Demonstration of microglial cells in and around senile (neuritic) plaques in the Alzheimer brain

Summary A monoclonal antibody, termed AD11/8, reactive to microglial cells, was produced by immunization of mice with partially purified amyloid fibrils of senile (neuritic) plaques. With immunoperoxidase staining on human tissues, AD11/8 also recognized macrophages in the red pulp of the spleen, Kupffer cells in the liver, and macrophages in the bone marrow. The results show that AD11/8 recognizes the antigens associated with mononuclear phagocytes lineage. In normal brains a few resting microglial cells were stained in gray matter, and less frequently in white matter. In senile dementia of the Alzheimer type numerous microglial cells were stained intensively and they often formed clusters in gray matter. By double immunostaining with AD11/8 and a polyclonal antibody against synthetic amyloid -protein, clustered microglial cells were observed in and around senile plaques with amyloid deposits. Some amyloid plaque cores were surrounded by microglial cell processes. These results indicate that microglial cells may play an important role in senile plaque formation.Supported in part by the Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture, the grants for Research of Dementia and for Primary Amyloidosis from the Ministry of Health and Welfare, Japan     

Inducible nitric oxide synthase activity of cloned murine microglial cells

Nitric oxide (NO) is a short-lived diffusable molecule now believed to participate in multiple physiologic functions in the CNS including neurotransmission and the maintenance of vascular tone. Previously, we reported that cell lines obtained by retroviral immortalization of tissue macrophages (M?;) could be induced to synthesize nitrite (NO), a stable end product of the NO synthetic pathway. We have further characterized the induction and activity of this pathway in a panel of seven microglial clones derived from primary embryonic mouse brain cultures. Like M?;, these clones were found to release high levels of NO_-² in response to recombinant interferon-γ (rIFN-γ) as a priming signal together with either bacterial lipopolysaccharide (LPS) or exogenous recombinant tumor necrosis factor-α (rTNF-α). As previously demonstrated for M?;, phagocytosis of zymosan particles during induction of enzyme activity enhanced subsequent NO production, which is of interest in light of the postulated phagocytic role of microglia within the CNS. Biochemical characterization of enzyme activity in intact microglial clones and in isolated cytosolic fractions indicates that the microglial NO synthase present in these murine cell clones represents the M?;-like isotype. These findings suggest that microglial cells could represent a major source of NO within the CNS.     

Brief postpartum separation from offspring promotes resilience to lipopolysaccharide challenge-induced anxiety and depressive-like behaviors and inhibits neuroinflammation in C57BL/6J dams

Emerging evidence indicates an important role for neuroinflammation in depression. Brief maternal separation promotes resilience to depression in offspring, but relatively little is known about the effects of different durations of postpartum separation (PS) from offspring on anxiety and depressive-like behaviors in dams following immune challenge. Lactating C57BL/6J mice were subjected to no separation (NPS), brief PS (15 min/day, PS15) or prolonged PS (180 min/day, PS180) from postpartum day (PPD) 1 to PPD21 and then injected with lipopolysaccharide (LPS). Behavioral tests, including the open field test (OFT) and forced swimming test (FST), were carried out at 24 h after the injection. LPS resulted in anxiety and depressive-like behaviors in NPS dams and activated ionized calcium-binding adaptor molecule (Iba1), an important biomarker of microglia, in the hippocampus. However, compared with NPS + LPS dams, PS15 + LPS dams spent significantly more time in the center of the OFT (anxiety-like behavior) and exhibited lower immobility time in the FST (depressive-like behavior), which indicated a phenomenon of resilience. Furthermore, the activation of neuroinflammation was inhibited in PS15 dams. Specifically, levels of the Iba1 mRNA and protein were decreased, while the mRNA expression of NLR family pyrin domain containing 3 (NLRP3) inflammasome/interleukin-18 (IL-18)/nuclear factor kappa-B (NF-κB) was decreased in the hippocampus. Furthermore, positive linear correlations were observed between microglial activation and LPS-induced depressive-like behaviors in dams. Collectively, the findings of this study confirm that brief PS from offspring promotes resilience to LPS immune challenge-induced behavioral deficits and inhibits neuroinflammation in dams separated from their offspring during lactation.     

