Journal of Neuroinflammation

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Online ISSN: 1742-2094
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Quantitative analysis of size A and representative photomicrographs of epididymal adipocytes B from wild-type (WT) and TLR4-mut mice fed a normolipid diet (white bars) or a high-fat diet (black bars) for 24 weeks. The data are represented as the mean ± SEM, n = 6. Magnification 400x, scale bar 50 μm. **p < 0.01 vs. WT normal diet
Spatial memory impairment A and time to find the platform, trial in the absence of the platform B by the MWM test of wild-type (WT) and TLR4-mut mice fed a normolipid diet (white bars) or a high-fat diet (black bars) for 24 weeks. The data are expressed as the mean ± SEM *p < 0.05, n = 8-15/group
Western blot analysis A and quantification of the expression of synaptophysin B and PSD-95 C in the whole brain from wild-type (WT) and TLR4-mut mice fed a normolipid diet (white bars) or a high-fat diet (black bars) for 24 weeks. Values represent the mean ± SEM of 4-7 animals per group. Quantitative protein expression of synaptophysin and PSD-95 bands detected by western blot using beta-tubulin as a loading control. *p < 0.05 versus WT-HFD
Graphical representation of the AngioTool analysis in the cerebral cortex. Total vessel length A and lacunarity (B). The results of AngioTool analysis of confocal images of brain slices of cortex from wild type (WT) and TLR4-mut mice fed a normolipid diet (white bars) or a high-fat diet (black bars) for 24 weeks. Skeleton in red and branching points in blue. Vessels labeled with IB4, scale bar 50 µm. n = 6. *p < 0.05 versus WT-ND and # p < 0.05 versus WT-HFD
Effects of high fat diet consumption on the metabolic hormones of WT or TLR4-mut mice
Background Metabolic syndrome (MS) is defined as a low-grade proinflammatory state in which abnormal metabolic and cardiovascular factors increase the risk of developing cardiovascular disease and neuroinflammation. Events, such as the accumulation of visceral adipose tissue, increased plasma concentrations of free fatty acids, tissue hypoxia, and sympathetic hyperactivity in MS may contribute to the direct or indirect activation of Toll-like receptors (TLRs), specifically TLR4, which is thought to be a major component of this syndrome. Activation of the innate immune response via TLR4 may contribute to this state of chronic inflammation and may be related to the neuroinflammation and neurodegeneration observed in MS. In this study, we investigated the role of TLR4 in the brain microcirculation and in the cognitive performance of high-fat diet (HFD)-induced MS mice. Methods Wild-type (C3H/He) and TLR4 mutant (C3H/HeJ) mice were maintained under a normal diet (ND) or a HFD for 24 weeks. Intravital video-microscopy was used to investigate the functional capillary density, endothelial function, and endothelial–leukocyte interactions in the brain microcirculation. Plasma concentrations of monocyte chemoattractant protein-1 (MCP-1), adipokines and metabolic hormones were measured with a multiplex immunoassay. Brain postsynaptic density protein-95 and synaptophysin were evaluated by western blotting; astrocytic coverage of the vessels, microglial activation and structural capillary density were evaluated by immunohistochemistry. Results The HFD-induced MS model leads to metabolic, hemodynamic, and microcirculatory alterations, as evidenced by capillary rarefaction, increased rolling and leukocyte adhesion in postcapillary venules, endothelial dysfunction, and less coverage of astrocytes in the vessels, which are directly related to cognitive decline and neuroinflammation. The same model of MS reproduced in mice deficient for TLR4 because of a genetic mutation does not generate such changes. Furthermore, the comparison of wild-type mice fed a HFD and a normolipid diet revealed differences in inflammation in the cerebral microcirculation, possibly related to lower TLR4 activation. Conclusions Our results demonstrate that TLR4 is involved in the microvascular dysfunction and neuroinflammation associated with HFD-induced MS and possibly has a causal role in the development of cognitive decline.
IL-13 secreting Mφs show upregulation of CCR2 and CCR5 gene expression but not C5aR. a-d Mφs were isolated from C57BL/6J mice. a IL-13 secretion by IL-13 Mφs was significantly higher than M0, M1, or M2 Mφs as determined by an ELISA. n = 9-13. b-d qPCR analyses showed that the gene expression of C5aR (b) was not changed, whereas IL-13 Mφs showed significant upregulation of CCR2 (c) and CCR5 (d) expression compared to both M0 and M2 Mφs. Data were normalized to M0 Mφs and represent mean ± SEM. n = 4-7. Kruskal-Wallis test with Dunn's correction.*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001
IL-13 Mφs have an anti-inflammatory phenotype and secrete functional IL-13. a-i Mφs were isolated from C57BL/6J mice. a-d Gene expression analysis by qPCR showed that IL-13 Mφs have an upregulation of Arg1 (a) and the conserved genes IRF4 (b) and KLF4 (c), while no increased expression of iNOS (d) was observed relative to M0 Mφs. Data were normalized to M0 Mφs and represent mean ± SEM. n = 8-14. e, f Western blot quantification showed increased expression of Arg1 (e) but no upregulation of iNOS (f) by IL-13 Mφs compared to M0 Mφs. Data were normalized to M0 Mφs and represent mean ± SEM. n = 4-5. g Representative immunoblot of Arg1 and iNOS expression. h, i Representative image (h) and quantification (i) of the western blot analysis of Arg1 expression. Following incubation with the conditioned medium (CM) of IL-13 Mφs, M1 Mφs showed Arg1 protein expression. Data were normalized to M1 Mφs and represent mean ± SEM. n = 3-4. Kruskal-Wallis test with Dunn's correction. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001
Transplantation of IL-13 Mφs induces a more anti-inflammatory environment at the lesion site. a-l Immediately following injury, C57BL/6j mice received vehicle, M2 Mφs or IL-13 Mφs. a, b Number of Arg1 + (a) at the lesion site was increased, while the amount of MHCII + (b) cells was significantly decreased in IL-13 Mφ-treated mice compared to vehicle. Data were normalized to vehicle and are shown as mean ± SEM. n = 4-7 mice/group. c The number of ORO + lipid droplets at the lesion site was significantly reduced in IL-13 Mφ-treated mice compared to vehicle. Data were normalized to vehicle and are shown as mean ± SEM. n = 9 mice/group. d-l Representative images of the Arg1 (d-f), MHCII (g-i), and ORO (j-l) staining at the lesion. Arg1 + or MHCll + cells, as well as ORO droplets, are indicated by white arrows. Scale bar = 100 µm. Kruskal-Wallis test with Dunn's correction (a, b) or one-way ANOVA with Bonferroni post hoc test (c). **P < 0.01
IL-13 is the key factor behind the beneficial effects of IL-13 Mφs in vivo. a IL-4Rα WT or KO BALB/c mice received vehicle or IL-13 Mφs immediately following injury. Functional recovery was improved by transplantation of IL-13 Mφs in WT mice but not in IL-4Rα KO mice. Data are shown as mean ± SEM. n = 6-12 mice/group. *WT + IL-13 Mφs versus vehicle. Two independent experiments. b C57BL/6J mice received vehicle or Arg-1 Mφs immediately following injury. No difference in functional recovery was observed between experimental groups. Data are shown as mean ± SEM. n = 10-12 mice/group. One independent experiment. Two-way ANOVA with Bonferroni post hoc test. *P < 0.05, **P < 0.01
Abbreviations Arg1: Arginase-1; BMS: Basso Mouse Scale; CA: Cornu ammonis; CNS: Central nervous system; CCR : C-C chemokine receptor; Dpi: Days post-injury; FCS: Fetal calf serum; FIZZ1: Found in inflammatory zone 1; GFAP: Glial fibrillary acidic protein; GFP: Green fluorescent protein; hiPSC-NSC: Human-induced pluripotent stem cell-derived neural stem cells; Iba-1: Ionized calcium binding adaptor molecule 1; IL-4Rα KO: Interleukin-4 receptor alpha knockout; IL-13 Mɸs: Interleukin-13 macrophages; iNOS: Inducible nitric oxide synthase; IRF4: Interferon regulatory factor 4; KLF4: Kruppel-like factor 4; KO: Knockout; LCM: L929 conditioned medium; LPS: Lipopolysaccharide; MBP: Myelin basic protein; MHCll: Major histocompatibility complex class ll; MSCs: Mesenchymal stem cells; MTT: 3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Mɸs: Macrophages; NF: Neurofilament; NMDA: N-methyl-d-aspartic acid; NO: Nitric oxide; OGD: Oxygen-glucose deprivation; ORO: Oil red O; PBS: Phosphate-buffered saline; PFA: Paraformaldehyde; PI: Propidium iodide; rIL-13: Recombinant IL-13; RT: Room temperature; SCI: Spinal cord injury; SEM: Standard error of mean; SNAP: S-nitroso-N-acetylpenicillamine; TNF-α: Tumor necrosis factor-alpha; WT: Wild type; Ym1: Chitinase-3-like protein 3.
Background Spinal cord injury (SCI) elicits a robust neuroinflammatory reaction which, in turn, exacerbates the initial mechanical damage. Pivotal players orchestrating this response are macrophages (Mφs) and microglia. After SCI, the inflammatory environment is dominated by pro-inflammatory Mφs/microglia, which contribute to secondary cell death and prevent regeneration. Therefore, reprogramming Mφ/microglia towards a more anti-inflammatory and potentially neuroprotective phenotype has gained substantial therapeutic interest in recent years. Interleukin-13 (IL-13) is a potent inducer of such an anti-inflammatory phenotype. In this study, we used genetically modified Mφs as carriers to continuously secrete IL-13 (IL-13 Mφs) at the lesion site. Methods Mφs were genetically modified to secrete IL-13 (IL-13 Mφs) and were phenotypically characterized using qPCR, western blot, and ELISA. To analyze the therapeutic potential, the IL-13 Mφs were intraspinally injected at the perilesional area after hemisection SCI in female mice. Functional recovery and histopathological improvements were evaluated using the Basso Mouse Scale score and immunohistochemistry. Neuroprotective effects of IL-13 were investigated using different cell viability assays in murine and human neuroblastoma cell lines, human neurospheroids, as well as murine organotypic brain slice cultures. Results In contrast to Mφs prestimulated with recombinant IL-13, perilesional transplantation of IL-13 Mφs promoted functional recovery following SCI in mice. This improvement was accompanied by reduced lesion size and demyelinated area. The local anti-inflammatory shift induced by IL-13 Mφs resulted in reduced neuronal death and fewer contacts between dystrophic axons and Mφs/microglia, suggesting suppression of axonal dieback. Using IL-4Rα-deficient mice, we show that IL-13 signaling is required for these beneficial effects. Whereas direct neuroprotective effects of IL-13 on murine and human neuroblastoma cell lines or human neurospheroid cultures were absent, IL-13 rescued murine organotypic brain slices from cell death, probably by indirectly modulating the Mφ/microglia responses. Conclusions Collectively, our data suggest that the IL-13-induced anti-inflammatory Mφ/microglia phenotype can preserve neuronal tissue and ameliorate axonal dieback, thereby promoting recovery after SCI.
Spatiotemporal distribution of PDGFB and PDGFRβ after SCI. Immunofluorescence staining of PDGFB (red), PDGFRβ (green) and nuclei (blue) in sagittal sections before SCI and at 3, 7, 14 and 28 dpi. The region of interest (ROI) represents the boxed region on the left. Asterisks indicate the injured core. Scale bars: 200 μm in S and 20 μm in T
Intrathecal injection of SU16f reduces fibrotic scar after SCI. A-F Immunofluorescence staining of fibronectin (red) and PDGFRβ (green) in sagittal sections of the Control and SU16f groups at 28 dpi. G-L Immunofluorescence staining of laminin (red) and PDGFRβ (green) in sagittal sections of the Control and SU16f groups at 28 dpi. M-O Quantification of the percentage of PDGFRβ + area, fibronectin + area or laminin + area in the area of the spinal cord segment spanning the injured core at 28 dpi. Asterisks indicate the injured core. Scale bars: 200 μm. ***P < 0.001 and ****P < 0.0001 by Student's t test, n = 5 animals per group (See figure on next page.)
Intrathecal injection of SU16f promotes axon regeneration after SCI. A-H Immunofluorescence staining of NF (red) and GFAP (green) in sagittal sections of the Control and SU16f groups at 28 dpi. The region of interest (ROI) represents the boxed region on the left. I-N Immunofluorescence staining of 5-HT (red) and GFAP (green) in sagittal sections of the Control and SU16f groups at 28 dpi. O-Q Higher magnification images of the boxed region in J and M. R Quantification of the density of NF + axons in the GFAP − area at 28 dpi. S Quantification of the percentage of 5-HT + area in the area of the spinal cord segment spanning the injured core at 28 dpi. Asterisk indicates the injured core. Scale bars: 200 μm in G and N, 20 μm in H and 50 μm in Q. *P < 0.05 and ****P < 0.0001 by Student's t test, n = 5 animals per group
Background Excessively deposited fibrotic scar after spinal cord injury (SCI) inhibits axon regeneration. It has been reported that platelet-derived growth factor receptor beta (PDGFRβ), as a marker of fibrotic scar-forming fibroblasts, can only be activated by platelet-derived growth factor (PDGF) B or PDGFD. However, whether the activation of the PDGFRβ pathway can mediate fibrotic scar formation after SCI remains unclear. Methods A spinal cord compression injury mouse model was used. In situ injection of exogenous PDGFB or PDGFD in the spinal cord was used to specifically activate the PDGFRβ pathway in the uninjured spinal cord, while intrathecal injection of SU16f was used to specifically block the PDGFRβ pathway in the uninjured or injured spinal cord. Immunofluorescence staining was performed to explore the distributions and cell sources of PDGFB and PDGFD, and to evaluate astrocytic scar, fibrotic scar, inflammatory cells and axon regeneration after SCI. Basso Mouse Scale (BMS) and footprint analysis were performed to evaluate locomotor function recovery after SCI. Results We found that the expression of PDGFD and PDGFB increased successively after SCI, and PDGFB was mainly secreted by astrocytes, while PDGFD was mainly secreted by macrophages/microglia and fibroblasts. In addition, in situ injection of exogenous PDGFB or PDGFD can lead to fibrosis in the uninjured spinal cord, while this profibrotic effect could be specifically blocked by the PDGFRβ inhibitor SU16f. We then treated the mice after SCI with SU16f and found the reduction of fibrotic scar, the interruption of scar boundary and the inhibition of lesion and inflammation, which promoted axon regeneration and locomotor function recovery after SCI. Conclusions Our study demonstrates that activation of PDGFRβ pathway can directly induce fibrotic scar formation, and specific blocking of this pathway would contribute to the treatment of SCI.
Background Diabetic retinopathy and retinal vein occlusion are vision threatening retinal vascular diseases. Current first-line therapy targets the vascular component, but many patients are treatment-resistant due to unchecked inflammation. Non-invasive inflammatory imaging biomarkers are a significant unmet clinical need for patients. Imaging of macrophage-like cells on the surface of the retina using clinical optical coherence tomography (OCT) is an emerging field. These cells are increased in patients with retinal vascular disease, and could be a potential inflammatory biomarker. However, since OCT is limited by an axial resolution of 5–10 microns, the exact location and identity of these retinal cells is currently unknown. Methods We performed OCT followed by confocal immunofluorescence in wild-type mice to identify macrophages within 5–10 microns of the vitreoretinal interface. Next, we used Cx3cr1CreER/+; Rosa26zsGreen/+ mice to fate map retinal surface macrophages. Using confocal immunofluorescence of retinal sections and flatmounts, we quantified IBA1⁺Tmem119⁺CD169neg microglia, IBA1⁺Tmem119negCD169neg perivascular macrophages, and IBA1⁺Tmem119negCD169⁺ vitreal hyalocytes. Finally, we modeled neuroinflammation with CCL2 treatment and characterized retinal surface macrophages using flow cytometry, OCT, and confocal immunofluorescence. Results We were able to detect IBA1⁺ macrophages within 5–10 microns of the vitreoretinal interface in wild-type mice using OCT followed by confirmatory confocal immunofluorescence. Retinal surface macrophages were 83.5% GFP⁺ at Week 1 and 82.4% GFP⁺ at Week 4 using fate mapping mice. At steady state, these macrophages included 82% IBA1⁺Tmem119⁺CD169neg microglia, 9% IBA1⁺Tmem119negCD169⁺ vitreal hyalocytes, and 9% IBA1⁺Tmem119negCD169neg perivascular macrophages. After CCL2-driven neuroinflammation, many Ly6C⁺ cells were detectable on the retinal surface using OCT followed by confocal immunofluorescence. Conclusions Macrophages within close proximity to the vitreoretinal interface are self-renewing cells, and predominantly microglia with minor populations of perivascular macrophages and vitreal hyalocytes at steady state. In the context of neuroinflammation, monocytes and monocyte-derived macrophages are a significant component of retinal surface macrophages. Human OCT-based imaging of retinal surface macrophages is a potential biomarker for inflammation during retinal vascular disease.
