Interleukin-12 induces mild experimental allergic encephalomyelitis following local central nervous system injury in the Lewis rat

The major pathological feature in the central nervous system (CNS) following traumatic brain injury is activation of microglia both around and distant from the injury site. Intraperitoneal administration of interleukin-12 (IL-12) after brain injury resulted in a 7% weight loss, clinical signs of mild EAE and significant myelin basic protein (MBP)-specific splenic cell proliferation. The extent of pathology, in terms of the number of inflammatory perivascular cuffs and activation of microglia was greatest if IL-12 was administered immediately compared to a week following brain injury, whether at one or two sites. Specifically immunostaining for MHC class II and iNOS on macrophages and microglia, ICAM-1 on endothelial cells and macrophages was observed around the site of injury. A degree of myelin processing was apparent from immunostaining of MBP in inflammatory cells distant from the lesion. Inflammatory cuffs comprising macrophages, activated microglia, CD4⁺ T cells and iNOS⁺ cells were also detected distant to the injury site in the medulla and spinal cord of animals treated with IL-12. These results suggest that immune-mediated events in which IL-12 production is stimulated as for example viral infection, superimposed on a brain injury, could provide a trigger for a MS-like pathology.     

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.     

AIF-1 expression defines a proliferating and alert microglial/macrophage phenotype following spinal cord injury in rats

Microglial cells are among the first and dominant cell types to respond to CNS injury. Following calcium influx, microglial activation leads to a variety of cellular responses, such as proliferation and release of cytotoxic and neurotrophic mediators. Allograft inflammatory factor-1, AIF-1 is a highly conserved EF-handed, putative calcium binding peptide, associated with microglia activation in the brain. Here, we have analyzed the expression of AIF-1 following spinal cord injury at the lesion site and at remote brain regions. Following spinal cord injury, AIF-1+ cells accumulated in parenchymal pan-necrotic areas and perivascular Virchow-Robin spaces. Subsequent to culmination at day 3--a situation characterized by infiltrating blood borne macrophages and microglia activation--AIF-1+ cell numbers decreased until day 7. In remote areas of Wallerian degeneration and delayed neuronal death, a more discrete and delayed activation pattern of AIF-1+ microglia/macrophages reaching maximum levels at day 14 was observed. There was a considerable match between AIF-1+ cells and PCNA (proliferating cell nuclear antigen) or Ki-67+ labeled cells. AIF-1 expression preceded the expression of ED1, thus indicating a pre-phagocytic role. It appears that AIF-1+ microglia/macrophages are among the earliest cells to respond to spinal cord injury. Our results suggest a role of AIF-1 in the initiation of the early microglial response leading to activation and proliferation essential for the acute response to CNS injury. AIF-1 might modulate microgliosis influencing the efficacy of tissue debris removal, myelin degradation, recruitment of oligodendrocytes and re-organisation of the CNS architecture.     

Microglial voltage-gated proton channel Hv1 in spinal cord injury

After spinal cord injury,microglia as the first responders to the lesion display both beneficial and detrimental characteristics.Activated microglia phagocyte and eliminate cell debris,release cytokines to recruit peripheral immune cells to the injury site.Excessively activated microglia can aggravate the secondary damage by producing extravagant reactive oxygen species and pro-inflammatory cytokines.Recent studies demonstrated that the voltage-gated proton channel Hv1 is selectively expressed in microglia and regulates microglial activation upon injury.In mouse models of spinal cord injury,Hv1 deficiency ameliorates microglia activation,resulting in alleviated production of reactive oxygen species and pro-inflammatory cytokines.The reduced secondary damage subsequently decreases neuronal loss and correlates with improved locomotor recovery.This review provides a brief historical perspective of advances in investigating voltage-gated proton channel Hv1 and home in on microglial Hv1.We discuss recent studies on the roles of Hv1 activation in pathophysiological activities of microglia,such as production of NOX-dependent reactive oxygen species,microglia polarization,and tissue acidosis,particularly in the context of spinal cord injury.Further,we highlight the rationale for targeting Hv1 for the treatment of spinal cord injury and related disorders.     