Lipopolysaccharide-induced sepsis-like state compromises post-ischemic neurological recovery,brain tissue survival and remodeling via mechanisms involving microvascular thrombosis and brain T cell infiltration

Sepsis predisposes for poor stroke outcome. This association suggests that sepsis disturbs post-ischemic tissue survival and brain remodeling. To elucidate this link, we herein exposed mice to 30 min intraluminal middle cerebral artery occlusion (MCAO) and induced a sepsis-like state at 72 h post-ischemia by intraperitoneal delivery of Escherichia coli lipopolysaccharide (LPS; three doses of 0.1 or 1 mg/kg, separated by 6 h), a major component of the bacterium’s outer membrane. Neurological recovery, ischemic injury, brain remodeling and immune responses were evaluated over up to 56 days post-sepsis (dps) by behavioral tests, immunohistochemistry and flow cytometry. Delivery of 1 mg/kg but not 0.1 mg/kg LPS reduced rectal temperature over 48 h by up to 3.4 ± 3.1 °C, increased general and focal neurological deficits in the Clark score over 72 h and increased motor-coordination deficits in the tight rope test over up to 21 days. Notably, 1 mg/kg, but not 0.1 mg/kg LPS increased intercellular adhesion molecule-1 abundance on ischemic microvessels, increased microvascular thrombosis and increased patrolling monocyte and T cell infiltrates in ischemic brain tissue at 3 dps. Infarct volume was increased by 1 mg/kg, but not 0.1 mg/kg LPS at 3 dps (that is, 6 days post-MCAO), as was brain atrophy at 28 and 56 dps. Microglial activation in ischemic brain tissue, evaluated by morphology analysis of Iba-1 immunostainings, was transiently increased by 0.1 and 1 mg/kg LPS at 3 dps. Our data provide evidence that neurological recovery and brain remodeling are profoundly compromised in the ischemic brain post-sepsis as a consequence of cerebral thromboinflammation.     

Tenascin-C preserves microglia surveillance and restricts leukocyte and,more specifically,T cell infiltration of the ischemic brain

As an endogenous activator of toll-like receptor-4 (Tlr4), the extracellular matrix glycoprotein tenascin-C (TnC) regulates chemotaxis, phagocytosis and proinflammatory cytokine production in microglia. The role of TnC for ischemic brain injury, post-ischemic immune responses and stroke recovery has still not been evaluated. By comparing wild type and TnC^−/− mice exposed to transient intraluminal middle cerebral artery occlusion (MCAO), we examined the effects of TnC deficiency for ischemic injury, neurological deficits, microglia/macrophage activation and brain leukocyte infiltration using behavioural tests, histochemical studies, Western blot, polymerase chain reaction and flow cytometry. Histochemical studies revealed that TnC was de novo expressed in the ischemic striatum, which contained the infarct core, and overlapped with the area of strongest accumulation of Iba1 + microglia/macrophages. TnC deficiency increased overall Iba1 immunoreactivity in the perilesional cortex, suggesting that TnC might restrict the distribution of microglial cells to the infarct core. By analysing microglial morphology in 3D we found that the post-ischemic loss of microglial cell territory, branching and volume at 3 and 7 days post-ischemia was amplified in the brains of TnC deficient compared with wild type mice. Microglial cell number was not different between genotypes. Hence, TnC deficiency reduced tissue surveillance by microglial cells. Concomitantly, the number of infiltrating leukocytes and, more specifically, T cells was increased in the ischemic brain parenchyma of TnC deficient compared with wild type mice. Ischemic injury and neurological deficits were not affected by TnC deficiency. We propose that the reduced microglia surveillance in TnC deficient mice might favour leukocyte accumulation in the ischemic brain.     

益气活血通络方通过抑制P2X7介导的脊髓小胶质细胞活化改善糖尿病大鼠神经病理性疼痛

目的 探讨益气活血通络方对糖尿病神经病理性疼痛（diabetic neuropathic pain,DNP）的影响及其作用机制。方法 高脂喂养大鼠4周后,35 mg/kg链脲佐菌素（streptozotocin,STZ）腹腔注射复制2型糖尿病大鼠模型,通过观测大鼠机械痛阈和热缩足反应时间作为判定DNP模型复制成功的标准。将DNP大鼠随机分为模型组,益气活血通络方高、中、低剂量组和阳性对照组（甲钴胺）,另设空白对照组。Western blot法和免疫荧光双标检测脊髓中补体受体- 3的单克隆抗体（monoclonal antibody of complement receptor type 3,OX42）和P2X7（purinergic 2X7）的表达水平;ELISA法检测血清肿瘤坏死因子- α（tumor necrosis factor- α,TNF- α）、白细胞介素- 1β（interleukin- 1β,IL- 1β）和趋化因子配体3（CC- chemokine ligand 3,CCL3）水平。结果 益气活血通络方干预后,DNP大鼠的机械痛阈和热缩足反应时间均得到显著的改善;大鼠血清TNF- α、IL- 1β和CCL3水平显著降低（P<0.05）;脊髓组织中OX42和P2X7表达水平均显著降低（P<0.05）。结论 益气活血通络方对DNP大鼠具有显著的治疗作用,其作用机制可能是通过抑制脊髓小胶质细胞活性,调节P2X7的表达,从而减轻神经炎症反应。     