Background Apoptosis signal-regulating kinase 1 (ASK1) not only causes neuronal programmed cell death via the mitochondrial pathway but also is an essential component of the signalling cascade during microglial activation. We hypothesize that ASK1 selective deletion modulates inflammatory responses in microglia/macrophages(Mi/Mϕ) and attenuates seizure severity and long-term cognitive impairments in an epileptic mouse model. Methods Mi/Mϕ-specific ASK1 conditional knockout (ASK1 cKO) mice were obtained for experiments by mating ASK1 flox/flox mice with CX3CR1 creER mice with tamoxifen induction. Epileptic seizures were induced by intrahippocampal injection of kainic acid (KA). ASK1 expression and distribution were detected by western blotting and immunofluorescence staining. Seizures were monitored for 24 h per day with video recordings. Cognition, social and stress related activities were assessed with the Y maze test and the three-chamber social novelty preference test. The heterogeneous Mi/Mϕ status and inflammatory profiles were assessed with immunofluorescence staining and real-time polymerase chain reaction (q-PCR). Immunofluorescence staining was used to detect the proportion of Mi/Mϕ in contact with apoptotic neurons, as well as neuronal damage. Results ASK1 was highly expressed in Mi/Mϕ during the acute phase of epilepsy. Conditional knockout of ASK1 in Mi/Mϕ markedly reduced the frequency of seizures in the acute phase and the frequency of spontaneous recurrent seizures (SRSs) in the chronic phase. In addition, ASK1 conditional knockout mice displayed long-term neurobehavioral improvements during the Y maze test and the three-chamber social novelty preference test. ASK1 selective knockout mitigated neuroinflammation, as evidenced by lower levels of Iba1 ⁺ /CD16 ⁺ proinflammatory Mi/Mϕ. Conditional knockout of ASK1 increased Mi/Mϕ proportion in contact with apoptotic neurons. Neuronal loss was partially restored by ASK1 selective knockout. Conclusion Conditional knockout of ASK1 in Mi/Mϕ reduced seizure severity, neurobehavioral impairments, and histological damage, at least via inhibiting proinflammatory microglia/macrophages responses. ASK1 in microglia/macrophages is a potential therapeutic target for inflammatory responses in epilepsy.
Background Histone deacetylases (HDACs) are believed to exacerbate traumatic brain injury (TBI) based on studies using pan-HDAC inhibitors. However, the HDAC isoform responsible for the detrimental effects and the cell types involved remain unknown, which may hinder the development of specific targeting strategies that boost therapeutic efficacy while minimizing side effects. Microglia are important mediators of post-TBI neuroinflammation and critically impact TBI outcome. HDAC3 was reported to be essential to the inflammatory program of in vitro cultured macrophages, but its role in microglia and in the post-TBI brain has not been investigated in vivo. Methods We generated HDAC3 LoxP mice and crossed them with CX3CR1 CreER mice, enabling in vivo conditional deletion of HDAC3. Microglia-specific HDAC3 knockout (HDAC3 miKO) was induced in CX3CR1 CreER :HDAC3 LoxP mice with 5 days of tamoxifen treatment followed by a 30-day development interval. The effects of HDAC3 miKO on microglial phenotype and neuroinflammation were examined 3–5 days after TBI induced by controlled cortical impact. Neurological deficits and the integrity of white matter were assessed for 6 weeks after TBI by neurobehavioral tests, immunohistochemistry, electron microscopy, and electrophysiology. Results HDAC3 miKO mice harbored specific deletion of HDAC3 in microglia but not in peripheral monocytes. HDAC3 miKO reduced the number of microglia by 26%, but did not alter the inflammation level in the homeostatic brain. After TBI, proinflammatory microglial responses and brain inflammation were markedly alleviated by HDAC3 miKO, whereas the infiltration of blood immune cells was unchanged, suggesting a primary effect of HDAC3 miKO on modulating microglial phenotype. Importantly, HDAC3 miKO was sufficient to facilitate functional recovery for 6 weeks after TBI. TBI-induced injury to axons and myelin was ameliorated, and signal conduction by white matter fiber tracts was significantly enhanced in HDAC3 miKO mice. Conclusion Using a novel microglia-specific conditional knockout mouse model, we delineated for the first time the role of microglial HDAC3 after TBI in vivo. HDAC3 miKO not only reduced proinflammatory microglial responses, but also elicited long-lasting improvement of white matter integrity and functional recovery after TBI. Microglial HDAC3 is therefore a promising therapeutic target to improve long-term outcomes after TBI.
Background The close interaction and interdependence of astrocytes and neurons allows for the possibility that astrocyte dysfunction contributes to and amplifies neurodegenerative pathology. Molecular pathways that trigger reactive astrocytes may represent important targets to preserve normal homeostatic maintenance and modify disease progression. Methods Semaphorin 4D (SEMA4D) expression in the context of disease-associated neuropathology was assessed in postmortem brain sections of patients with Huntington’s (HD) and Alzheimer’s disease (AD), as well as in mouse models of HD (zQ175) and AD (CVN; APPSwDI/NOS2 −/− ) by immunohistochemistry. Effects of SEMA4D antibody blockade were assessed in purified astrocyte cultures and in the CVN mouse AD model. CVN mice were treated weekly from 26 to 38 weeks of age; thereafter mice underwent cognitive assessment and brains were collected for histopathology. Results We report here that SEMA4D is upregulated in neurons during progression of neurodegenerative diseases and is a trigger of reactive astrocytes. Evidence of reactive astrocytes in close proximity to neurons expressing SEMA4D is detected in brain sections of patients and mouse models of HD and AD. We further report that SEMA4D-blockade prevents characteristic loss of GABAergic synapses and restores spatial memory and learning in CVN mice, a disease model that appears to reproduce many features of AD-like pathology including neuroinflammation. In vitro mechanistic studies demonstrate that astrocytes express cognate receptors for SEMA4D and that ligand binding triggers morphological variations, and changes in expression of key membrane receptors and enzymes characteristic of reactive astrocytes. These changes include reductions in EAAT-2 glutamate transporter and glutamine synthetase, key enzymes in neurotransmitter recycling, as well as reduced GLUT-1 glucose and MCT-4 lactate transporters, that allow astrocytes to couple energy metabolism with synaptic activity. Antibody blockade of SEMA4D prevented these changes and reversed functional deficits in glucose uptake. Conclusions Collectively, these results suggest that SEMA4D blockade may ameliorate disease pathology by preserving normal astrocyte function and reducing the negative consequences of reactive astrogliosis.
Serum levels of main cytokines across persistent CE. CE cerebral edema. *P value < 0.05 was evaluated by the multivariable logistic regression after adjusting age, sex, risk factors and comorbidities
Clinical course according to the persistent CE (A) and change of CE (B). CE cerebral edema; DCI delayed cerebral ischemia, mRS modified Rankin scale. *P value < 0.05 was evaluated by Pearson chi-square test
Background: Cerebral edema (CE) at admission is a surrogate marker of 'early brain injury' (EBI) after subarachnoid hemorrhage (SAH). Only recently has the focus on the changes in CE after SAH such as delayed resolution or newly developed CE been examined. Among several factors, an early systemic inflammatory response has been shown to be associated with CE. We investigate inflammatory markers in subjects with early CE which does not resolve, i.e., persistent CE after SAH. Methods: Computed tomography scans of SAH patients were graded at admission and at 7 days after SAH for CE using the 0-4 'subarachnoid hemorrhage early brain edema score' (SEBES). SEBES ≤ 2 and SEBES ≥ 3 were considered good and poor grade, respectively. Serum samples from the same subject cohort were collected at 4 time periods (at < 24 h [T1], at 24 to 48 h [T2]. 3-5 days [T3] and 6-8 days [T4] post-admission) and concentration levels of 17 cytokines (implicated in peripheral inflammatory processes) were measured by multiplex immunoassay. Multivariable logistic regression analyses were step-wisely performed to identify cytokines independently associated with persistent CE adjusting for covariables including age, sex and past medical history (model 1), and additional inclusion of clinical and radiographic severity of SAH and treatment modality (model 2). Results: Of the 135 patients enrolled in the study, 21 of 135 subjects (15.6%) showed a persistently poor SEBES grade. In multivariate model 1, higher Eotaxin (at T1 and T4), sCD40L (at T4), IL-6 (at T1 and T3) and TNF-α (at T4) were independently associated with persistent CE. In multivariate model 2, Eotaxin (at T4: odds ratio [OR] = 1.019, 95% confidence interval [CI] = 1.002-1.035) and possibly PDGF-AA (at T4), sCD40L (at T4), and TNF-α (at T4) was associated with persistent CE. Conclusions: We identified serum cytokines at different time points that were independently associated with persistent CE. Specifically, persistent elevations of Eotaxin is associated with persistent CE after SAH.
Background Pyroptosis is a programmed cell death mediated by inflammasomes. Previous studies have reported that inhibition of neurokinin receptor 1 (NK1R) exerted neuroprotection in several neurological diseases. Herein, we have investigated the role of NK1R receptor inhibition using Aprepitant to attenuate NLRC4-dependent neuronal pyroptosis after intracerebral hemorrhage (ICH), as well as the underlying mechanism. Methods A total of 182 CD-1 mice were used. ICH was induced by injection of autologous blood into the right basal ganglia. Aprepitant, a selective antagonist of NK1R, was injected intraperitoneally at 1 h after ICH. To explore the underlying mechanism, NK1R agonist, GR73632, and protein kinase C delta (PKCδ) agonist, phorbol 12-myristate 13-acetate (PMA), were injected intracerebroventricularly at 1 h after ICH induction, and small interfering ribonucleic acid (siRNA) for NLRC4 was administered via intracerebroventricular injection at 48 h before ICH induction, respectively. Neurobehavioral tests, western blot, and immunofluorescence staining were performed. Results The expression of endogenous NK1R and NLRC 4 were gradually increased after ICH. NK1R was expressed on neurons. Aprepitant significantly improved the short- and long-term neurobehavioral deficits after ICH, which was accompanied with decreased neuronal pyroptosis, as well as decreased expression of NLRC4, Cleaved-caspase-1, GSDMD (gasdermin D), IL-1β, and IL-18. Activation of NK1R or PKCδ abolished these neuroprotective effects of Aprepitant after ICH. Similarly, knocking down NLRC4 using siRNA produced similar neuroprotective effects. Conclusion Aprepitant suppressed NLRC4-dependent neuronal pyroptosis and improved neurological function, possibly mediated by inhibition of NK1R/PKCδ signaling pathways after ICH. The NK1R may be a promising therapeutic target for the treatment of ICH.
ITPR1 protein expression, as detected by immunohistochemistry, in the cerebellum (a), cerebral cortex (b), hippocampus (c), and lateral ventricle wall/basal ganglia (d), together with protein expression scores (e)
(modified images from the Human Protein Atlas image database [40];; licensed under the Creative Commons Attribution-ShareAlike 3.0 International License)
Background In 2014, we first described novel autoantibodies to the inositol 1,4,5-trisphosphate receptor type 1 (ITPR1-IgG/anti-Sj) in patients with autoimmune cerebellar ataxia (ACA) in this journal. Here, we provide a review of the available literature on ITPR1-IgG/anti-Sj, covering clinical and paraclinical presentation, tumour association, serological findings, and immunopathogenesis. Methods Review of the peer-reviewed and PubMed-listed English language literature on ITPR1-IgG/anti-Sj. In addition, we provide an illustrative report on a new patient with ITPR1-IgG-associated encephalitis with cognitive decline and psychosis. Results So far, at least 31 patients with serum ITPR1-IgG/anti-Sj have been identified (clinical information available for 21). The most common manifestations were ACA, encephalopathy with seizures, myelopathy, and (radiculo)neuropathy, including autonomic neuropathy. In 45% of cases, an underlying tumour was present, making the condition a facultative paraneoplastic neurological disorder. The neurological syndrome preceded tumour diagnosis in all but one case. In most cases, immunotherapy had only moderate or no effect. The association of ITPR1-IgG/anti-Sj with manifestations other than ACA is corroborated by the case of a 48-year-old woman with high-titre ITPR1-IgG/anti-Sj antibodies and rapid cognitive decline, affecting memory, attention and executive function, and psychotic manifestations, including hallucinations, investigated here in detail. FDG-PET revealed right-temporal glucose hypermetabolism compatible with limbic encephalitis. Interestingly, ITPR1-IgG/anti-Sj mainly belonged to the IgG2 subclass in both serum and cerebrospinal fluid (CSF) in this and further patients, while it was predominantly IgG1 in other patients, including those with more severe outcome, and remained detectable over the entire course of disease. Immunotherapy with intravenous methylprednisolone, plasma exchange, and intravenous immunoglobulins, was repeatedly followed by partial or complete recovery. Long-term treatment with cyclophosphamide was paralleled by relative stabilization, although the patient noted clinical worsening at the end of each treatment cycle. Conclusions The spectrum of neurological manifestations associated with ITPR1 autoimmunity is broader than initially thought. Immunotherapy may be effective in some cases. Studies evaluating the frequency of ITPR1-IgG/anti-Sj in patients with cognitive decline and/or psychosis of unknown aetiology are warranted. Tumour screening is essential in patients presenting with ITPR1-IgG/anti-Sj.
Background Valproic acid (VPA) is a clinically used antiepileptic drug, but it is associated with a significant risk of a low verbal intelligence quotient (IQ) score, attention-deficit hyperactivity disorder and autism spectrum disorder in children when it is administered during pregnancy. Prenatal VPA exposure has been reported to affect neurogenesis and neuronal migration and differentiation. In addition, growing evidence has shown that microglia and brain immune cells are activated by VPA treatment. However, the role of VPA-activated microglia remains unclear. Methods Pregnant female mice received sodium valproate on E11.5. A microglial activation inhibitor, minocycline or a CCR5 antagonist, maraviroc was dissolved in drinking water and administered to dams from P1 to P21. Measurement of microglial activity, evaluation of neural circuit function and expression analysis were performed on P10. Behavioral tests were performed in the order of open field test, Y-maze test, social affiliation test and marble burying test from the age of 6 weeks. Results Prenatal exposure of mice to VPA induced microglial activation and neural circuit dysfunction in the CA1 region of the hippocampus during the early postnatal periods and post-developmental defects in working memory and social interaction and repetitive behaviors. Minocycline, a microglial activation inhibitor, clearly suppressed the above effects, suggesting that microglia elicit neural dysfunction and behavioral disorders. Next-generation sequencing analysis revealed that the expression of a chemokine, C–C motif chemokine ligand 3 (CCL3), was upregulated in the hippocampi of VPA-treated mice. CCL3 expression increased in microglia during the early postnatal periods via an epigenetic mechanism. The CCR5 antagonist maraviroc significantly suppressed neural circuit dysfunction and post-developmental behavioral disorders induced by prenatal VPA exposure. Conclusion These findings suggest that microglial CCL3 might act during development to contribute to VPA-induced post-developmental behavioral abnormalities. CCR5-targeting compounds such as maraviroc might alleviate behavioral disorders when administered early.
Background Demyelinating diseases in central nervous system (CNS) are a group of diseases characterized by myelin damage or myelin loss. Transforming growth factor beta1 (TGF-β1) is widely recognized as an anti-inflammatory cytokine, which can be produced by both glial and neuronal cells in CNS. However, the effects of TGF-β1 on demyelinating diseases and its underlying mechanisms have not been well investigated. Methods A demyelinating mouse model using two-point injection of lysophosphatidylcholine (LPC) to the corpus callosum in vivo was established. Exogenous TGF-β1 was delivered to the lesion via brain stereotactic injection. LFB staining, immunofluorescence, and Western blot were applied to examine the severity of demyelination and pyroptosis process in microglia. Morris water maze test was used to assess the cognitive abilities of experimental mice. Furthermore, lipopolysaccharide (LPS) was applied to induce pyroptosis in primary cultured microglia in vitro, to explore potential molecular mechanism. Results The degree of demyelination in LPC-modeling mice was found improved with supplement of TGF-β1. Besides, TGF-β1 treatment evidently ameliorated the activated proinflammatory pyroptosis of microglia, with downregulated levels of the key pyroptosis effector Gasdermin D (GSDMD), inflammasomes, and cleaved-IL-1β, which effectively attenuated neuroinflammation in vivo. Evaluated by behavioral tests, the cognitive deficit in LPC-modeling mice was found mitigated with application of TGF-β1. Mechanistically, TGF-β1 could reverse pyroptosis-like morphology in LPS-stimulated primary cultured microglia observed by scanning electron microscopy, as well as decrease the protein levels of cleaved-GSDMD, inflammasomes, and cleaved-IL-1β. Activation of ERK1/2 and NF-κB pathways largely abolished the protective effects of TGF-β1, which indicated that TGF-β1 alleviated the pyroptosis possibly via regulating NF-κB/ERK1/2 signal pathways. Conclusions Our studies demonstrated TGF-β1 notably relieved the demyelinating injury and cognitive disorder in LPC-modeling mice, by attenuating the inflammatory pyroptosis of microglia via ERK1/2 and NF-κB pathways. Targeting TGF-β1 activity might serve as a promising therapeutic strategy in demyelinating diseases.