Microglia activation in rat spinal cord by systemic injection of TLR3 and TLR7/8 agonists

Here we describe activation of microglia in the rat spinal cord by systemic injections of toll-like receptor agonist polyinosine-polycytidylic acid (poly(I:C), a TLR3 ligand) and R848 (a TLR 7/8 ligand). A significant but transient increase of ED-1+ spinal cord microglia was observed 4 days after a single intraperitoneal (i.p.) injection. Immunostainings by different microglial markers, AIF-1, EMAPII, OX6, P2X(4) receptor (P2X4R), indicated that microglia were not fully activated and tracing of cell proliferation by 5-bromo-2 -deoxyuridine revealed that only a small fraction of proliferating cells were microglia (less than 5%). Thus, these stimulators of the innate immune system have, after peripheral administration, clearly effects on the innate immune system of the spinal cord. This should be considered in the design of clinical trials, as both TLR ligands have been used in patients. As injections of TLR ligands can be used to modulate immune activity in the spinal cord, such agents might be tools to modulate local regenerative processes in the spinal cord.     

Ion channels on microglia: therapeutic targets for neuroprotection

Under pathological conditions microglia (resident CNS immune cells) become activated, and produce reactive oxygen and nitrogen species and pro-inflammatory cytokines: molecules that can contribute to axon demyelination and neuron death. Because some microglia functions can exacerbate CNS disorders, including stroke, traumatic brain injury, progressive neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis, and several retinal diseases, controlling their activation might ameliorate immune-mediated CNS disorders. A growing body of evidence now points to ion channels on microglia as contributing to the above neuropathologies. For example, the ATP-gated P2X7 purinergic receptor cation channel is up-regulated around amyloid β-peptide plaques in transgenic mouse models of Alzheimer's disease and co-localizes to microglia and astrocytes. Upregulation of the P2X7 receptor subtype on microglia occurs also following spinal cord injury and after ischemia in the cerebral cortex of rats, while P2X7 receptor-like immunoreactivity is increased in activated microglial cells of multiple sclerosis and amyotrophic lateral sclerosis spinal cord. Utilizing neuron/microglia co-cultures as an in vitro model for neuroinflammation, P2X7 receptor activation on microglia appears necessary for microglial cell-mediated injury of neurons. A second example can be found in the chloride intracellular channel 1 (CLIC1), whose expression is related to macrophage activation, undergoes translocation from the cytosol to the plasma membrane (activation) of microglia exposed to amyloid β-peptide, and participates in amyloid β-peptide-induced neurotoxicity through the generation of reactive oxygen species. A final example is the small-conductance Ca2+/calmodulin-activated K+ channel KCNN4/KCa3.1/SK4/IK1, which is highly expressed in rat microglia. Lipopolysaccharide-activated microglia are capable of killing adjacent neurons in co-culture, and show markedly reduced toxicity when treated with an inhibitor of KCa3.1 channels. Moreover, blocking KCa3.1 channels mitigated the neurotoxicity of amyloid β-peptide-stimulated microglia. Excessive microglial cell activation and production of potentially neurotoxic molecules, mediated by ion channels, may thus constitute viable targets for the discovery and development of neurodegenerative disease therapeutics. This chapter will review recent data that reflect the prevailing approaches targeting neuroinflammation as a pathophysiological process contributing to the onset or progression of neurodegenerative diseases, with a focus on microglial ion channels and their neuroprotective potential.     

Characterization of microglia induced from mouse embryonic stem cells and their migration into the brain parenchyma

We derived microglia from mouse embryonic stem cells (ES cells) at very high density. Using the markers Mac1⁺/CD45^low and Mac1⁺/CD45^high to define microglia and macrophages, respectively, we show that Mac1⁺ cells are induced by GM-CSF stimulation following neuronal differentiation of mouse ES cells using a five-step method. CD45^low expression was high and CD45^high expression was low on induced cells. We used a density gradient method to obtain a large amount of microglia-like cells, approximately 90% of Mac1⁺ cells. Microglia-like cells expressed MHC class I, class II, CD40, CD80, CD86, and IFN-γR. The expression level of these molecules on microglia-like cells was barely enhanced by IFN-γ. Intravenously transferred GFP⁺ microglia derived from GFP⁺ ES cells selectively accumulated in brain but not in peripheral tissues such as spleen and lymph node. GFP⁺ cells were detected mainly in corpus callosum and hippocampus but were rarely seen in cerebral cortex, where Iba1, another marker of microglia, is primarily expressed. Furthermore, both GFP⁺ and Iba1⁺ cells exhibited a ramified morphology characteristic of mature microglia. These studies suggest that ES cell-derived microglia-like cells obtained using our protocol are functional and migrate selectively into the brain but not into peripheral tissues after intravenous transplantation.     