Novel mGluR5 Positive Allosteric Modulator Improves Functional Recovery,Attenuates Neurodegeneration,and Alters Microglial Polarization after Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) causes microglial activation and related neurotoxicity that contributes to chronic neurodegeneration and loss of neurological function. Selective activation of metabotropic glutamate receptor 5 (mGluR5) by the orthosteric agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), is neuroprotective in experimental models of TBI, and has potent anti-inflammatory effects in vitro. However, the therapeutic potential of CHPG is limited due to its relatively weak potency and brain permeability. Highly potent, selective and brain penetrant mGluR5 positive allosteric modulators (PAMs) have been developed and show promise as therapeutic agents. We evaluated the therapeutic potential of a novel mGluR5 PAM, VU0360172, after controlled cortical impact (CCI) in mice. Vehicle, VU0360172, or VU0360172 plus mGluR5 antagonist (MTEP), were administered systemically to CCI mice at 3 h post-injury; lesion volume, hippocampal neurodegeneration, microglial activation, and functional recovery were assessed through 28 days post-injury. Anti-inflammatory effects of VU0360172 were also examined in vitro using BV2 and primary microglia. VU0360172 treatment significantly reduced the lesion, attenuated hippocampal neurodegeneration, and improved motor function recovery after CCI. Effects were mediated by mGluR5 as co-administration of MTEP blocked the protective effects of VU0360172. VU0360172 significantly reduced CD68 and NOX2 expression in activated microglia in the cortex at 28 days post-injury, and also suppressed pro-inflammatory signaling pathways in BV2 and primary microglia. In addition, VU0360172 treatment shifted the balance between M1/M2 microglial activation states towards an M2 pro-repair phenotype. This study demonstrates that VU0360172 confers neuroprotection after experimental TBI, and suggests that mGluR5 PAMs may be promising therapeutic agents for head injury.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0298-6) contains supplementary material, which is available to authorized users.     

Resveratrol alleviates temporomandibular joint inflammatory pain by recovering disturbed gut microbiota

Patients with temporomandibular disorders (TMDs) often experience persistent facial pain. However, the treatment of TMD pain is still inadequate. In recent years, the disturbance of gut microbiota has been shown to play an important role in the pathogenesis of different neurological diseases including chronic pain. In the present study, we investigated the involvement of gut microbiota in the development of temporomandibular joint (TMJ) inflammation. Intra-temporomandibular joint injection of complete Freund's adjuvant (CFA) was employed to induce TMJ inflammation. Resveratrol (RSV), a natural bioactive compound with anti-inflammatory property, was used to treat the CFA-induced TMJ inflammation. We observed that CFA injection not only induces persistent joint pain, but also causes the reduction of short-chain fatty acids (SCFAs, including acetic acid, propionic acid and butyric acid) in the gut as well as decreases relevant gut bacteria Bacteroidetes and Lachnospiraceae. Interestingly, systemic administration of RSV (i.p.) dose-dependently inhibits CFA-induced TMJ inflammation, reverses CFA-caused reduction of SCFAs and these gut bacteria. Moreover, CFA injection causes blood–brain barrier (BBB) leakage, activates microglia and enhances tumor necrosis factor alpha (TNFα) release in the spinal trigeminal nucleus caudalis (Sp5C). The RSV treatment restores the BBB integrity, inhibits microglial activation and decreases the release of TNFα in the Sp5C. Furthermore, fecal microbiota transplantation with feces from RSV-treated mice significantly diminishes the CFA-induced TMJ inflammation. Taken together, our results suggest that gut microbiome perturbation is critical for the development of TMJ inflammation and that recovering gut microbiome to normal levels could be a new therapeutic approach for treating such pain.     

Aβ1-42作用的小胶质细胞对体外培养的神经干细胞生存的影响

目的: 探讨β-淀粉样蛋白(Aβ_1-42)作用的小胶质细胞对体外培养的神经干细胞(NSCs)生存的影响。方法: 利用Transwell小室在体外建立NSCs与小胶质细胞共培养体系,检测Aβ_1-42蛋白作用小胶质细胞前后NSCs增殖、分化及凋亡情况。结果: 与单纯共培养组相比,Aβ_1-42干预共培养组的增殖率降低,且微管相关蛋白2(MAP-2)和胆碱乙酰转化酶(CHAT)阳性表达率也均降低。结论: Aβ_1-42介导的炎症反应抑制了神经干细胞的增殖,促进其凋亡,并能显著降低其分化成神经元尤其是胆碱能神经元的能力。