Glyphosate and its major metabolite are detectable in brain tissue. A C57BL/6J mice were orally gavaged for 14 days, with urine being collected on the last 3 days. Blood was collected at endpoint, 4 h after the last dosage on day 14, followed by perfusion and postmortem analysis. B Levels of glyphosate detected in the brain tissue revealed a significant dose-dependent response between the four groups. C Levels of AMPA detected in the brain tissue are elevated in the highest two doses. D Level of glyphosate detected in mouse urine is elevated in the 500 mg/kg groups compared to the lower doses. E Positive correlation between levels of glyphosate and AMPA in the brain (p < 0.0001). F Positive correlation between brain and urine glyphosate (p = 0.0182). Data in A–C are presented as boxplots. The center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution. *p < .05 **p < .01, ***p < .001, ****p < .0001
Levels of TNFα are elevated with glyphosate exposure. A Plasma concentration of TNFα are significantly elevated after 500 mg/kg daily exposure compared to all other doses. B TNFα levels in cohort 1 whole brain homogenates are significantly increased after glyphosate exposure in all three doses. C Levels of TNFα are elevated in isolated hippocampal homogenates from cohort 2 mice in a dose-dependent manner. D Levels of TNFα in isolated cortical homogenates from cohort 2 mice are elevated in a dose-dependent manner. Data are presented as boxplots. The center line represents the median value, the limits represent the 25th and 75th percentile, and the whiskers represent the minimum and maximum value of the distribution. **p < .01, ****p < .0001
Correlations between urine and brain glyphosate and peripheral blood plasma and brain TNFα measures. A Significant positive correlation between brain glyphosate and brain TNFα levels (p < 0.0001). B Significant positive correlation between brain glyphosate and peripheral blood plasma TNFα levels (p < 0.0001). C Trending correlation of urine glyphosate and brain TNFα levels (p = 0.0604). D Significant positive correlation of urine glyphosate and peripheral blood plasma TNFα levels (p = 0.0206)
Glyphosate exposure increases Aβ40-42 and increase cell death in vitro. A Primary cortical neurons were derived from APP/PS1 pups and incubated. Glyphosate was introduced at dosages of 0 µg/mL (vehicle), 10 µg/mL, 20 µg/mL and 40 µg/mL. After 24 h of glyphosate incubation, media was collected, and cell viability was examined. B Soluble Aβ40 levels are increased in the 40 µg/mL (p < 0.0001) and 20 µg/mL (p = 0.001) glyphosate treated groups compared to the 0 µg/mL (vehicle) group. C Soluble Aβ42 levels are increased in a dose-dependent manner in the 40 µg/mL (p < 0.0001), 20 µg/mL (p = 0.001), and 10 µg/mL (p = 0.0092) glyphosate treated groups compared to compared to the 0 µg/mL (vehicle) group. D The 40 µg/mL group had a reduced cell viability when compared to the 20 µg/mL (p = 0.0007), 10 µg/mL (p < 0.0001), and the 0 µg/mL (p < 0.0001) group. Scatterplots with bar graphs are means ± SE. *p < .05 **p < .01, ***p < .001
Glyphosate exposure alters the brain transcriptome. A Volcano plot showing the differential expression analysis results after dosage regression. Genes in blue and red were downregulated and upregulated, respectively, after glyphosate exposure (adj-p < 0.05). B Volcano plots showing differential expression in individual cell class following deconvolution analysis. A: astrocytes; EC: endothelial cells; GB_N: GABAergic neurons; GL_N: glutamatergic Neurons; M: microglia; NoEnrichment: unclassified cell type; O: oligodendrocytes; OPC: oligodendrocyte progenitor cells. Colored dots represent dysregulated gene expression. C Boxplot of normalized counts for Plp1 showing significant dose-dependent upregulation (p adj. = 0.034). D Boxplot of normalized counts for Cntn2 showing significant dose-dependent upregulation (p adj. = 0.029). E Boxplot of normalized counts for Arhgef10 showing significant dose-dependent upregulation (p adj. = 0.010). F Boxplot of normalized counts for Abca2 showing significant dose-dependent upregulation (p adj. = 0.008)
Background Herbicides are environmental contaminants that have gained much attention due to the potential hazards they pose to human health. Glyphosate, the active ingredient in many commercial herbicides, is the most heavily applied herbicide worldwide. The recent rise in glyphosate application to corn and soy crops correlates positively with increased death rates due to Alzheimer’s disease and other neurodegenerative disorders. Glyphosate has been shown to cross the blood–brain barrier in in vitro models, but has yet to be verified in vivo. Additionally, reports have shown that glyphosate exposure increases pro-inflammatory cytokines in blood plasma, particularly TNFα. Methods Here, we examined whether glyphosate infiltrates the brain and elevates TNFα levels in 4-month-old C57BL/6J mice. Mice received either 125, 250, or 500 mg/kg/day of glyphosate, or a vehicle via oral gavage for 14 days. Urine, plasma, and brain samples were collected on the final day of dosing for analysis via UPLC–MS and ELISAs. Primary cortical neurons were derived from amyloidogenic APP/PS1 pups to evaluate in vitro changes in Aβ 40-42 burden and cytotoxicity. RNA sequencing was performed on C57BL/6J brain samples to determine changes in the transcriptome. Results Our analysis revealed that glyphosate infiltrated the brain in a dose-dependent manner and upregulated TNFα in both plasma and brain tissue post-exposure. Notably, glyphosate measures correlated positively with TNFα levels. Glyphosate exposure in APP/PS1 primary cortical neurons increases levels of soluble Aβ 40-42 and cytotoxicity. RNAseq revealed over 200 differentially expressed genes in a dose-dependent manner and cell-type-specific deconvolution analysis showed enrichment of key biological processes in oligodendrocytes including myelination, axon ensheathment, glial cell development, and oligodendrocyte development. Conclusions Collectively, these results show for the first time that glyphosate infiltrates the brain, elevates both the expression of TNFα and soluble Aβ, and disrupts the transcriptome in a dose-dependent manner, suggesting that exposure to this herbicide may have detrimental outcomes regarding the health of the general population.
Background No reports exist as to neuroprotective effects associated with topical activation of transient receptor potential melastatin 8 (TRPM8), a noted cold receptor. In the present study, we identified whether activating peripheral TRPM8 can be an adjuvant therapy for ischemic stroke. Methods Menthol, an agonist of TRPM8, was applied orally or topically to all paws or back of the mouse after middle cerebral artery occlusion (MCAO). We used Trpm8 gene knockout (Trpm8−/−) mice or TRPM8 antagonist and lidocaine to validate the roles of TRPM8 and peripheral nerve conduction in menthol against ischemic stroke. Results Application of menthol 16% to paw derma attenuated infarct volumes and ameliorated sensorimotor deficits in stroke mice induced by MCAO. The benefits of topically applied menthol were associated with reductions in oxidative stress, neuroinflammation and infiltration of monocytes and macrophages in ischemic brains. Antagonizing TRPM8 or Trpm8 knockout dulls the neuroprotective effects of topically application of menthol against MCAO. Immunohistochemistry analyses revealed significantly higher TRPM8 expression in skin tissue samples obtained from the paws compared with skin from the backs, which was reflected by significantly smaller infarct lesion volumes and better sensorimotor function in mice treated with menthol on the paws compared with the back. Blocking conduction of peripheral nerve in the four paws reversed the neuroprotective effects of topical menthol administrated to paws. On the other hand, oral menthol dosing did not assist with recovery from MCAO in our study. Conclusion Our results suggested that activation of peripheral TRPM8 expressed in the derma tissue of limbs with sufficient concentration of menthol is beneficial to stroke recovery. Topical application of menthol on hands and feet could be a novel and simple-to-use therapeutic strategy for stroke patients.
Siglec-E suppresses prototypical proinflammatory mediators in LPS-activated microglia. A Mouse primary brain microglia from C57BL/6 wild type and Siglec-E knockout pups were prepared in 48-well plates (4 × 10⁴ cells/well). The purity of microglia in these cultures was assessed by immunostaining for the microglial marker Iba-1 (red fluorescence) and compared (n = 9, p = 0.678, Mann–Whitney U test). Note that cell nuclei were stained with DAPI (blue fluorescence) to illustrate all cell types. Scale bar: 50 μm. The cultured cells were then stimulated with LPS (0, 1, 10, or 100 ng/mL) for 16 h. A number of key proinflammatory mediators that were secreted by LPS-activated microglia into the culture medium, such as PGE2 (B), IL-1β (C), IL-6 (D), and TNF-α (E), were measured by ELISA. Note that all these conventional proinflammatory mediators produced by microglia were induced by LPS treatment in a concentration-dependent manner and were further dramatically increased in the absence of Siglec-E (n = 8–12, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, two-way ANOVA with post hoc Šidák multiple comparisons). All data are presented as mean ± SEM
Ablation of Siglec-E exacerbates microglial activation in the brain. A C57BL/6 wild type and Siglec-E knockout mice were systemically treated by LPS (0, 3, or 5 mg/kg, i.p.), and 24 h later the mRNA expression of Siglec-E in the hippocampus was measured by qPCR (n = 5–8, *p = 0.01, Kruskal–Wallis test with post hoc Dunn’s multiple comparisons). Data are visualized using box plot. B Reverse transcription PCR was performed to examine the Siglec-E mRNA expression in the hippocampal tissues of wild type and Siglec-E knockout mice treated by LPS, with GAPDH as control. C Immunostaining for Siglec-E (green fluorescence) and Iba1 (red fluorescence) indicating the hippocampal microglial activation in wild type and Siglec-E knockout mice was performed 24 h after LPS treatment (5 mg/kg, i.p.). Representative images are presented here to exemplify the induction of Siglec-E and Iba1 as well as their colocalization in activated microglia. Scale bar: 50 μm. D Iba1-positive (Iba1⁺) microglia in the hippocampus were counted (Left) and their Iba1 expression levels were quantified by measuring the fluorescence intensity (Right) (n = 4–6, *p < 0.05, **p < 0.01, Mann–Whitney U test). Data are shown as mean ± SEM
Siglec-E regulates the morphology of brain microglia. A The morphological analyses of brain microglia (Iba1⁺) in mice with 26 measurements (Table 1) were performed 24 h after LPS treatment (5 mg/kg, i.p.) using ImageJ/Fiji software. A radar chart was generated to show the morphological changes of brain microglia in mice by LPS treatment and deletion of Siglec-E. The statistical p values less than 0.1 were labeled (n = 4–6, *p < 0.05, **p < 0.01, Mann–Whitney U test). Note that the 26 measurements can be categorized into three clusters indicated by purple, green, and yellow arcs, showing that the measurements in wild-type control mice, when compared to other two groups, were higher, equivalent, and lower, respectively. B Principal component analysis of microglial morphology was performed using IBM SPSS Statistics software with 3000 microglia randomly sampled from each group. The observable core cluster of brain microglia in wild-type control group (black dots) is shown by an ellipse (~ 90%). The remaining cells were scattered either along the positive axis of principal component 1 (~ 6%) or along the negative axis of principal component 2 (~ 4%). Treatment with LPS (blue dots) increased the cells scattered out of the core to ~ 17%, which was further increased in LPS-treated Siglec-E knockout mice (red dots) to ~ 25%
Induced Siglec-E is neuroprotective after ischemia–reperfusion injuries. A Primary neuron–glia cultures derived from cortices of wild type or Siglec-E knockout mouse embryos were subjected to oxygen–glucose deprivation (OGD) for 1.5, 3, or 4.5 h. Following reoxygenation with full nutrition supply for 16 h, the cell viability in these cultures was measured and compared (n = 8–24, *p = 0.0172, two-way ANOVA with post hoc Šidák multiple comparisons). Data are presented as mean ± SEM. B Intraluminal filament-based middle cerebral artery occlusion (MCAO) model was utilized to examine the effects of genetic ablation of Siglec-E on cerebral ischemia. In this study, adult wild type and Siglec-E knockout male mice (12–14 weeks old) were subjected to transient MCAO for 30 min, which was followed by reperfusion for 72 h. C Siglec-E mRNA expression in the ipsilateral brain tissues of mice subjected to 30-min MCAO and 72-h reperfusion was measured by qPCR and compared to that of sham cohort (n = 7–14, ****p < 0.0001, Mann–Whitney U test). Data are visualized using box plot. D Neurological deficits of wild type and Siglec-E knockout mice after MCAO were evaluated at multiple time points using Bederson’s scale (n = 12–16, ***p < 0.001, two-way ANOVA). Data are presented as mean ± SEM. E Deficiency of Siglec-E in mice exacerbated post-stroke weight loss (n = 12–16, ***p < 0.001, two-way ANOVA with post hoc Šidák multiple comparisons). Data are presented as mean ± SEM. F Triphenyltetrazolium chloride (TTC) staining was performed to measure the brain infarction in wild type and Siglec-E knockout mice 72 h after MCAO. Representative images from each cohort are shown. The viable brain parenchyma appeared reddish, whereas the infarcted areas were pale and highlighted. G The volumes of brain infarcts in wild type and Siglec-E knockout mice were quantified and compared (n = 10–14, *p = 0.022, Mann–Whitney U test). Data are presented as mean ± SEM. H Animal mortality over 72 h following transient MCAO for 30 min (n = 14–17, p = 0.5734, log-rank test)
Sialic acid immunoglobulin-like lectin E (Siglec-E) is a subtype of pattern recognition receptors found on the surface of myeloid cells and functions as a key immunosuppressive checkpoint molecule. The engagement between Siglec-E and the ligand α 2,8 -linked disialyl glycans activates the immunoreceptor tyrosine-based inhibitory motif (ITIM) in its intracellular domain, mitigating the potential risk of autoimmunity amid innate immune attacks on parasites, bacteria, and carcinoma. Recent studies suggest that Siglec-E is also expressed in the CNS, particularly microglia, the brain-resident immune cells. However, the functions of Siglec-E in brain inflammation and injuries under many neurological conditions largely remain elusive. In this study, we first revealed an anti-inflammatory role for Siglec-E in lipopolysaccharide (LPS)-triggered microglial activation. We then found that Siglec-E was induced within the brain by systemic treatment with LPS in mice in a dose-dependent manner, while its ablation exacerbated hippocampal reactive microgliosis in LPS-treated animals. The genetic deficiency of Siglec-E also aggravated oxygen–glucose deprivation (OGD)-induced neuronal death in mouse primary cortical cultures containing both neurons and glial cells. Moreover, Siglec-E expression in ipsilateral brain tissues was substantially induced following middle cerebral artery occlusion (MCAO). Lastly, the neurological deficits and brain infarcts were augmented in Siglec-E knockout mice after moderate MCAO when compared to wild-type animals. Collectively, our findings suggest that the endogenous inducible Siglec-E plays crucial anti-inflammatory and neuroprotective roles following ischemic stroke, and thus might underlie an intrinsic mechanism of resolution of inflammation and self-repair in the brain.