Distinct types of microglial activation in white and grey matter of rat lumbosacral cord after mid-thoracic spinal transection

The inflammatory response has been characterized in the lumbosacral segments (L4-S1) of rats after spinal transection at T8. Immune cells were identified immunohistochemically using antibodies to complement type 3 receptor, CD11b (OX-42), the macrophage lysosomal antigen, CD68 (ED1), major histocompatibility complex class II (MHC II), and CD163 (ED2), a marker of perivascular cells. One week after cord transection, OX-42+ microglial density had nearly doubled. In the white matter, microglia became enlarged, often with retracted processes. In contrast, microglia in the grey matter remained ramified although nearly half of those lying medially contained clusters of ED1+ granules. After 8 weeks, ED1+ (+/-MHC II) macrophages were prominent in regions of Wallerian degeneration extending from dorsolateral to ventral funiculi. Microglial density remained raised in grey matter, particularly in the ventral horns of L4/5. Ramified microglia expressing MHC II+ (+/-ED1) extended from deep in the dorsal columns and around the central canal to the ventral columns. More ED2+ (+/-MHC II) perivascular and meningeal cells were recruited and expressed ED1. Thus, distinct from their conversion into macrophages in the white matter, the activation of ramified microglia after degeneration in the grey matter involves expression of ED1 without morphologic transformation.     

Transforming growth factor beta expression in reactive spinal cord microglia and meningeal inflammatory cells during experimental allergic neuritis

Experimental allergic neurities (EAN), an inflammatory demyelinating disorder of the peripheral nervous system, is preceded and accompanied by a massive microglial reaction in the spinal cord which occurs in the absence of inflammatory cells infiltrating the cord parenchyma. Since transforming growth factor beta (TGF-β) has been shown to play a beneficial role in experimental autoimmune disease and might be involved in the regulation of glial activity, we have investigated the expression of TGF-β in EAN spinal cord and nerve root tissue. Adoptive transfer EAN was induced by the injection of neurotogenic T-cells specific for the P₂ myelin protein. In normal spinal cord tissue, both TGF-β1 and TGF-β3 mRNA were constitutively expressed at low levels. Already 3 days following injection of P₂-specific T-cells, TGF-β1 mRNA levels began to increase, peaked at day 6 at levels about tenfold above normal, and thereafter declined. TGF-β3 was induced even earlier with a sharp rise at day 3 and a peak fourfold above normal at day 4. In situ hybridization for TGF-β1 performed on spinal cord sections 6 days after injection of cells localized TGF-β1 mRNA to many nonneuronal cells with the typical morphology of microglia. In addition, TGF-β1 mRNA was observed in the meninges, and massive accumulation of signal was seen over inflammatory cells infiltrating the nerve roots. Our data indicate that TGF-β1 and -β3 are involved in regulating the glial response in EAN and that activated microglial cells might control their own activity state by expressing TGF-β1. The expression of TGF-β1 by infiltrating inflammatory cells in the nerve roots might represent an important endogenous mechanism to limit the extent of inflammation. © 1993 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.     

PKH67示踪骨髓间充质干细胞迁移至损伤脊髓的实验研究

目的 探讨SCI后体外移植PKH67标记的BMSCs迁移至脊髓损伤处并进行增值和分化的动员情况。方法 用梯度离心法分离和培养出SD 大鼠第3代BMSCs,用绿色荧光染料PKH67标记; 采用钳夹法制备脊髓损伤(SCI)模型,分为实验组(n=15)、对照组(n=16)、假手术组(n=16); SCI术后对脊髓损伤组织进行HE染色,实验组和假手术组于术后尾静脉移植含有1×10⁷个BMSCs的0.5 mL生理盐水,对照组注射等量生理盐水; 分别于术后1、7、14、21 d观察大鼠后肢运动功能恢复情况,并做BBB分; 术后21 d后取脊髓组织,行免疫荧光染色,观察BMSCs的迁移,增值和分化情况。结果(1)镜下可见损伤脊髓形成的空洞、坏死及炎性细胞的增多;(2)共聚焦荧光显微镜观察显示术后21 d实验组脊髓损伤部位可见移植的BMSCs, 部分BMSCs呈GFAP和Nestin阳性表达; 假手术组无 PKH67标记的BMSCs; 实验组GFAP和Nestin阳性细胞数较对照组和假手术组明显增加(P<0.05),对照组较假手术组增加不明显(P>0.05);(3)实验组和对照组BBB评分均有增加,但实验组BBB评分显著高于对照组(P<0.05)。结论 PKH67示踪的BMSCs可迁移至损伤脊髓部位,进行增值并分化为神经元样细胞,促进损伤脊髓的神经功能恢复。     

