dominant phenotypes for both T cell infiltration and neurodegeneration, whereas both strains contributed significantly to the phenotype for MHC2 expression. Our findings suggest that (1) T cells do not appear to modify measures of microglial reactivity in the axotomized FMN; and (2) the impact of T cells on injured motoneurons in immunologically intact mice and in immunodeficient mice grafted with T cells by adoptive transfer may be different. Further study is required to understand the role of T cells following motoneuron injury in immunologically intact mice and how the seemingly divergent effects of T cells in intact and immunodeficient mice might provide insight into their role in neuronal injury and repair.     

Development of microglia in the chick embryo spinal cord: Implications in the regulation of motoneuronal survival and death

The role of microglia during normal development of the nervous system is still not well understood. In the present study, a chick embryo model was used to examine the development of microglia in the spinal cord and characterize their changes in response to naturally occurring and pathological death of motoneurons (MNs). The microglial response to MN axotomy and the effects of microglial activation on MN survival were also studied. We found that: 1) macrophages/microglial cells were present in the spinal cord at early developmental stages (E3) and that they were recruited after normal and induced MN apoptosis; 2) although many microglial cells were seen phagocytosing apoptotic bodies, a proportion of dying cells were devoid of engulfing microglia; 3) axotomy of mature MNs was accompanied by microglial activation in the absence of MN death; 4) excitotoxic (necrotic) MN death provoked a rapid and massive microglial recruitment with phagocytic activity; 5) lipopolysaccharide‐induced microglial activation in vivo resulted in the death of immature, but not mature, microglia; and 6) overactivation of microglia modulated the survival of mature MNs, either by killing them or by enhancing their vulnerability to die in response to a mild injury. Taken together, these observations indicate that normal microglia do not play an active role in triggering apoptosis of developing MNs. Rather, they act as phagocytes for the removal of dying cells during the process of programmed cell death. © 2009 Wiley‐Liss, Inc.     

Rapid estimates of neuron number in the confocal microscope combined with in situ hybridisation and immunocytochemistry.

A rapid counting protocol is described which combines the optical disector and Cavalieri methods on non-embedded slices of fixed tissue viewed in the confocal laser scanning microscope. By eliminating the embedding stage and avoiding the need to align adjacent sections in the z plane for counting, considerable time savings are gained over physical disector methods. It also allows the remaining non-embedded sections to be used for other purposes, such as in situ hybridisation and immunocytochemistry. Starting with fixed brainstem, it was possible in less than 2 h to determine the total number of motoneurons in both facial nuclei of an adult Sprague-Dawley rat. This method revealed that the normal facial nucleus contained approximately 3200 motoneurons (n=12 rats). One month following facial nerve avulsion (n=4 rats), mean numbers of motoneurons were reduced by 75%. Using intervening sections, changes in neuronal number were compared with changes in in situ hybridisation signal and immunostaining.