Analyses of CD45 subsets in MDMs and microglia at 7 days the post-ischemic brain. a Gating strategy for cell counts of CD11b + /CD45 + / NK1.1 − /Ly6G − microglia and MDMs in R1'-R4' . b Cell counts for CD45 Low and CD45 High subsets within R1'-R4' . c Gating strategy for analysis of Ki-67 expression in R1'-R4' . d Cell counts of Ki67 + /GFP − cells in R1' and R3' , as well as Ki67 + /GFP + cells in R2' and R4' . e Counts of cells in d divided into CD45 Low and CD45 High expression. f Gating strategy for analysis of CD11c expression in R1'-R4' . g Cell counts of CD11c + /GFP − cells in R1' and R3' , as well as CD11c + /GFP + cells in R2' and R4' . h Counts of cells in g divided into CD45 Low and CD45 High expression. Statistical significance was assessed using one-way ANOVA with post hoc Bonferroni comparison test in d and g, whereas Student T-test was used in b, e, and h (with * , ** , and *** , respectively, indicating p < 0.05, 0.01, and 0.001)
Assessment of MDM phenotype conversion upon phagocytosis. a Gating strategy for measuring phagocytic activity of CD45 + /CD11b + / NK1.1 − /Ly6G − microglia and MDMs isolated from the contra-and ipsi-lateral hemispheres in asplenic, stroked mice 1 day following an infusion of GFP + splenocytes that were incubated for 4 h with bead 580/605 . b Distribution of CD45 + subsets in GFP + MDMs with or without bead 580/605 , counts of CD45 Low and CD45 High with or without bead 580/605 , and CD45 Low /CD45 High ratio of MDMs in cultures either with or without bead 580/605 (n = 5/ group). Statistical significance was determined with Student T-test ( * and ** , respectively, indicate p < 0.05 and 0.01). c In vivo phagocytic activity of CD45 + /CD11b + /NK1.1 − /Ly6G − microglia and MDMs in asplenic mice receiving infusions of GFP + splenocytes and bead 580/605 on the 6th day after stroke. Phagocytic activities were determined for CD45 Low (L) and CD45 High (H) cells (n = 8/group). Statistical significance was determined using Student T-test between CD45 subsets ( * , ** , and **** , respectively, indicate p < 0.05, 0.01, and 0.0001)
Background Monocyte-derived macrophages (MDMs) and microglia elicit neural inflammation and clear debris for subsequent tissue repair and remodeling. The role of infiltrating MDMs in the injured brain, however, has been controversial due to overlapping antigen expression with microglia. In this study, we define the origin and function of MDMs in cerebral ischemia. Methods Using adoptive transfer of GFP ⁺ splenocytes into adult asplenic mice subjected to transient middle cerebral artery occlusion, we compared the role of CD11b + /CD45 ⁺ /NK1.1 − /Ly6G − MDMs and microglia in the ischemic brain. The phagocytic activities of MDMs and microglia were measured by the uptake of fluorescent beads both in vivo with mice infused with GFP ⁺ splenocytes and ex vivo with cultures of isolated brain immune cells. Results Stroke induced an infiltration of MDMs [GFP+] into the ipsilateral hemisphere at acute (3 days) and sub-acute phases (7 days) of post-stroke. At 7 days, the infiltrating MDMs contained both CD45 High and CD45 Low subsets. The CD45 High MDMs in the injured hemisphere exhibited a significantly higher proliferation capacity (Ki-67 expression levels) as well as higher expression levels of CD11c when compared to CD45 Low MDMs. The CD45 High and CD45 Low MDM subsets in the injured hemisphere were approximately equal populations, indicating that CD45 High MDMs infiltrating the ischemic brain changes their phenotype to CD45 Low microglia-like phenotype. Studies with fluorescent beads reveal high levels of MDM phagocytic activity in the post-stroke brain, but this phagocytic activity was exclusive to post-ischemic brain tissue and was not detected in circulating monocytes. By contrast, CD45 Low microglia-like cells had low levels of phagocytic activity when compared to CD45 High cells. Both in vivo and ex vivo studies also show that the phagocytic activity in CD45 High MDMs is associated with an increase in the CD45 Low /CD45 High ratio, indicating that phagocytosis promotes MDM phenotype conversion. Conclusions This study demonstrates that MDMs are the predominant phagocytes in the post-ischemic brain, with the CD45 High subset having the highest phagocytic activity levels. Upon phagocytosis, CD45 High MDMs in the post-ischemic brain adopt a CD45 Low phenotype that is microglia-like. Together, these studies reveal key roles for MDMs and their phagocytic function in tissue repair and remodeling following cerebral ischemia.
Background Nafamostat mesylate (nafamostat, NM) is an FDA-approved serine protease inhibitor that exerts anti-neuroinflammation and neuroprotective effects following rat spinal cord injury (SCI). However, clinical translation of nafamostat has been limited by an unclear administration time window and mechanism of action. Methods Time to first dose of nafamostat administration was tested on rats after contusive SCI. The optimal time window of nafamostat was screened by evaluating hindlimb locomotion and electrophysiology. As nafamostat is a serine protease inhibitor known to target thrombin, we used argatroban (Arg), a thrombin-specific inhibitor, as a positive control in the time window experiments. Western blot and immunofluorescence of thrombin expression level and its enzymatic activity were assayed at different time points, as well its receptor, the protease activated receptor 1 (PAR1) and downstream protein matrix metalloproteinase-9 (MMP9). Blood–spinal cord barrier (BSCB) permeability leakage indicator Evans Blue and fibrinogen were analyzed along these time points. The infiltration of peripheral inflammatory cell was observed by immunofluorescence. Results The optimal administration time window of nafamostat was 2–12 h post-injury. Argatroban, the thrombin-specific inhibitor, had a similar pattern. Thrombin expression peaked at 12 h and returned to normal level at 7 days post-SCI. PAR1, the thrombin receptor, and MMP9 were significantly upregulated after SCI. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). Nafamostat and argatroban significantly downregulated thrombin and MMP9 expression as well as thrombin activity in the spinal cord. Nafamostat inhibited thrombin enrichment in endothelial cells. Nafamostat administration at 2–12 h after SCI inhibited the leakage of Evans Blue in the epicenter and upregulated tight junction proteins (TJPs) expression. Nafamostat administration 8 h post-SCI effectively inhibited the infiltration of peripheral macrophages and neutrophils to the injury site. Conclusions Our study provides preclinical information of nafamostat about the administration time window of 2–12 h post-injury in contusive SCI. We revealed that nafamostat functions through inhibiting the thrombin-mediated BSCB breakdown and subsequent peripheral immune cells infiltration.
Background: There is growing evidence that neuroinflammation may contribute to schizophrenia neuropathology. Elevated pro-inflammatory cytokines are evident in the midbrain from schizophrenia subjects, findings that are driven by a subgroup of patients, characterised as a "high inflammation" biotype. Cytokines trigger the release of antibodies, of which immunoglobulin G (IgG) is the most common. The level and function of IgG is regulated by its transporter (FcGRT) and by pro-inflammatory IgG receptors (including FcGR3A) in balance with the anti-inflammatory IgG receptor FcGR2B. Testing whether abnormalities in IgG activity contribute to the neuroinflammatory abnormalities schizophrenia patients, particularly those with elevated cytokines, may help identify novel treatment targets. Methods: Post-mortem midbrain tissue from healthy controls and schizophrenia cases (n = 58 total) was used to determine the localisation and abundance of IgG and IgG transporters and receptors in the midbrain of healthy controls and schizophrenia patients. Protein levels of IgG and FcGRT were quantified using western blot, and gene transcript levels of FcGRT, FcGR3A and FcGR2B were assessed using qPCR. The distribution of IgG in the midbrain was assessed using immunohistochemistry and immunofluorescence. Results were compared between diagnostic (schizophrenia vs control) and inflammatory (high vs low inflammation) groups. Results: We found that IgG and FcGRT protein abundance (relative to β-actin) was unchanged in people with schizophrenia compared with controls irrespective of inflammatory subtype. In contrast, FcGRT and FcGR3A mRNA levels were elevated in the midbrain from "high inflammation" schizophrenia cases (FcGRT; p = 0.02, FcGR3A; p < 0.0001) in comparison to low-inflammation patients and healthy controls, while FcGR2B mRNA levels were unchanged. IgG immunoreactivity was evident in the midbrain, and approximately 24% of all individuals (control subjects and schizophrenia cases) showed diffusion of IgG from blood vessels into the brain. However, the intensity and distribution of IgG was comparable across schizophrenia cases and control subjects. Conclusion: These findings suggest that an increase in the pro-inflammatory Fcγ receptor FcGR3A, rather than an overall increase in IgG levels, contribute to midbrain neuroinflammation in schizophrenia patients. However, more precise information about IgG-Fcγ receptor interactions is needed to determine their potential role in schizophrenia neuropathology.
Background In chronic myelogenous leukemia, reciprocal translocation between chromosome 9 and chromosome 22 generates a chimeric protein, Bcr-Abl, that leads to hyperactivity of tyrosine kinase-linked signaling transduction. The therapeutic agent nilotinib inhibits Bcr-Abl/DDR1 and can cross the blood–brain barrier, but its potential impact on neuroinflammatory responses and cognitive function has not been studied in detail. Methods The effects of nilotinib in vitro and in vivo were assessed by a combination of RT-PCR, real-time PCR, western blotting, ELISA, immunostaining, and/or subcellular fractionation. In the in vitro experiments, the effects of 200 ng/mL LPS or PBS on BV2 microglial cells, primary microglia or primary astrocytes pre- or post-treated with 5 µM nilotinib or vehicle were evaluated. The in vivo experiments involved wild-type mice administered a 7-day course of daily injections with 20 mg/kg nilotinib (i.p.) or vehicle before injection with 10 mg/kg LPS (i.p.) or PBS. Results In BV2 microglial cells, pre- and post-treatment with nilotinib altered LPS-induced proinflammatory/anti-inflammatory cytokine mRNA levels by suppressing AKT/P38/SOD2 signaling. Nilotinib treatment also significantly downregulated LPS-stimulated proinflammatory cytokine levels in primary microglia and primary astrocytes by altering P38/STAT3 signaling. Experiments in wild-type mice showed that nilotinib administration affected LPS-mediated microglial/astroglial activation in a brain region-specific manner in vivo. In addition, nilotinib significantly reduced proinflammatory cytokine IL-1β, IL-6 and COX-2 levels and P38/STAT3 signaling in the brain in LPS-treated wild-type mice. Importantly, nilotinib treatment rescued LPS-mediated spatial working memory impairment and cortical dendritic spine number in wild-type mice. Conclusions Our results indicate that nilotinib can modulate neuroinflammatory responses and cognitive function in LPS-stimulated wild-type mice.
Inflammasome-dependent pathway of pyroptosis. A Inflammasome sensors are cytosolic proteins that contain a PYD and/or a CARD. They may also contain a LRR, NACHT, HIN-200 domain, B30.2 domain, C–C, B-box domain (B), BIR, or FIIND. Upon detection of specific stimuli, sensors with a PYD recruit adaptor protein ASC to mediate CARD–CARD interactions with the effector cysteine protease caspase-1. Of note, NLRC4 and murine NLRP1b can interact directly with caspase-1 without ASC recruiting them. Nek7 is an important component of the murine NLRP3 inflammasome, binding to the LRR and NACHT of NLRP3. B Canonical pathway: inflammasome sensors can be activated by various signals followed by oligomerization with ASC and pro-caspase-1. Activated caspase-1 cleaves GSDMD to release the N-terminal domain (GSDMD-N), which then induces pyroptosis. Caspase-1 also cleaves pro-IL-1β and pro-IL-18 into their active forms, which are released through GSDMD pores. C Noncanonical pathway: caspase-4/5/11 can be directly activated by LPS, leading to GSDMD cleavage and cell contents and K⁺ release. K⁺ efflux further promotes the activation of caspase-1. PYD pyrin domain, CARD caspase activation and recruitment domain, LRR leucine-rich repeat domain, NACHT nucleotide-binding NACHT domain, C–C coiled–coil domain, BIR baculovirus inhibitor of apoptosis repeat, FIIND function-to-find domain, Nek7 NIMA-related kinase 7, GSDMD gasdermin D, IL interleukin, LPS lipopolysaccharide
Inflammasome-independent pathway of pyroptosis. YopJ expressed during Yersinia infection inhibits TGF-β-activated kinase 1 (TAK1) and induces caspase-8-related cleavage of GSDMD and GSDMC. Neutrophil elastase, porcine epidemic diarrhea virus, and cathepsin G cleave GSDMD. Caspase-8 acts upstream of caspase-3 through the Bax/Bak signaling, while caspase-3 mediates GSDME cleavage. GZMA and GZMB activate GSDMB and GSDME, respectively. Streptococcal pyrogenic exotoxin B (SpeB) could cleave GSDMA and trigger pyroptosis
Molecular signaling pathway of pyroptosis in diabetic retinopathy. A Retinal microvascular endothelial cell (RMEC): increased extracellular ATP binds to P2X7R then activates NLRP3 inflammasome by causing K⁺ efflux and Ca²⁺ influx. TXNIP expression is induced through increased intracellular Ca²⁺ level, miR-590-3p/NOX4/ROS/TXNIP axis, or miR-20b-3p downregulation. After activation, the TXNIP shuttles to mitochondria, competes with apoptosis signal-regulating kinase 1 (ASK1), binds to NLRP3 and activates it; miR-590-3p also targets NLRP1. HG suppresses the voltage-dependent anion channel (VDAC1) expression, causing VDAC1/PINK1/Parkin-mediated mitophagy inhibition. Damaged mitochondria and mtROS accumulation results from impaired mitophagy-activated NLRP3 inflammasome. B Retinal pigment epithelium: hyperglycemia triggers the connexin43/ATP/P2X7R/Ca²⁺ influx pathway, mitochondrial ROS, miR-130a/TNF-α/SOD1/ROS pathway, or METTL3/miR-25-3p/PTEN/Akt/NLRP3 signaling cascade to activate NLRP3 inflammasome. C Pericyte: increased lncRNA MIAT competes with CASP1 mRNA for binding to miR-342-3p, blocking the CASP1 translation and CASP1-dependent pyroptosis. D Microglia (M1): MiR-30a downregulates NLRP3 expression. E Müller cell: hyperglycemia triggers the ROS/TXNIP axis to activate NLRP3 and downregulates the transcription factor nuclear receptor subfamily 4 group A member 2 (Nurr1) to suppress NLRP3 activation
Molecular signaling pathway of pyroptosis in age-related macular degeneration. A Priming of NLRP3 with TLR. B Activation of the NF‑κB pathway induces the transcription of NLRP3 and pro‑IL‑1β. C–H Components of drusen activate the NLRP3 inflammasome. C Ox-LDL can induce a large amount of ROS, targeting the CD36 receptor, thereby causing lysosomal disruption following cathepsin release, or promoting the ATP/P2X7R/Ca²⁺ influx pathway to activate NLRP3. D Complement components: C1Q initiates lysosomal rupture and release of cathepsin B to activate NLRP3 assembly, while C5a and C3 send priming signals to NLRP3 without a clear mechanism. E, F Aβ1-40 increases intracellular ROS via NOX4 and mitochondrial electron transport chain to activate NLRP3. G Aβ1-40 acts as a priming signal to activate the NF-κB pathway, which upregulates the transcription of NLRP3, pro-IL-18, and pro-IL-1β. H MicroRNAs: miR-191-5p is downregulated after Aβ1-40 stimulation, subsequently leading to an increase in C/EBPβ levels, resulting in the upregulation of NLRP3; miR-223 and miR-22-3p suppress NLRP3 expression. I Alu RNA due to DICER1 deficiency increases ROS production; Alu RNA-induced NF-κB-mediated NLRP3 activation and P2X7R signaling control NLRP3 inflammasome priming and activation, respectively. J ATP outflow via connexin43 hemichannels acts as a NLRP3 inflammasome signal 2 activator. K A2E, a major fluorophore in lipofuscin, activates NLRP3 by causing lysosomal damage and release of cathepsins into the cytoplasm. L Membrane attack complex (MAC) deposition triggers the assembly and activation of the NLRP3 inflammasome downstream of the Aβ1-40 priming signal. M The mature form of IL-18 mediates the activation of interleukin-1 receptor-associated kinases 1 and 4 (IRAK1 and IRAK4), which contributes to RPE cell death
Molecular signaling pathway of pyroptosis in glaucoma. A Elevated IOP results in the inhibition of hypoxia-induced mitophagy. As dysfunctional and fragmented mitochondria accumulate, the subsequent oxidative stress promotion induces NLRP3 activation. B Elevated IOP triggers pannexin and connexin hemichannels, induces ATP efflux, and promotes the ATP/P2X7R/Ca²⁺ influx pathway to activate the NLRP1/NLRP3/AIM2 inflammasome. C Retinal ischemic reperfusion injury triggers the release of high-mobility group box 1 (HMGB1) in the retina, which binds to TLR4, promotes the activation of caspase-8, subsequently regulating the activation of NLRP3 via NF-κB pathway. D CASP8-HIF-1α signaling is an upstream regulator of NLRP3/NLRP12/NLRC4 in high IOP-induced retinal ischemic injury and may initiate pyroptosis. E AMP-activated protein kinase (AMPK) activates NF-κB signaling and induces NLRP3 inflammasome assembly. F Elevated glutamate binds to the glutamate receptors on RGCs, causing a large influx of Ca²⁺ and mitochondrial dysfunction, thus triggering NLRP3 inflammasome activation. G Ocular hypertension drives astrocytes or Müller cells to release ATP to activate microglial cells via the ATP/P2X7R/NLRP3 pathway, which contributes to RGC death. H PM2.5, triggers oxidative stress, which activates NLRP3-mediated pyroptosis in trabecular meshwork cells and results in ocular hypertension
Pyroptosis is a programmed cell death characterized by swift plasma membrane disruption and subsequent release of cellular contents and pro-inflammatory mediators (cytokines), including IL‐1β and IL‐18. It differs from other types of programmed cell death such as apoptosis, autophagy, necroptosis, ferroptosis, and NETosis in terms of its morphology and mechanism. As a recently discovered form of cell death, pyroptosis has been demonstrated to be involved in the progression of multiple diseases. Recent studies have also suggested that pyroptosis is linked to various ocular diseases. In this review, we systematically summarized and discussed recent scientific discoveries of the involvement of pyroptosis in common ocular diseases, including diabetic retinopathy, age-related macular degeneration, AIDS-related human cytomegalovirus retinitis, glaucoma, dry eye disease, keratitis, uveitis, and cataract. We also organized new and emerging evidence suggesting that pyroptosis signaling pathways may be potential therapeutic targets in ocular diseases, hoping to provide a summary of overall intervention strategies and relevant multi-dimensional evaluations for various ocular diseases, as well as offer valuable ideas for further research and development from the perspective of pyroptosis.