Cellular inflammatory response after spinal cord injury in sprague-dawley and lewis rats

The distribution of microglia, macrophages, T-lymphocytes, and astrocytes was characterized throughout a spinal contusion lesion in Sprague-Dawley and Lewis rats by using immunohistochemistry. The morphology, spatial localization, and activation state of these inflammatory cells were described both qualitatively and quantitatively at 12 hours, 3, 7, 14, and 28 days after injury. By use of OX42 and ED1 antibodies, peak microglial activation was observed within the lesion epicenter of both rat strains between three and seven days post-injury preceding the bulk of monocyte influx and macrophage activation (seven days). Rostral and caudal to the injury site, microglial activation plateaued between two and four weeks post-injury in the dorsal and lateral funiculi as indicated by morphological transformation and the de-novo expression of major histocompatibility class II (MHC II) molecules. Similar to the timing of microglial reactions, T-lymphocytes maximally infiltrated the lesion epicenter between three and seven days post-injury. Reactive astrocytes, while present in the acute lesion, were more prominent at later survival times (7–28 days). These cells were interspersed with activated microglia but appeared to surround and enclose tissue sites occupied by reactive microglia and phagocytic macrophages. Thus, trauma-induced central nervous system (CNS) inflammation, regardless of strain, occurs rapidly at the site of injury and involves the activation of resident and recruited immune cells. In regions rostral or caudal to the epicenter, prolonged activation of inflammatory cells occurs preferentially in white matter and primarily consists of activated microglia and astrocytes. Differences were observed in the magnitude and duration of macrophage activation between Sprague-Dawley (SD) and Lewis (LEW) rats throughout the lesion. Increased expression of complement type 3 receptors (OX42) and macrophage-activation antigens (ED1) persisted for longer times in LEW rats while expression of MHC class II molecules was attenuated in LEW compared to SD rats at all times examined. Variations in the onset and duration of T-lymphocyte infiltration also were observed between strains with twice as many T-cells present in the lesion epicenter of Lewis rats by 3 days post-injury. These strain-specific findings potentially represent differences in corticosteroid regulation of immunity and may help predict a range of functional neurologic consequences affected by neuroimmune interactions. J. Comp. Neurol. 377:443–464, 1997. © 1997 Wiley-Liss, Inc.     

Microglial involvement in experimental autoimmune inflammation of the central and peripheral nervous system

Microglial cells form a network of potential antigen presenting cells throughout the nervous system. Much progress has recently been made towards a better understanding of their immunological properties. This study examines their activation in 2 models of T cell-mediated autoimmune inflammation of the nervous system, experimental autoimmune encephalomyelitis (EAE) and its peripheral counterpart, experimental autoimmune neuritis (EAN), induced by the transfer of antigen-specific T cell lines. In both models microglial activation occurs at early stages of the disease. Activated microglial cells show an increased expression of MHC class I and II antigens. In EAE ultrastructural analysis revealed that MHC antigen expression is pronounced on perivascular microglial cells, suggesting this cell population may be important for antigen presentation at a site close to the blood-brain barrier. In contrast to EAE, the microglial reaction in EAN occurs at sites remote from the inflammatory response in the peripheral nerve, not only in the spinal cord but also in the terminal projection fields of primary sensory neurons in the lower brainstem. This early microglial activation in EAN suggests that a rapid and remote signaling mechanism can operate following peripheral inflammation. Immuno-electron microscopy revealed that activated microglial cells are also involved in the synaptic deafferentation of spinal cord motoneurons during autoimmune reactions. The rapid involvement of microglial cells in experimental autoimmune inflammation of the nervous system further points to their role as the main intrinsic immuneffector cell population of the central nervous system.     

Up-regulation of CD81 (target of the antiproliferative antibody; TAPA) by reactive microglia and astrocytes after spinal cord injury in the rat