Background We previously reported higher plasma levels of complement fragments C3a and C5a in neovascular Age-related Macular Degeneration (nAMD) patients with macular fibrosis. This study aimed to understand whether complement activation contributes to the development of macular fibrosis and the underlying mechanisms involved. Methods Complement activation was blocked using a C5 neutralizing antibody (BB5.1) in C57BL/6J mice after induction of subretinal fibrosis using the two-stage laser protocol. Fibrotic lesions were examined 10 days after the 2nd laser through fundus examination and immunohistochemistry. The expression of C5aR in fibrotic lesions and retinal pigment epithelial (RPE) cultures were examined by confocal microscopy. Primary murine RPE cells were treated with C3a or C5a (10–100 ng/mL) or TGF-β2 (10 ng/mL). Epithelial-to-mesenchymal transition (EMT) was assessed through various readouts. The expression of E-cadherin, vimentin, fibronectin, α-SMA, Slug, ERK/AKT and pSMAD2/3 were determined by Western blot and immunocytochemistry. Collagen contraction and wound-healing assays were used as functional readouts of EMT. The production of IL-6, TGF-β1, TGF-β2 and VEGF by RPE cells were determined by ELISA. PMX53 was used to block C5aR in RPE cultures and in vivo in mice with subretinal fibrosis. Results Extensive C5b-9 deposition was detected at the site of subretinal fibrosis. BB5.1 treatment completely abrogated complement activation and significantly reduced subretinal fibrosis. C5aR was detected in RPE and infiltrating MHC-II ⁺ cells in subretinal fibrosis. In vitro, RPE cells constitutively express C5/C5a and C5aR, and their expression was increased by TGF-β2 treatment. C5a but not C3a increased fibronectin, α-SMA, vimentin and Slug expression, and decreased E-cadherin expression in RPE cells. C5a treatment also increased the contractility and migration of RPE cells and enhanced the production of VEGF and TGF-β1/2. C5a treatment induced pSmad2/3 and pERK1/2 expression in RPE cells and this was blocked by PMX53. PMX53 treatment significantly reduced sodium fluorescein leakage in the subretinal fibrosis model, while collagen-I ⁺ lesions only mildly reduced. Conclusions Complement activation is critically involved in the development of subretinal fibrosis, partially through C5a–C5aR-mediated EMT in RPE cells. Targeting complement activation rather than C5a may be a novel approach for the management of macular fibrosis.
Background Depression is a recurrent and devastating mental disease that is highly prevalent worldwide. Prolonged exposure to stressful events or a stressful environment is detrimental to mental health. In recent years, an inflammatory hypothesis has been implicated in the pathogenesis of stress-induced depression. However, less attention has been given to the initial phases, when a series of stress reactions and immune responses are initiated. Peripheral CD4 ⁺ T cells have been reported as the major contributors to the occurrence of mental disorders. Chronic stress exposure-evoked release of cytokines can promote the differentiation of peripheral CD4 ⁺ cells into various phenotypes. Among them, Th17 cells have attracted much attention due to their high pathogenic potential in central nervous system (CNS) diseases. Thus, we intended to determine the crucial role of CD4 ⁺ Th17 cells in the development of specific subtypes of depression and unravel the underpinnings of their pathogenetic effect. Methods In the present research, a daily 6-h restraint stress paradigm was employed in rats for 28 successive days to mimic the repeated mild and predictable, but inevitable environmental stress in our daily lives. Then, depressive-like symptoms, brain–blood barrier (BBB) permeability, neuroinflammation, and the differentiation and functional changes of CD4 ⁺ cells were investigated. Results We noticed that restrained rats showed significant depressive-like symptoms, concomitant BBB disruption and neuroinflammation in the dorsal striatum (DS). We further observed a time-dependent increase in thymus- and spleen-derived naïve CD4 ⁺ T cells, as well as the aggregation of inflammatory Th17 cells in the DS during the period of chronic restraint stress (CRS) exposure. Moreover, increased Th17-derived cytokines in the brain can further impair the BBB integrity, thus allowing more immune cells and cytokines to gain easy access to the CNS. Our findings suggested that, through a complex cascade of events, peripheral immune responses were propagated to the CNS, and gradually exacerbated depressive-like symptoms. Furthermore, inhibiting the differentiation and function of CD4 ⁺ T cells with SR1001 in the early stages of CRS exposure ameliorated CRS-induced depressive-like behaviour and the inflammatory response. Conclusions Our data demonstrated that inflammatory Th17 cells were pivotal in accelerating the onset and exacerbation of depressive symptoms in CRS-exposed rats. This subtype of CD4 ⁺ T cells may be a promising therapeutic target for the early treatment of stress-induced depression.
MSC treatment attenuates persistent cognitive deficits induced by rmTBI in mice. A Representative immunofluorescent images of CD90 + cells were used to identify MSCs. Scale bar = 50 µm. B Representative immunofluorescent images of MAB1281 + cells were used to observe the migration of MSCs. Scale bar (left) = 100 µm; Scale bar (right) = 15 µm. C The rotarod test was tested on days 3 and 7 post-rmTBI. D The NOR test was tested on days 28-30 post-rmTBI. E-I The MWM test was performed on days 28-32 post-rmTBI. Representative training and spatial probe traces of mice in the sham, vehicle, MSCs, and Lip-1 groups (E). Escape latencies during the training period of MWM (F). The frequency of crossing the hidden platform during the probe trial of MWM (G). The first latency time to reach the platform during the probe trial of MWM (H). Swim speed during the probe trial of MWM (I). Data are expressed as mean ± SD and analyzed using one-way ANOVA with Dunnett's T3 post hoc test and Tukey's post hoc test (C, D, G, H, I). Repeated measures ANOVA with multiple comparisons was used for comparisons of escape latency (F). For vehicle group: **P < 0.01, ***P < 0.001 vs sham group; for MSCs group: # P < 0.05, ## P < 0.01, ### P < 0.001 vs vehicle group; for Lip-1 group: ## P < 0.01,
MSC treatment reduced pathological protein deposition and promoted glucose metabolism after rmTBI. A-D Western blot for APP, Aβ1-42, p-Tau, Tau-5 was performed on day 28 after rmTBI. Protein expression was normalized to GAPDH and expressed as a fold change in sham. Data are expressed as mean ± SD (n = 6) and analyzed using one-way ANOVA with Tukey's post hoc test (B) and one-way ANOVA with Dunnett's T3 post hoc test (D). E-G Reconstructed S16-TAU PET/CT images and [ 18 F] FDG PET/CT images of mice in the sham, vehicle, MSCs and Lip-1 groups (E). Bar graph showing maximum SUV values in different regions of the brain of mice, including the cortex and hippocampi. Data are expressed as mean ± SD (n = 3) and analyzed using one-way ANOVA with the LSD post hoc test (F, G). For vehicle group: **P < 0.01, and ***P < 0.001 vs sham group; for MSCs group: # P < 0.05, ## P < 0.01, and ### P < 0.001 vs vehicle group; for Lip-1 group: ## P < 0.01, ### P < 0.001 vs vehicle group
MSCs treatment rescued rmTBI-induced neurodegeneration and neuronal loss. A Representative immunofluorescence staining of FJC-positive cells in the cortex and hippocampus of sham, vehicle, MSCs, and Lip-1 groups. Green indicates FJC-positive staining, and blue indicates positive DAPI nuclear staining. Scale bar = 25 μm. B Quantitative analysis of FJC-positive cells. C, D Western blot analysis of NeuN proteins in the sham, vehicle, MSCs, Lip-1 groups. Protein expression was normalized to β-actin and expressed as a fold change of sham. Data are expressed as mean ± SD (n = 6) and analyzed using one-way ANOVA with Tukey's post hoc test. For vehicle group: **P < 0.01, and ***P < 0.001 vs sham group; for MSCs group: ## P < 0.01, and ### P < 0.001 vs vehicle group; for Lip-1 group: # P < 0.05, ### P < 0.001 vs vehicle group
MSCs treatment inhibit rmTBI-induced ferroptosis. A, B Western blot analysis of the Fpn, Tfr1 proteins in sham, vehicle, MSCs, Lip-1 groups. Protein expression was normalized to GAPDH and expressed as fold change of sham. C, D The levels of Fe 2+ content and GPx activity in the sham, vehicle, MSCs, Lip-1 groups. E, F Western blot analysis of the 4-HNE and GPx4 proteins in sham, vehicle, MSCs, Lip-1 groups. Protein expression was normalized to GAPDH and expressed as fold change of sham. Data are expressed as mean ± SD (n = 6) and analyzed using one-way ANOVA with Tukey's post hoc test. For vehicle group: ***P < 0.001 vs sham group; for MSCs group: # P < 0.05, ## P < 0.01, ### P < 0.001 vs vehicle group; for Lip-1 group: # P < 0.05, ## P < 0.01, ### P < 0.001 vs vehicle group
Details of the reagents used
The incidence of repetitive mild traumatic brain injury (rmTBI), one of the main risk factors for predicting neurodegenerative disorders, is increasing; however, its underlying mechanism remains unclear. As suggested by several studies, ferroptosis is possibly related to TBI pathophysiology, but its effect on rmTBI is rarely studied. Mesenchymal stromal cells (MSCs), the most studied experimental cells in stem cell therapy, exert many beneficial effects on diseases of the central nervous system, yet evidence regarding the role of MSCs in ferroptosis and post-rmTBI neurodegeneration is unavailable. Our study showed that rmTBI resulted in time-dependent alterations in ferroptosis-related biomarker levels, such as abnormal iron metabolism, glutathione peroxidase (GPx) inactivation, decrease in GPx4 levels, and increase in lipid peroxidation. Furthermore, MSC treatment markedly decreased the aforementioned rmTBI-mediated alterations, neuronal damage, pathological protein deposition, and improved cognitive function compared with vehicle control. Similarly, liproxstatin-1, a ferroptosis inhibitor, showed similar effects. Collectively, based on the above observations, MSCs ameliorate cognitive impairment following rmTBI, partially via suppressing ferroptosis, which could be a therapeutic target for rmTBI.
Background Reactive oxygen species (ROS) often promote acute brain injury after stroke, but their roles in the recovery phase have not been well studied. We tested the hypothesis that ROS activity mediated by NADPH oxidase 2 (NOX2) contributes to acute brain injury but promotes functional recovery during the delayed phase, which is linked with neuroinflammation, autophagy, angiogenesis, and the PI3K/Akt signaling pathway. Methods We used the NOX2 inhibitor apocynin to study the role of NOX2 in brain injury and functional recovery in a middle cerebral artery occlusion (MCAO) stroke mouse model. Infarct size, neurological deficits and behavior were evaluated on days 3, 7, 10 and 14 after reperfusion. In addition, dynamic NOX2-induced ROS levels were measured by dihydroethidium (DHE) staining. Autophagy, inflammasomes, and angiogenesis were measured by immunofluorescence staining and western blotting. RNA sequencing was performed, and bioinformatics technology was used to analyze differentially expressed genes (DEGs), as well as the enrichment of biological functions and signaling pathways in ischemia penumbra at 7 days after reperfusion. Then, Akt pathway-related proteins were further evaluated by western blotting. Results Our results showed that apocynin injection attenuated infarct size and mortality 3 days after stroke but promoted mortality and blocked functional recovery from 5 to 14 days after stroke. DHE staining showed that ROS levels were increased at 3 days after reperfusion and then gradually declined in WT mice, and these levels were significantly reduced by the NOX2 inhibitor apocynin. RNA-Seq analysis indicated that apocynin activated the immune response under hypoxic conditions. The immunofluorescence and western blot results demonstrated that apocynin inhibited the NLRP3 inflammasome and promoted angiogenesis at 3 days but promoted the NLRP3 inflammasome and inhibited angiogenesis at 7 and 14 days after stroke, which was mediated by regulating autophagy activation. Furthermore, RNA-Seq and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that apocynin injection resulted in PI3K–Akt signaling pathway enrichment after 7 days of MCAO. We then used an animal model to show that apocynin decreased the protein levels of phosphorylated PI3K and Akt and NF-κB p65, confirming that the PI3K–Akt–NF-κB pathway is involved in apocynin-mediated activation of inflammation and inhibition of angiogenesis. Conclusions NOX2-induced ROS production is a double-edged sword that exacerbates brain injury in the acute phase but promotes functional recovery. This effect appears to be achieved by inhibiting NLRP3 inflammasome activation and promoting angiogenesis via autophagy activation.
Background Perioperative neurocognitive disorders (PNDs) are considered the most common postoperative complication in geriatric patients. However, its pathogenesis is not fully understood. Surgery-triggered neuroinflammation is a major contributor to the development of PNDs. Neuroinflammation can influence N-methyl-D-aspartate receptor (NMDAR) expression or function which is closely associated with cognition. We, therefore, hypothesized that the persistent changes in NMDAR expression or function induced by transient neuroinflammation after surgery were involved in the development of PNDs. Methods Eighteen-month-old male Sprague–Dawley rats were subjected to abdominal surgery with sevoflurane anesthesia to establish the PNDs animal model. Then, we determined the transient neuroinflammation by detecting the protein levels of proinflammatory cytokines and microglia activation using ELISA, western blot, immunohistochemistry, and microglial morphological analysis from postoperative days 1–20. Persistent changes in NMDAR expression were determined by detecting the protein levels of NMDAR subunits from postoperative days 1–59. Subsequently, the dysfunction of synaptic NMDAR was evaluated by detecting the structural plasticity of dendritic spine using Golgi staining. Pull-down assay and western blot were used to detect the protein levels of Rac1-GTP, phosphor-cofilin, and Arp3, which contribute to the regulation of the structural plasticity of dendritic spine. Finally, glycyrrhizin, an anti-inflammatory agent, was administered to further explore the role of synaptic NMDAR dysfunction induced by transient neuroinflammation in the neuropathogenesis of PNDs. Results We showed that transient neuroinflammation induced by surgery caused sustained downregulation of synaptic NR2A and NR2B subunits in the dorsal hippocampus and led to a selective long-term spatial memory deficit. Meanwhile, the detrimental effect of neuroinflammation on the function of synaptic NMDARs was shown by the impaired structural plasticity of dendritic spines and decreased activity of the Rac1 signaling pathways during learning. Furthermore, anti-inflammatory treatment reversed the downregulation and hypofunction of synaptic NR2A and NR2B and subsequently rescued the long-term spatial memory deficit. Conclusions Our results identify sustained synaptic NR2A and NR2B downregulation and hypofunction induced by transient neuroinflammation following surgery as important contributors to the development of PNDs in elderly rats.
Background Studies have suggested that many down-regulated miRNAs identified in the brain tissue or serum of Alzheimer’s disease (AD) patients were involved in the formation of senile plaques and neurofibrillary tangles. Specifically, our previous study revealed that microRNA-22-3p (miR-22-3p) was significantly down-regulated in AD patients. However, the molecular mechanism underlying the down-regulation of miR-22-3p has not been comprehensively investigated. Methods The ameliorating effect of miR-22-3p on apoptosis of the Aβ-treated HT22 cells was detected by TUNEL staining, flow cytometry, and western blotting. The cognition of mice with stereotaxic injection of agomir or antagomir of miR-22-3p was assessed by Morris water maze test. Pathological changes in the mouse hippocampus were analyzed using hematoxylin and eosin (HE) staining, Nissl staining, and immunohistochemistry. Proteomics analysis was performed to identify the targets of miR-22-3p, which were further validated using dual-luciferase reporter analysis and western blotting analysis. Results The miR-22-3p played an important role in ameliorating apoptosis in the Aβ-treated HT22 cells. Increased levels of miR-22-3p in the mouse hippocampus improved the cognition in mice. Although the miR-22-3p did not cause the decrease of neuronal loss in the hippocampus, it reduced the Aβ deposition. Proteomics analysis revealed Sox9 protein as the target of miR-22-3p, which was verified by the luciferase reporter experiments. Conclusion Our study showed that miR-22-3p could improve apoptosis and reduce Aβ deposition by acting on Sox9 through the NF-κB signaling pathway to improve the cognition in AD mice. We concluded that miR-22-3p ameliorated AD by targeting Sox9 through the NF-κB signaling pathway in the hippocampus.
Male and female B6 (WT) mice have indistinguishable DRG macrophage accumulation and activation, and conditioned and unconditioned in vivo regeneration. A Diagram of our in vivo conditioning lesion (CL) and regeneration paradigm. B Macrophage quantification in Naïve, 2 day post-crush (Sh CL), and 9 day post-transection plus 2 day post-crush (CL) DRGs from male and female mice. Macrophages were quantified as the percent area stained by anti-CD68. Means were compared with a two-way ANOVA followed by Tukey's post-hoc tests. C-H Representative images of macrophages in the cell body area of L4 DRGs from the indicated sex and injury condition. Scale bar is 50 μm. I Axon regeneration expressed as the fraction of axons relative to the crush site every 200 µm. Each point is the mean fraction ± SEM. Pairs of curves were compared using non-linear regression assuming one phase decay, initial Y = 1, and the plateau = 0 and significance was determined by comparing the decay constants of the fitted curves. J-M Representative images of regenerating axons stained for SCG10, a marker of regenerating sensory axons. Unconditioned growth (J, K), representing neuron intrinsic regeneration rate, and conditioned growth (L, M) were the same between sexes. Conditioned growth was increased compared to unconditioned growth in both sexes. The dotted line indicates the center of the crush site which was considered to be 500 μm wide, and the solid line is 3000 μm from the crush site. Scale bar is 500 μm. n = 10 for all groups and * indicates significance differences (**p < 0.01, ***p < 0.001) between indicated means
Degeneration and regeneration are functionally normal in the absence of CCL2. Conditioning and in vivo regeneration were done as in Fig. 4. A Axon regeneration expressed as the fraction of axons relative to the crush site every 100 μm. Each point is the mean fraction ± SEM. B Mean regeneration determined by integrating regenerating axon fluorescence to find the average axon length for each nerve. Average axon lengths are plotted as mean ± SEM. For A and B, the analysis ends at 3000 μm from the crush, because that is the length of the shortest nerve segment. n = 7 nerves per injury condition per genotype, except WT Sh CL n = 8. One WT CL nerve was excluded from analysis for violating assumption 5 (see "Materials and methods"). C-E Representative images of regenerating nerves stained for SCG10. Unconditioned growth (C), representing neuron intrinsic regeneration rate, was the same in both genotypes. Conditioned growth (D, E) was also the same in Ccl2 KOs as in WT and increased compared to unconditioned controls (compare to C). The dotted line indicates the center of the crush site which was considered to be 500 μm wide, and the solid line is 3000 μm from the crush. Scale bar is 500 μm. F Myelin clearance, quantified by LFB area and plotted as mean ± SEM. WT n = 8, Ccl2 KO n = 9. G-I Representative images of myelin stained with LFB in uninjured (G) and injured (H, I) sciatic nerves. In both genotypes, myelin has almost completely degenerated and been cleared by 7 DPI. # Indicates a significant (p < 0.05) difference between the Sh CL (unconditioned) and CL (conditioned) regeneration within a genotype
Background Peripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve. Methods Ccl2 fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2–RFP fl/fl mice. CCL2–CCR2 signaling was further examined using global Ccl2 KOs and Ccr2 gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining. Results Surprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2–RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12 , were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2 . Finally, Ccr2 gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. C cr2 gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages. Conclusions CCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation.
Background The complement system is part of the innate immune system that clears pathogens and cellular debris. In the healthy brain, complement influences neurodevelopment and neurogenesis, synaptic pruning, clearance of neuronal blebs, recruitment of phagocytes, and protects from pathogens. However, excessive downstream complement activation that leads to generation of C5a, and C5a engagement with its receptor C5aR1, instigates a feed-forward loop of inflammation, injury, and neuronal death, making C5aR1 a potential therapeutic target for neuroinflammatory disorders. C5aR1 ablation in the Arctic (Arc) model of Alzheimer’s disease protects against cognitive decline and neuronal injury without altering amyloid plaque accumulation. Methods To elucidate the effects of C5a–C5aR1 signaling on AD pathology, we crossed Arc mice with a C5a-overexpressing mouse (ArcC5a+) and tested hippocampal memory. RNA-seq was performed on hippocampus and cortex from Arc, ArcC5aR1KO, and ArcC5a+ mice at 2.7–10 months and age-matched controls to assess mechanisms involved in each system. Immunohistochemistry was used to probe for protein markers of microglia and astrocytes activation states. Results ArcC5a+ mice had accelerated cognitive decline compared to Arc. Deletion of C5ar1 delayed or prevented the expression of some, but not all, AD-associated genes in the hippocampus and a subset of pan-reactive and A1 reactive astrocyte genes, indicating a separation between genes induced by amyloid plaques alone and those influenced by C5a–C5aR1 signaling. Biological processes associated with AD and AD mouse models, including inflammatory signaling, microglial cell activation, and astrocyte migration, were delayed in the ArcC5aR1KO hippocampus. Interestingly, C5a overexpression also delayed the increase of some AD-, complement-, and astrocyte-associated genes, suggesting the possible involvement of neuroprotective C5aR2. However, these pathways were enhanced in older ArcC5a+ mice compared to Arc. Immunohistochemistry confirmed that C5a–C5aR1 modulation in Arc mice delayed the increase in CD11c-positive microglia, while not affecting other pan-reactive microglial or astrocyte markers. Conclusion C5a–C5aR1 signaling in AD largely exerts its effects by enhancing microglial activation pathways that accelerate disease progression. While C5a may have neuroprotective effects via C5aR2, engagement of C5a with C5aR1 is detrimental in AD models. These data support specific pharmacological inhibition of C5aR1 as a potential therapeutic strategy to treat AD.
Background Traumatic brain injury (TBI) is characterized by a primary mechanical injury and a secondary injury associated with neuroinflammation, blood–brain barrier (BBB) disruption and neurodegeneration. We have developed a novel cannabidiol aminoquinone derivative, VCE-004.8, which is a dual PPARγ/CB 2 agonist that also activates the hypoxia inducible factor (HIF) pathway. VCE-004.8 shows potent antifibrotic, anti-inflammatory and neuroprotective activities and it is now in Phase II clinical trials for systemic sclerosis and multiple sclerosis. Herein, we investigated the mechanism of action of VCE-004.8 in the HIF pathway and explored its efficacy in a preclinical model of TBI. Methods Using a phosphoproteomic approach, we investigated the effects of VCE-004.8 on prolyl hydroxylase domain-containing protein 2 (PHD2) posttranslational modifications. The potential role of PP2A/B55α in HIF activation was analyzed using siRNA for B55α. To evaluate the angiogenic response to the treatment with VCE-004.8 we performed a Matrigel plug in vivo assay. Transendothelial electrical resistance (TEER) as well as vascular cell adhesion molecule 1 (VCAM), and zonula occludens 1 (ZO-1) tight junction protein expression were studied in brain microvascular endothelial cells. The efficacy of VCE-004.8 in vivo was evaluated in a controlled cortical impact (CCI) murine model of TBI. Results Herein we provide evidence that VCE-004.8 inhibits PHD2 Ser125 phosphorylation and activates HIF through a PP2A/B55α pathway. VCE-004.8 induces angiogenesis in vivo increasing the formation of functional vessel (CD31/α-SMA) and prevents in vitro blood–brain barrier (BBB) disruption ameliorating the loss of ZO-1 expression under proinflammatory conditions. In CCI model VCE-004.8 treatment ameliorates early motor deficits after TBI and attenuates cerebral edema preserving BBB integrity. Histopathological analysis revealed that VCE-004.8 treatment induces neovascularization in pericontusional area and prevented immune cell infiltration to the brain parenchyma. In addition, VCE-004.8 attenuates neuroinflammation and reduces neuronal death and apoptosis in the damaged area. Conclusions This study provides new insight about the mechanism of action of VCE-004.8 regulating the PP2A/B55α/PHD2/HIF pathway. Furthermore, we show the potential efficacy for TBI treatment by preventing BBB disruption, enhancing angiogenesis, and ameliorating neuroinflammation and neurodegeneration after brain injury.
Background: Metabolic dysregulation and disruption of immune homeostasis have been widely associated with perioperative complications including perioperative ischemic stroke. Although immunometabolite S-2-hydroxyglutarate (S-2HG) is an emerging regulator of immune cells and thus triggers the immune response, it is unclear whether and how S-2HG elicits perioperative ischemic brain injury and exacerbates post-stroke cognitive dysfunction. Methods: Perioperative ischemic stroke was induced by transient middle cerebral artery occlusion for 60 min in C57BL/6 mice 1 day after ileocecal resection. CD8+ T lymphocyte activation and invasion of the cerebrovascular compartment were measured using flow cytometry. Untargeted metabolomic profiling was performed to detect metabolic changes in sorted CD8+ T lymphocytes after ischemia. CD8+ T lymphocytes were transfected with lentivirus ex vivo to mobilize cell proliferation and differentiation before being transferred into recombination activating gene 1 (Rag1-/-) stroke mice. Results: The perioperative stroke mice exhibit more severe cerebral ischemic injury and neurological dysfunction than the stroke-only mice. CD8+ T lymphocyte invasion of brain parenchyma and neurotoxicity augment cerebral ischemic injury in the perioperative stroke mice. CD8+ T lymphocyte depletion reverses exacerbated immune-mediated cerebral ischemic brain injury in perioperative stroke mice. Perioperative ischemic stroke triggers aberrant metabolic alterations in peripheral CD8+ T cells, in which S-2HG is more abundant. S-2HG alters CD8+ T lymphocyte proliferation and differentiation ex vivo and modulates the immune-mediated ischemic brain injury and post-stroke cognitive dysfunction by enhancing CD8+ T lymphocyte-mediated neurotoxicity. Conclusion: Our study establishes that S-2HG signaling-mediated activation and neurotoxicity of CD8+ T lymphocytes might exacerbate perioperative ischemic brain injury and may represent a promising immunotherapy target in perioperative ischemic stroke.
The cerebrospinal fluid (CSF) space is convoluted. CSF flow oscillates with a net flow from the ventricles towards the cerebral and spinal subarachnoid space. This flow is influenced by heartbeats, breath, head or body movements as well as the activity of the ciliated epithelium of the plexus and ventricular ependyma. The shape of the CSF space and the CSF flow preclude rapid equilibration of cells, proteins and smaller compounds between the different parts of the compartment. In this review including reinterpretation of previously published data we illustrate, how anatomical and (patho)physiological conditions can influence routine CSF analysis. Equilibration of the components of the CSF depends on the size of the molecule or particle, e.g., lactate is distributed in the CSF more homogeneously than proteins or cells. The concentrations of blood-derived compounds usually increase from the ventricles to the lumbar CSF space, whereas the concentrations of brain-derived compounds usually decrease. Under special conditions, in particular when distribution is impaired, the rostro-caudal gradient of blood-derived compounds can be reversed. In the last century, several researchers attempted to define typical CSF findings for the diagnosis of several inflammatory diseases based on routine parameters. Because of the high spatial and temporal variations, findings considered typical of certain CNS diseases often are absent in parts of or even in the entire CSF compartment. In CNS infections, identification of the pathogen by culture, antigen detection or molecular methods is essential for diagnosis.
Repopulation of microglia does not ameliorate Aβ pathology and neuritic damage in male 3xTg mice. Experimental paradigm depicting duration of PLX5622 treatment and subsequent microglial repopulation (A). Representative confocal immunofluorescent 20× images of the subiculum in control versus PLX-repopulated group, showing Aβ plaque (6E10, red), microglia (Iba1, magenta), neuritic damage (LAMP1, green) (B). Scale bar represents 200 µm. There were no significant differences in the total area (C, G), size (D, H) and number (E, I) of plaques between the control and PLX-repopulated group in both cohorts. Ratio of plaque-associated LAMP1⁺ neuritic damage to plaque load was similar between the control and PLX-repopulated groups in both cohorts (F, J). Representative confocal immunofluorescent 20× images of the subiculum in control versus PLX-repopulated group, showing Aβ plaque labeled with MeX04 (blue) and 6E10 (red) (K). There was no difference in the total plaque area labeled with MeX04 (L) and the ratio of MeX04/6E10 (M) between the two treatment groups. Student’s t-test. Data are presented as mean ± SEM (Cohort 1: n = 9–11; Cohort 2: n = 7)
Repopulation of microglia does not ameliorate Aβ pathology and neuritic damage in APP/PS1 male mice. Experimental paradigm depicting duration of PLX5622 treatment and subsequent microglial repopulation (A). Representative immunofluorescent 20× images of the subiculum in control versus PLX-repopulated group, showing Aβ plaque (6E10, red), microglia (Iba1, magenta), and neuritic damage (LAMP1, green) (B). Scale bar represents 200 µm. There was no difference in the total area (C), size (D) and number (E) of plaques between the control and PLX-repopulated groups in Subiculum or CA1. Ratio of plaque-associated LAMP1⁺ neuritic damage to plaque load was similar between control and PLX-repopulated group (F). Student’s t-test. Data are presented as mean ± SEM (n = 6)
Repopulated microglia show similar recruitment to plaques but exhibit changes in homeostatic markers. Flow cytometry revealed no significant difference in microglia internalization of MeX04 in the two groups but showed a trend toward a higher percentage of MeX04⁺ microglia with PLX treatment (A). Microglial levels of P2RY12 (B) were significantly lower, while TMEM119 levels (C) were mildly elevated in PLX-repopulated group compared to the control. Representative immunofluorescent 40× images of the subiculum in the control and PLX-repopulated groups showing microglia (Iba1, magenta) and CD68 (green) in the first cohort of 3xTg mice (D). Representative immunofluorescent 20× images of the CA1 in the control and PLX-repopulated groups showing P2RY12 (red) in the second cohort of 3xTg mice (E). Scale bars represent 200 µm. Microglia in both cohorts showed no difference in their recruitment to plaque (F, J) as well as their total coverage (G, K) between control and PLX-repopulated groups. Levels of CD68, a marker of activated microglia, were slightly lower in the PLX-treated group in the subiculum (H) but not in the CA1 (I). The intensity of P2RY12, a homeostatic maker of microglia, was similar between treatments in the subiculum (L) but showed a trend toward being lower in the PLX-repopulated group in the CA1 region (M) as analyzed by immunohistochemistry. Student’s t-test (Welch’s corrections for A and C), ***p < 0.001. Data are presented as mean ± SEM (Cohort 1: n = 9–11; Cohort 2: n = 7)
Repopulation of microglia impacts phosphorylation of different Tau epitopes in male 3xTg mice. Representative immunofluorescent 20× images of the CA1 hippocampal region in control and PLX-repopulated groups showing total Tau (HT7, green), pT205 (magenta), pS409 (cyan) and pS396 (red) (A). All images are from Cohort 1 and the scale bar represents 200 µm. Quantification of normalized pT205 revealed a significant increase in the PLX-repopulated group of Cohort 1 (B) but not Cohort 2 (F). On the other hand, normalized pS409 levels were found to be significantly decreased in Cohort 1 (C) and Cohort 2 (G). Levels of normalized pS396 were unchanged between control and PLX-repopulated groups of Cohort 1 (D), but showed a trend towards decreased levels in PLX-repopulated group of Cohort 2 (H). Levels of total Tau remained unchanged in both of the cohorts (E, I). Student’s t-test, *p < 0.05. Data are presented as mean ± SEM (Cohort 1: n = 9–11; Cohort 2: n = 7)
Repopulation increases Cxcl13 expression in hippocampal microglia of male 3xTg mice. Schematic of experimental design (A, B). FACS-sorted cells were used as the input for scRNAseq (A). UMAP plots of control and PLX-repopulated groups illustrate clustering of cells (B). The inset shows the output of scMCA-based cell annotations. An overwhelming majority of sequenced cells were microglia (B). Proportions of different cell clusters out of all sequenced cells are depicted in C. Panel of genes identified through Seurat’s statistical framework or established literature is shown in D for annotation purposes. Red boxes highlight genes that are associated with their corresponding clusters. Increased Cxcl13 expression can be noted across the majority of repopulated microglia but in particular for PLX-mg and ARM/DAM clusters (E). Increasing tones of green denote increased Cxcl13 expression. ELISA on hippocampal lysates confirmed the upregulation of CXCL13 at the protein level (F). Representative immunofluorescent 10× images of in situ analysis of Cxcl13 expression (G, H). Scale bar represents 100 µm in G or 200 µm in H. The resultant yellow signal from green Cxcl13 in situ hybridization and red Iba1 immunohistochemistry suggests that all of the Cxcl13 transcripts we observed are associated with microglia (G). This representative image is from a PLX-repopulated sample. Cxcl13 staining showed strong trends towards increased expression in PLX-repopulated groups in Subiculum and CA1 (H–J), however did not change in the cortex (K). Student’s t-test (F, I–K), *p < 0.05. Data are presented as mean ± SEM
Background Adult microglia rely on self-renewal through division to repopulate and sustain their numbers. However, with aging, microglia display morphological and transcriptional changes that reflect a heightened state of neuroinflammation. This state threatens aging neurons and other cells and can influence the progression of Alzheimer’s disease (AD). In this study, we sought to determine whether renewing microglia through a forced partial depletion/repopulation method could attenuate AD pathology in the 3xTg and APP/PS1 mouse models. Methods We pharmacologically depleted the microglia of two cohorts of 21- to 22-month-old 3xTg mice and one cohort of 14-month-old APP/PS1 mice using PLX5622 formulated in chow for 2 weeks. Following depletion, we returned the mice to standard chow diet for 1 month to allow microglial repopulation. We assessed the effect of depletion and repopulation on AD pathology, microglial gene expression, and surface levels of homeostatic markers on microglia using immunohistochemistry, single-cell RNAseq and flow cytometry. Results Although we did not identify a significant impact of microglial repopulation on amyloid pathology in either of the AD models, we observed differential changes in phosphorylated-Tau epitopes after repopulation in the 3xTg mice. We provide evidence that repopulated microglia in the hippocampal formation exhibited changes in the levels of homeostatic microglial markers. Lastly, we identified novel subpopulations of microglia by performing single-cell RNAseq analysis on CD45 int/+ cells from hippocampi of control and repopulated 3xTg mice. In particular, one subpopulation induced after repopulation is characterized by heightened expression of Cxcl13 . Conclusion Overall, we found that depleting and repopulating microglia causes overexpression of microglial Cxcl13 with disparate effects on Tau and amyloid pathologies.