We examined the expression of CD81 (also known as TAPA, or target of the antiproliferative antibody) after traumatic spinal cord injury in the rat. CD81, a member of the tetraspanin family of proteins, is thought to be involved in reactive gliosis. This is based on the antiproliferative and antiadhesive effects of antibodies against CD81 on cultured astrocytes, as well as its up-regulation after penetrating brain injury. CD81 expression following dorsal hemisection of the spinal cord was determined immunohistochemically at time points ranging from 1 day to 2 months postlesion (p.l.). In the unlesioned cord a low background level of CD81 was observed, with the exception of the ependyma of the central canal and the pia mater, which were strongly CD81-positive. One day p.l., CD81 was diffusely up-regulated in the spinal cord parenchyma surrounding the lesion site. From 3 days onward, intensely CD81-positive round cells entered the lesion site, completely filling it by 7 days p.l. Staining with the microglial markers OX-42 and Iba1 revealed that these cells were reactive microglia/macrophages. At this time, no significant CD81 expression by GFAP-positive reactive astrocytes was noted. From the second week onward, CD81 was gradually down-regulated; i.e., its spatial distribution became more restricted. The CD81-positive microglia/macrophages disappeared from the lesion site, leaving behind large cavities. After 2 months, astrocytes that formed the wall of these cavities were strongly CD81-positive. In addition, CD81 was present on reactive astrocytes in the dorsal funiculus distal from the lesion in degenerated white matter tracts. In conclusion, the spatiotemporal expression pattern of CD81 by reactive microglia and astrocytes indicates that CD81 is involved in the glial response to spinal cord injury.     

Differential microglial regulation in the human spinal cord under normal and pathological conditions

As the primary intrinsic immune effector cells of the central nervous system, microglia are involved in virtually all pathological processes of the brain and spinal cord including inflammatory, neurodegenerative, traumatic, neoplastic and vascular diseases. Despite this important role, there is a lack of data concerning microglial distribution and protein expression in the human spinal cord. In this study, we immunohistochemically investigated 10 normal human spinal cords to establish reference data and compared these results with 15 pathological human spinal cords deriving from distinct pathologies. Each spinal cord was evaluated at eight different levels for three white and two grey matter areas for both constitutive (MHC-II, CD68, IL-16, AIF-1, LCA, CD4) and reactive (MRP-8, MRP-14) microglial antigens. Whereas previous studies revealed significant regional differences in microglial distribution and protein expression in human brain, normal spinal cord displayed a uniform expression pattern, reaching levels of up to 17% MHC-II positive cells of the total cell population. This datum formed the basis for the further evaluation of microglia expression levels in pathological spinal cords, where levels of up to 45% positive cells were observed. Our results represent important reference values for future neuropathological diagnostic and therapeutical approaches in spinal cord pathologies.     

Microglial control of neuronal death and synaptic properties

Microglia have long been characterized by their immune function in the nervous system and are still mainly considered in a beneficial versus detrimental dialectic. However a review of literature enables to shed novel lights on microglial function under physiological conditions. It is now relevant to position these cells as full time partners of neuronal function and more specifically of synaptogenesis and developmental apoptosis. Indeed, microglia can actively control neuronal death. It has actually been shown in retina that microglial nerve growth factor (NGF) is necessary for the developmental apoptosis to occur. Similarly, in cerebellum, microglia induces developmental Purkinje cells death through respiratory burst. Furthermore, in spinal cord, microglial TNFalpha commits motoneurons to a neurotrophic dependent developmental apoptosis. Microglia can also control synaptogenesis. This is suggested by the fact that a mutation in KARAP/DAP12, a key protein of microglial activation impacts synaptic functions in hippocampus, and synapses protein content. In addition it has been now demonstrated that microglial brain-derived neurotrophin factor (BDNF) directly regulates synaptic properties in spinal cord. In conclusion, microglia can control neuronal function under physiological conditions and it is known that neuronal activity reciprocally controls microglial activation. We will discuss the importance of this cross-talk which allows microglia to orchestrate the balance between synaptogenesis and neuronal death occurring during development or injuries.     

Poly(I:C)与R848对大鼠脊髓小胶质细胞活性的影响

目的 研究Toll样受体3(TLR3)配体多聚肌胞苷酸[Poly(I:C)]与TLR7/8配体R848对小胶质细胞(MG)活性的影响.方法 6～8周龄Lewis大鼠30只分为Poly(I:C)组(12只)、R848组(15只)和对照组(3只),前两组大鼠根据处死时间的不同分别分为4、5个亚组,每亚组3只,分别单次腹腔注射1 mL Poly(I:C)(5 mg/kg)和R848(1 mg/kg),对照组注射等量PBS;应用免疫组化检测大鼠脊髓内ED-1、同种异体移植炎性因子-1(AW-1)、血管内皮单核细胞活化肽Ⅱ(EMAPⅡ)、OX-6和P2X4R的表达,应用免疫组化双染染色检测增殖的MG.结果 与对照组相比,Poly(I:C)组和R848组注药后第4天ED-1+的MG明显增加,差异有统计学意义(P<0.05),Poly(I:C)组与R848组仅观察到少量EMAP Ⅱ+细胞,未观察到AIF-1+、OX6+,P2X4R+细胞,对照组未观察到EMAPII+、AIF-1+、OX6+及P2X4R+细胞;免疫组化双染显示Poly(I:C)组和R848组大鼠注射后第4天脊髓有少量BrdU+/ED-1+双染细胞,提示仅有少量MG增殖.结论 TLR配体外周给药后可作用于脊髓内的固有免疫系统,进而影响脊髓内MG的免疫活性,提示这类制剂对调节脊髓损伤组织的再生修复有一定价值.     