Spermidine reduced soluble Aβ and induced transcriptomic alterations in microglia of APPPS1 mice. a APPPS1 mice were treated with 3 mM spermidine via their drinking water starting at 30 days (d) until mice reached an age of 120 days or 290 days according to the depicted treatment scheme. Spermidine-treated APPPS1 mice were compared to non-treated controls (H2O). The Aβ40 and Aβ42 content was measured in the TBS (soluble) fraction of brain homogenates of 120-day-old or 290-day-old spermidine-treated mice and water controls (mixed sex) using electrochemiluminescence (MesoScale Discovery panel). Values were normalized to water controls. 120d APPPS1 H2O (n = 14), 120d APPPS1 spermidine (n = 14), 290d APPPS1 H2O (n = 14), 290d APPPS1 spermidine (n = 12); two‐tailed t‐test, Aβ42 in 120d mice: Mann–Whitney U test. b Single nuclei sequencing of hemispheres harvested from 180-day-old male spermidine-treated APPPS1, H2O APPPS1 and H2O control mice was performed of FACS-sorted DAPI-stained nuclei using the 10x Genomics platform (n = 3). c UMAP embedding and clustering of the snRNA-seq data, together with annotation of the major cell types. d Heatmap showing the top 5 marker genes for 300 cells in each of the major cell types. e Dot plot for the top 50 genes in a cell-type-specific differential expression analysis between spermidine-treated APPPS1 and H2O APPPS1 mice. Color scale indicates log2 fold change, dot size indicates adjusted p value. f Same as e, for selected genes differentially expressed in microglia clusters 1 or 2. Associated pathways are color-coded. g Expression of Plxna2 in APPPS1 spermidine and APPPS1 H2O mice. Color scale indicates normalized expression, grey dots represent no data (left panels). For validation, neonatal microglia were treated with the indicated concentrations of spermidine in combination with LPS (1 µg/ml) and ATP (2 mM) or with poly I:C (50 µg/ml) and the gene expression was assessed by RT-qPCR (right panels). Plxna2 expression was normalized to Actin and displayed as fold change compared to non-treated control cells; n = 5–6, one-way ANOVA, Dunnett’s post hoc test. h Neonatal microglia were pre-treated with 3 or 10 µM spermidine for 15 h. The confluent cell layer was scratched and the scratch area was imaged for 72 h at the indicated timepoints. The gap area normalized to timepoint 0 h is displayed; n = 5–6, two-way ANOVA, Dunnett’s post hoc test. i Neonatal microglia were non-treated or treated with 10 µM spermidine and their migration towards 300 µM ATP was quantified after 24 h using a transwell migration assay; two‐tailed t‐test. *p < 0.05, **p < 0.01, ***p < 0.001
Spermidine altered AD-associated microglia and their capacity to degrade Aβ. a Cluster abundance in snRNA-seq of male spermidine-treated APPPS1 and H2O APPPS1 mice with p values from mixed-effects binomial model. b Volcano plot of genes differentially expressed in microglia cluster 2 vs. 1. The top 5 up- and down-regulated genes are indicated as well as previously published markers for homeostatic (yellow) and disease-associated (blue) microglia [1]. Significance threshold of adj. p value < 0.01 was used. Axl as a gene of interest is highlighted in red. c Tissue sections of male 180-day-old mice were stained for the microglia cluster 2 marker AXL (red) and IBA1 (green). The AXL intensity normalized to the IBA1 area was determined by ImageJ analysis; n = 6–10, two‐tailed t‐test. d Neonatal microglia were pre-treated for 18 h with 10 µM spermidine and fluorescently labelled oligomeric (Aβo) Aβ (magenta) was added for further 24 h. Microglia cells were stained for IBA1 (green). The percentage of phagocytic cells and the Aβ mean intensity density per phagocytic cell was assessed by confocal microscopy. Representative images are shown; n = 7, two‐tailed t‐test. *p < 0.05, **p < 0.01, ***p < 0.001
Spermidine treatment reduced progressive neuroinflammation in APPPS1 mice. a Male APPPS1 mice were treated with 3 mM spermidine via their drinking water starting at 30 days (d) until mice reached an age of 180 days. Microglia were isolated by MACS and the proteome assessed by mass spectrometry. b Scatterplot of protein regulation in Contrast2 (APPPS1 spermidine—APPPS1 H2O, y axis) vs. its regulation in Contrast1 (APPPS1 H2O,—WT H2O, x axis). Contrast 2 shows regulation due to spermidine effect, Contrast 1 shows the regulation of proteins by Alzheimer disease. Proteins that are regulated by spermidine and show significant anti-APPPS1 effect were marked in red. As such we selected proteins that show significant (alpha = 0.04) regulation due to spermidine in APPPS1 mice (Contrast2) and simultaneously, show significant (alpha = 0.04) effect in Contrast5 = (Contrast2 − Contrast1)/2, in the direction, opposite to the effect of the AD-like model. c Volcano plot of GSEA enrichment of GO BP terms. x-axis shows normalized enrichment score of functional term, y-axis represent the − log10 of its false discovery rate. Labelled are only terms that have relation to neurodegeneration and inflammation. As such we selected terms that have in their names following strings: neuro, inflamm, Clathrin, interleukin, Caspase, TNF, ubiquitin, SUMO, Alzheimer, Parkinson, Huntington, lipoprotein, autophagy, cell migration, cell motility, microtubule, actin, actin-, glia, amyloid. Not all labels appear due to strong overlap, especially at high fdr > ~ 0.5 (− log10(fdr) < ~ 0.3). Long term names are truncated to 50 characters. d GSEA enrichment map using top 50 REACTOME terms from list of neurodegeneration and inflammation terms. As such we selected terms that have in their names following strings: neuro, inflamm, Clathrin, interleukin, Caspase, TNF, ubiquitin, SUMO, Alzheimer, Parkinson, Huntington, lipoprotein, autophagy, cell migration, cell motility, microtubule, actin, actin-, glia, amyloid. e Dot plot of selected functional terms related to neuroinflammation and degeneration. f APPPS1 mice were treated with 3 mM spermidine via their drinking water starting at 30 days until mice reached an age of 290 days. The content of the indicated pro-inflammatory cytokines was measured in the TBS (soluble) fraction of brain homogenates of male spermidine-treated mice and water controls using electrochemiluminescence (MesoScale Discovery panel). Values were normalized to water controls. 290d APPPS1 H2O (n = 14), 290d APPPS1 spermidine (n = 12); two‐tailed t‐test. *p < 0.05, **p < 0.01, ***p < 0.01
Spermidine exhibits direct anti-inflammatory effects on microglia. a Hemispheres of wild type (WT) and APPPS1 mice were coronally sliced and treated with the indicated spermidine concentration, LPS (10 µg/ml) and ATP (5 mM) as depicted. The IL-1β and IL-6 concentration in the supernatant was determined by ELISA; n = 3–5, two-way ANOVA, Tukey’s post hoc test. b–f Neonatal microglia (neoMG) were either treated with LPS (1 µg/ml) and ATP (2 mM), with poly I:C (50 µg/ml) or with oligomeric Aβ (Aβo, 5 µM) and the indicated spermidine concentrations as depicted in the schemes. b–d Amount of cytokines in the cell supernatant was determined by electrochemiluminescence (MesoScale Discovery panel); n = 4–5. b IFN-γ, IL-10, IL-12, IL-2: Kruskal–Wallis, Dunn’s multiple comparison; IL-1β, IL-4, IL-5, IL-6, KC/GRO, TNF-α: one-way ANOVA, Dunnett’s post hoc test. c INF-γ, IL-2, IL-4: Kruskal–Wallis, Dunn’s multiple comparison; IL-10, IL-12, IL-1β, IL-5, IL-6, KC/GRO, TNF-α: one-way ANOVA, Dunnett´s post hoc test. d IL-10, IL-12, IL-4, KC/GRO: Kruskal–Wallis, Dunn’s multiple comparison; INF-γ, IL-1β, IL-2, IL-5, IL-6, TNF-α: one-way ANOVA, Dunnett’s post hoc test. e The gene expression of Tnf-α and Il-6 was assessed by RT-qPCR after treatment of neonatal microglia as depicted in b. Their expression was normalized to Actin and displayed as fold change compared to non-treated control cells; n = 4. Il-6: one-way ANOVA, Dunnett’s post hoc test; Tnf-α: Kruskal–Wallis, Dunn’s multiple comparison. f Levels of phosphorylated NF-κB (pNF-κB) and NF-κB were determined by western blot in neonatal microglia treated as depicted in b. Representative images are shown and protein levels are displayed as fold changes compared to non-treated controls normalized to ACTIN; n = 7. NF-κB: Kruskal–Wallis, Dunn’s multiple comparison; pNF-κB: one-way ANOVA, Dunnett’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001
Spermidine regulates neuroinflammation beyond transcription by interfering with inflammasome assembly. Neonatal microglia (neoMG) were treated with LPS (1 µg/ml) and spermidine at indicated concentrations for 1.45 h and ATP (2 mM) as depicted in the scheme (a). b IL-1β concentration in the cell supernatant was determined by ELISA; n = 4–8; Kruskal–Wallis, Dunn´s multiple comparison. c IL-18 concentration in the cell supernatant was determined by ELISA; n = 3; Kruskal–Wallis, Dunn’s multiple comparison. d Pro-IL-1β protein levels were determined by western blot and normalized to ACTIN. Representative images are shown and values are displayed as fold changes compared to LPS/ATP-treated cells; n = 8–9; Kruskal–Wallis, Dunn’s multiple comparison. e Cellular Pro-CASP1 and cleaved CASP1 p20 levels in the supernatant were determined by western blot (* non-specific band). Pro-CASP1 was normalized to ACTIN (n = 4–8) and CASP1 p20 was normalized on whole protein content determined by Ponceau S staining (n = 3). Values are displayed as fold changes compared to LPS/ATP-treated cells; Pro-CASP1: Kruskal–Wallis, Dunn´s multiple comparison; cleaved CASP1: one-way ANOVA, Dunnett’s post hoc test. f Neonatal microglia were stimulated as shown in a and MCC950 was added 15 min before addition of ATP. Cells were stained for ASC to visualize inflammasomes and with DAPI for nuclear staining. The percentage of ASC specks in respect to the number of total cells (DAPI positive cells) was determined (left). The IL-1β concentration in the cell supernatant was assessed by ELISA (right); n = 3; one-way ANOVA, Dunnett’s post hoc test. g Neonatal WT and Casp1−/− microglia were stimulated as shown in a but with 4 mM ATP to increase the number of inflammasomes. Cells were stained for ASC (red) to visualize inflammasomes and with DAPI (blue) for nuclear staining as shown in the representative images (scale bar = 100 µm). Arrowheads highlight ASC specks within microglia. The percentage of ASC specks in respect to the number of total cells (DAPI positive cells) was determined (left). The IL-1β concentration in the cell supernatant was assessed by ELISA (right); WT: n = 8–16; Casp1−/−: n = 3. Kruskal–Wallis, Dunn’s multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.001
Background Deposition of amyloid beta (Aβ) and hyperphosphorylated tau along with glial cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimer’s disease (AD). In recent years, impairment of autophagy has been identified as another important feature contributing to AD progression. Therefore, the potential of the autophagy activator spermidine, a small body-endogenous polyamine often used as dietary supplement, was assessed on Aβ pathology and glial cell-mediated neuroinflammation. Results Oral treatment of the amyloid prone AD-like APPPS1 mice with spermidine reduced neurotoxic soluble Aβ and decreased AD-associated neuroinflammation. Mechanistically, single nuclei sequencing revealed AD-associated microglia to be the main target of spermidine. This microglia population was characterized by increased AXL levels and expression of genes implicated in cell migration and phagocytosis. A subsequent proteome analysis of isolated microglia confirmed the anti-inflammatory and cytoskeletal effects of spermidine in APPPS1 mice. In primary microglia and astrocytes, spermidine-induced autophagy subsequently affected TLR3- and TLR4-mediated inflammatory processes, phagocytosis of Aβ and motility. Interestingly, spermidine regulated the neuroinflammatory response of microglia beyond transcriptional control by interfering with the assembly of the inflammasome. Conclusions Our data highlight that the autophagy activator spermidine holds the potential to enhance Aβ degradation and to counteract glia-mediated neuroinflammation in AD pathology.
Background After traumatic brain injury (TBI), peripheral monocytes infiltrate into the central nervous system due to disruption of the blood–brain barrier, and play an important role in neuroinflammation. However, the mechanisms regulating the movement and function of peripheral monocytes after TBI have not been fully investigated. Methods TBI patients who underwent surgery at our hospital were recruited. CXCR2 expression in CD14⁺ monocytes from peripheral blood and cerebrospinal fluid (CSF) of TBI patients around surgery was analyzed by flow cytometry and compared with that of patients who suffered TBI 2–24 months prior and underwent cranioplasty. In vitro, serum or CSF from TBI/non-TBI patients were used to treat peripheral monocytes isolated from healthy volunteers to evaluate their effect on CXCR2 expression. Transwell experiments were performed to analyze the role of CXCR2 in monocyte chemotaxis toward the CSF. The role of CXCR2 in monocyte-mediated immunogenic cell death (ICD) of nerve cells was explored in an indirect co-culture system. Results Transient CXCR2 upregulation in monocytes from the peripheral blood and CSF of TBI patients was detected soon after surgery and was associated with unfavorable outcomes. TBI serum and CSF promoted CXCR2 expression in monocytes, and dexamethasone reversed this effect. Peripheral monocytes from TBI patients showed enhanced chemotaxis toward the CSF and increased inflammatory cytokine secretion. The CXCR2 antagonist SB225002 decreased monocyte chemotaxis toward TBI CSF, and lowered pro-inflammatory cytokine secretion in monocytes treated with TBI serum. SB225002 also relieved ICD in nerve cells co-cultured with TBI serum-treated monocytes. Conclusions CXCR2 is transiently overexpressed in the peripheral monocytes of TBI patients post-surgery, and drives peripheral monocyte chemotaxis toward CSF and monocyte-mediated ICD of nerve cells. Therefore, CXCR2 may be a target for monocyte-based therapies for TBI.
Background Gut microbiota has been found involved in neuronal functions and neurological disorders. Whether and how gut microbiota impacts chronic somatic pain disorders remain elusive. Methods Neuropathic pain was produced by different forms of injury or diseases, the chronic constriction injury (CCI) of the sciatic nerves, oxaliplatin (OXA) chemotherapy, and streptozocin (STZ)-induced diabetes in mice. Continuous feeding of antibiotics (ABX) cocktail was used to cause major depletion of the gut microbiota. Fecal microbiota, biochemical changes in the spinal cord and dorsal root ganglion (DRG), and the behaviorally expressed painful syndromes were assessed. Results Under condition of gut microbiota depletion, CCI, OXA, or STZ treatment-induced thermal hyperalgesia or mechanical allodynia were prevented or completely suppressed. Gut microbiota depletion also prevented CCI or STZ treatment-induced glial cell activation in the spinal cord and inhibited cytokine production in DRG in OXA model. Interestingly, STZ treatment failed to induce the diabetic high blood glucose and painful hypersensitivity in animals with the gut microbiota depletion. ABX feeding starting simultaneously with CCI, OXA, or STZ treatment resulted in instant analgesia in all the animals. ABX feeding starting after establishment of the neuropathic pain in CCI- and STZ-, but not OXA-treated animals produced significant alleviation of the thermal hyeralgesia or mechanical allodynia. Transplantation of fecal bacteria from SPF mice to ABX-treated mice partially restored the gut microbiota and fully rescued the behaviorally expressed neuropathic pain, of which, Akkermansia, Bacteroides, and Desulfovibrionaceae phylus may play a key role. Conclusion This study demonstrates distinct roles of gut microbiota in the pathogenesis of chronic painful conditions with nerve injury, chemotherapy and diabetic neuropathy and supports the clinical significance of fecal bacteria transplantation.