Inhibition of tumour necrosis factor-alpha by antisense targeting produces immunophenotypical and morphological changes in injury-activated microglia and macrophages

Microglia respond in a stereotypical pattern to a diverse array of pathological states. These changes are coupled to morphological and immunophenotypical alterations and the release of a variety of reactive species, trophic factors and cytokines that modify both microglia and their cellular environment. We examined whether a microglial-produced cytokine, tumour necrosis factor-alpha (TNF-alpha), was involved in the maintenance of microglial activation after spinal cord injury by selective inhibition using TNF-alpha antisense deoxyoligonucleotides (ASOs). Microglia and macrophages harvested from 3 d post-contused rat spinal cord were large and rounded (86.3 +/- 9.6%). They were GSA-IB4-positive (GSA-IB4(+)) (Griffonia simplicifolia lectin, microglia specific; 94.8 +/- 5.1%), strongly OX-42 positive (raised against a type 3 complement/integrin receptor, CD11b; 78.9 +/- 9.1%), ED-1 positive (a lysosomal marker shown to correlate well with immune cell activation; 97.2 +/- 2.6%) and IIA positive (antibody recognizes major histocompatibility complex II; 57.2 +/- 5.6%), indicative of fully activated cells, for up to 48 h after plating. These cells also secreted significant amounts of TNF-alpha (up to 436 pg/microg total protein, 16 h). Fluoroscein isothiocyanate-labelled TNF-alpha ASOs (5, 50 and 200 nm) added to the culture medium were taken up very efficiently into the cells (> 90% cells) and significantly reduced TNF-alpha production by up to 92% (26.5 pg/microg total protein, 16 h, 200 nm TNF-alpha ASOs). Furthermore, few of the treated cells at this time were round (5.4 +/- 2.7%), having become predominantly spindle shaped (74.9 +/- 6.3%) or stellate (21.4 +/- 2.7%); immunophenotypically, although all of them remained GSA-IB4 positive (91.6 +/- 6.2%), many were weakly OX-42 positive and few expressed either ED-1 (12.9 +/- 2.5%) or IIA (19.8 +/- 7.4%). Thus, the secretion of TNF-alpha early in spinal cord injury may be involved in autoactivating microglia/macrophages. However, at the peak of microglial activation after injury, the activation state of microglia/macrophages is not stable and this process may still be reversible by blocking TNF-alpha.     

Acute Inflammatory Response in Spinal Cord Following Impact Injury

Numerous factors are involved in the spread of secondary damage in spinal cord after traumatic injury, including ischemia, edema, increased excitatory amino acids, and oxidative damage to the tissue from reactive oxygen species. Neutrophils and macrophages can produce reactive oxygen species when activated and thus may contribute to the lipid peroxidation that is known to occur after spinal cord injury. This study examined the rostral–caudal distribution of neutrophils and macrophages/microglia at 4, 6, 24, and 48 h after contusion injury to the T10 spinal cord of rat (10 g weight, 50 mm drop). Neutrophils were located predominantly in necrotic regions, with a time course that peaked at 24 h as measured with assays of myeloperoxidase activity (MPO). The sharpest peak of MPO activity was localized between 4 mm rostral and caudal to the injury. Macrophages/microglia were visualized with antibodies against ED1 and OX-42. Numerous cells with a phagocytic morphology were present by 24 h, with a higher number by 48 h. These cells were predominantly located within the gray matter and dorsal funiculus white matter. The number of cells gradually declined through 6 mm rostral and caudal to the lesion. OX-42 staining also revealed reactive microglia with blunt processes, particularly at levels distant to the lesion. The number of macrophages/microglia was significantly correlated with the amount of tissue damage at each level. Treatments to decrease the inflammatory response are likely to be beneficial to recovery of function after traumatic spinal cord injury.