Examples of homeostatic reciprocal interactions between the nervous system, immune system, and microbiota, elucidated in the zebrafish model
Examples of pathological interactions involving CNS invasion. Pathogens (including but not limited to those listed) may infect immune cells or directly invade neurons or the CNS in zebrafish. Infected immune cells in the periphery may either elicit peripheral nerve damage or cross the blood–brain barrier and invade the CNS, causing neuroinflammation and subsequent neuronal damage. Infection can result in behavioral changes with some similarities to mammals
Animals rely heavily on their nervous and immune systems to perceive and survive within their environment. Despite the traditional view of the brain as an immunologically privileged organ, these two systems interact with major consequences. Furthermore, microorganisms within their environment are major sources of stimuli and can establish relationships with animal hosts that range from pathogenic to mutualistic. Research from a variety of human and experimental animal systems are revealing that reciprocal interactions between microbiota and the nervous and immune systems contribute significantly to normal development, homeostasis, and disease. The zebrafish has emerged as an outstanding model within which to interrogate these interactions due to facile genetic and microbial manipulation and optical transparency facilitating in vivo imaging. This review summarizes recent studies that have used the zebrafish for analysis of bidirectional control between the immune and nervous systems, the nervous system and the microbiota, and the microbiota and immune system in zebrafish during development that promotes homeostasis between these systems. We also describe how the zebrafish have contributed to our understanding of the interconnections between these systems during infection in fish and how perturbations may result in pathology.
Background Stress-induced mental illnesses (mediated by neuroinflammation) pose one of the world’s most urgent public health challenges. A reliable in vivo chemical biomarker of stress would significantly improve the clinical communities’ diagnostic and therapeutic approaches to illnesses, such as depression. Methods Male and female C57BL/6J mice underwent a chronic stress paradigm. We paired innovative in vivo serotonin and histamine voltammetric measurement technologies, behavioral testing, and cutting-edge mathematical methods to correlate chemistry to stress and behavior. Results Inflammation-induced increases in hypothalamic histamine were co-measured with decreased in vivo extracellular hippocampal serotonin in mice that underwent a chronic stress paradigm, regardless of behavioral phenotype. In animals with depression phenotypes, correlations were found between serotonin and the extent of behavioral indices of depression. We created a high accuracy algorithm that could predict whether animals had been exposed to stress or not based solely on the serotonin measurement. We next developed a model of serotonin and histamine modulation, which predicted that stress-induced neuroinflammation increases histaminergic activity, serving to inhibit serotonin. Finally, we created a mathematical index of stress, Si and predicted that during chronic stress, where Si is high, simultaneously increasing serotonin and decreasing histamine is the most effective chemical strategy to restoring serotonin to pre-stress levels. When we pursued this idea pharmacologically, our experiments were nearly identical to the model’s predictions. Conclusions This work shines the light on two biomarkers of chronic stress, histamine and serotonin, and implies that both may be important in our future investigations of the pathology and treatment of inflammation-induced depression.
Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal domain (BET) protein family, plays a crucial role in regulating inflammation and oxidative stress that are tightly related to stroke development and progression. Consequently, BRD4 blockade has attracted increasing interest for associated neurological diseases, including stroke. dBET1 is a novel and effective BRD4 degrader through the proteolysis-targeting chimera (PROTAC) strategy. We hypothesized that dBET1 protects against brain damage and neurological deficits in a transient focal ischemic stroke mouse model by reducing inflammation and oxidative stress and preserving the blood–brain barrier (BBB) integrity. Post-ischemic dBET1 treatment starting 4 h after stroke onset significantly ameliorated severe neurological deficits and reduced infarct volume 48 h after stroke. dBET1 markedly reduced inflammation and oxidative stress after stroke, indicated by multiple pro-inflammatory cytokines and chemokines including IL-1β, IL-6, TNF-α, CCL2, CXCL1 and CXCL10, and oxidative damage markers 4-hydroxynonenal (4-HNE) and gp91phox and antioxidative proteins SOD2 and GPx1. Meanwhile, stroke-induced BBB disruption, increased MMP-9 levels, neutrophil infiltration, and increased ICAM-1 were significantly attenuated by dBET1 treatment. Post-ischemic dBET1 administration also attenuated ischemia-induced reactive gliosis in microglia and astrocytes. Overall, these findings demonstrate that BRD4 degradation by dBET1 improves acute stroke outcomes, which is associated with reduced neuroinflammation and oxidative stress and preservation of BBB integrity. This study identifies a novel role of BET proteins in the mechanisms resulting in ischemic brain damage, which can be leveraged to develop novel therapies.
Background Little is known about how the obesogenic environment influences emotional states associated with glial responses and neuronal function. Here, we investigated glial reactivation and neuronal electrophysiological properties in emotion-related brain regions of high-fat diet (HFD) and ob/ob mice under chronic stress. Methods The glial reactivation and neuronal activities in emotion-related brain regions were analyzed among normal diet mice (ND), HFD mice, wild-type mice, and ob/ob mice. To further activate or inhibit astrocytes in medial prefrontal cortex (mPFC), we injected astrocytes specific Gq-AAV or Gi-AAV into mPFC and ongoing treated mice with CNO. Results The results showed that obesogenic factors per se had no significant effect on neuronal activities in emotion-related brain regions, or on behavioral performance. However, exposure to a chronic stressor profoundly reduced the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) in the mPFC; depressive-like behaviors were seen, accompanied by significant upregulation of astrocyte reactivation. We identified resilient and susceptible mice among chronic social defeat stress-exposed HFD mice. As expected, astrocyte reactivity was upregulated, while neuronal activity was depressed, in the mPFC of susceptible compared to resilient mice. Furthermore, activating astrocytes resulted in similar levels of neuronal activity and depressive-like behaviors between resilient and susceptible mice. Additionally, inhibiting astrocyte reactivation in the mPFC of HFD mice upregulated neuronal activities and inhibited depressive-like behaviors. Conclusions These observations indicate that obesogenic factors increase the risk of depression, and improve our understanding of the pathological relationship between obesity and depression. Graphical Abstract
Study design flowchart. The discovery cohort was followed for at least 1 year, except one lost for the wrong contact detail. The validation cohort with at least a 6-month follow-up was divided into two cohorts due to the consumption of CSF samples in the exploration of experimental conditions. * The two validation cohorts had some duplicated patients (n = 2) and controls (n = 1) but were different from the discovery cohort
Imaging findings of a patient with misdiagnosis. A 69-year-old woman complained of lower-extremity progressive weakness and numbness over one year. Magnetic resonance imaging showed a multisegmental intramedullary T2 hyperintensity (A) with diffuse contrast enhancement (B). There was no evidence of obvious fluid voids. C DSA revealed a fistula (white arrow) at the right T6 level, fed by the radicular artery rising from the segmental artery
Preliminary analysis of DEPs. Volcano plot of CSF (A) and paired plasma samples (B) comparing SDAVF patients with the controls. The red dots refer to significantly overexpressed proteins, and significantly downregulated proteins are coloured blue. Cluster heatmap of CSF (C) and paired plasma samples (D) comparing SDAVF patients with controls. The ‘red’ blocks refer to overexpression and downregulated proteins are coloured ‘blue’. The colour intensity indicates the degree of fold change. E The Venn diagram summarizes the relationship between the two datasets. F GO term/KEGG pathway enrichment analysis of overexpressed proteins in CSF samples. The size of the nodes denotes the number of proteins, and the colour represents the adjusted p value
PPI network and hub gene analysis. Five interactional subclusters (A) and the top 10 hub genes (B) were obtained from 98 DEPs in CSF samples. The node score cut-off was set as 0.2. Continuous colour variation from deep to shallow reveals the score (A) or rank (B) from high to low. C Enrichment and visualization of GO/KEGG annotation based on the subclusters and hub gene list. The size of the nodes denotes the number of proteins, and the colour represents the adjusted p value
Quantitative analysis and clinical relevance of potential biomarkers. A–C The specific protein levels were measured in CSF samples via ELISA kits. The concentration (Y-axis) was normalized to the total protein in CSF. Generally, data that passed the normality test were described by the mean with SD and compared by unpaired t test. *p < 0.05, “ns” refers to p > 0.05. D, E Simple linear regression was used to assess the correlation between clinical factors and proteins levels. Solid lines are the fit of linear regression, and dotted lines represent the 95% confidence interval. F ROC analysis of multiple variables. The diagonal dashed line reflects a random prediction (AUC = 0.5)
Background and purpose A major challenge in spinal dural arteriovenous fistula (SDAVF) is timely diagnosis, but no specific predictive biomarkers are known. Methods In the discovery cohort (case, n = 8 vs. control, n = 8), we used cerebrospinal fluid (CSF) and paired plasma samples to identify differentially expressed proteins by label-free quantitative proteomics. Further bioinformatics enrichment analyses were performed to screen target proteins. Finally, it was validated by ELISA in two of the new cohorts (case, n = 17 vs. control, n = 9), and univariate analysis, simple linear regression, and receiver operator characteristic (ROC) curve analysis were performed to evaluate the diagnostic potential. Results In the discovery cohort, the most overexpressed proteins were APOB and C4BPA in CSF samples of patients. The GO/KEGG enrichment analysis indicated that the upregulated proteins were mainly involved in the acute inflammatory response and complement activation. Hub-gene analysis revealed that APP might be the key protein in the molecular interaction network. In the validation cohort, C4BPA and C1QA were significantly overexpressed in the CSF of patients, averaging 3046.9 ng/ml and 2167.2 ng/ml, respectively. Simple linear regression demonstrated that levels of C1QA and C4 were positively correlated with total protein in CSF (R² = 0.8021, p = 0.0005; R² = 0.7447, p = 0.0013). The areas under the ROC curves of C4BPA and C1QA were 0.86 and 1.00, respectively. Conclusions This study was the first to identify C4BPA and C1QA as potential biomarkers for the diagnosis of SDAVF and revealed that complement pathway activation might be one of the molecular mechanisms for venous hypertension myelopathy.
Background Hydrocephalus is a severe complication of intracerebral hemorrhage with ventricular extension (ICH-IVH) and causes cerebrospinal fluid (CSF) accumulation. The choroid plexus epithelium plays an important role in CSF secretion and constitutes the blood–CSF barrier within the brain–immune system interface. Although the NLRP3 inflammasome, as a key component of the innate immune system, promotes neuroinflammation, its role in the pathogenesis of hydrocephalus after hemorrhage has not been investigated. Therefore, this study aimed to investigate the potential mechanism of NLRP3 in hydrocephalus to discover a potential marker for targeted therapy. Methods A rat model of hydrocephalus after ICH-IVH was developed through autologous blood infusion in wild-type and Nlrp3−/− rats. By studying the features and processes of the model, we investigated the relationship between the NLRP3 inflammasome and CSF hypersecretion in the choroid plexus. Results The ICH-IVH model rats showed ventricular dilation accompanied by CSF hypersecretion for 3 days. Based on the choroid plexus RNA-seq and proteomics results, we found that an inflammatory response was activated. The NLRP3 inflammasome was investigated, and the expression levels of NLRP3 inflammasome components reached a peak at 3 days after ICH-IVH. Inhibition of NLRP3 by an MCC950 inflammasome inhibitor or Nlrp3 knockout decreased CSF secretion and ventricular dilation and attenuated neurological deficits after ICH-IVH. The mechanism underlying the neuroprotective effects of NLRP3 inhibition involved decreased phosphorylation of NKCC1, which is a major protein that regulates CSF secretion by altering Na⁺- and K⁺-coupled water transport, via MCC950 or Nlrp3 knockout. In combination with the in vitro experiments, this experiment confirmed the involvement of the NLRP3/p-NKCC1 pathway and Na⁺ and K⁺ flux. Conclusions This study demonstrates that NKCC1 phosphorylation in the choroid plexus epithelium promotes NLRP3 inflammasome-mediated CSF hypersecretion and that NLRP3 plays an important role in the pathogenesis of hydrocephalus after hemorrhage. These findings provide a new therapeutic strategy for treating hydrocephalus.
Background The pathogenesis of neuropathic pain and the reasons for the prolonged unhealing remain unknown. Increasing evidence suggests that sex oestrogen differences play a role in pain sensitivity, but few studies have focused on the oestrogen receptor which may be an important molecular component contributing to peripheral pain transduction. We aimed to investigate the impact of oestrogen receptors on the nociceptive neuronal response in the dorsal root ganglion (DRG) and spinal dorsal horn using a spared nerve injury (SNI) rat model of chronic pain. Methods We intrathecally (i.t.) administered a class of oestrogen receptor antagonists and agonists intrathecal (i.t.) administrated to male rats with SNI or normal rats to identify the main receptor. Moreover, we assessed genes identified through genomic metabolic analysis to determine the key metabolism point and elucidate potential mechanisms mediating continuous neuronal sensitization and neuroinflammatory responses in neuropathic pain. The excitability of DRG neurons was detected using the patch-clamp technique. Primary culture was used to extract microglia and DRG neurons, and siRNA transfection was used to silence receptor protein expression. Immunofluorescence, Western blotting, RT-PCR and behavioural testing were used to assess the expression, cellular distribution, and actions of the main receptor and its related signalling molecules. Results Increasing the expression and function of G protein-coupled oestrogen receptor (GPER), but not oestrogen receptor-α (ERα) and oestrogen receptor-β (ERβ), in the DRG neuron and microglia, but not the dorsal spinal cord, contributed to SNI-induced neuronal sensitization. Inhibiting GPER expression in the DRG alleviated SNI-induced pain behaviours and neuroinflammation by simultaneously downregulating iNOS, IL-1β and IL-6 expression and restoring GABAα2 expression. Additionally, the positive interaction between GPER and β-alanine and subsequent β-alanine accumulation enhances pain sensation and promotes chronic pain development. Conclusion GPER activation in the DRG induces a positive association between β-alanine with iNOS, IL-1β and IL-6 expression and represses GABAα2 involved in post-SNI neuropathic pain development. Blocking GPER and eliminating β-alanine in the DRG neurons and microglia may prevent neuropathic pain development.
Background Peripheral nerve inflammation or lesion can affect contralateral healthy structures, and thus result in mirror-image pain. Supraspinal structures play important roles in the occurrence of mirror pain. The anterior cingulate cortex (ACC) is a first-order cortical region that responds to painful stimuli. In the present study, we systematically investigate and compare the neuroimmune changes in the bilateral ACC region using unilateral- (spared nerve injury, SNI) and mirror-(L5 ventral root transection, L5-VRT) pain models, aiming to explore the potential supraspinal neuroimmune mechanism underlying the mirror-image pain. Methods The up-and-down method with von Frey hairs was used to measure the mechanical allodynia. Viral injections for the designer receptors exclusively activated by designer drugs (DREADD) were used to modulate ACC glutamatergic neurons. Immunohistochemistry, immunofluorescence, western blotting, protein microarray were used to detect the regulation of inflammatory signaling. Results Increased expressions of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and chemokine CX3CL1 in ACC induced by unilateral nerve injury were observed on the contralateral side in the SNI group but on the bilateral side in the L5-VRT group, representing a stronger immune response to L5-VRT surgery. In remote ACC, both SNI and L5-VRT induced robust bilateral increase in the protein level of Nav1.6 (SCN8A), a major voltage-gated sodium channel (VGSC) that regulates neuronal activity in the mammalian nervous system. However, the L5-VRT-induced Nav1.6 response occurred at PO 3d, earlier than the SNI-induced one, 7 days after surgery. Modulating ACC glutamatergic neurons via DREADD-Gq or DREADD-Gi greatly changed the ACC CX3CL1 levels and the mechanical paw withdrawal threshold. Neutralization of endogenous ACC CX3CL1 by contralateral anti-CX3CL1 antibody attenuated the induction and the maintenance of mechanical allodynia and eliminated the upregulation of CX3CL1, TNF-α and Nav1.6 protein levels in ACC induced by SNI. Furthermore, contralateral ACC anti-CX3CL1 also inhibited the expression of ipsilateral spinal c-Fos, Iba1, CD11b, TNF-α and IL-6. Conclusions The descending facilitation function mediated by CX3CL1 and its downstream cascade may play a pivotal role, leading to enhanced pain sensitization and even mirror-image pain. Strategies that target chemokine-mediated ACC hyperexcitability may lead to novel therapies for the treatment of neuropathic pain.
Background and purpose: An aneurysmal subarachnoid hemorrhage is a devastating event. To establish an effective therapeutic strategy, its pathogenesis must be clarified, particularly the pathophysiology of brain harboring intracranial aneurysms (IAs). To elucidate the pathology in brain harboring IAs, we examined the significance of the receptor for advanced glycation end-products (RAGE)/mineralocorticoid receptor (MR) pathway and Na+/K+-ATPase (ATP1α3). Methods: Ten-week-old female rats were subjected to oophorectomy as well as hypertension and hemodynamic changes to induce IAs, and were fed a high-salt diet. Brain damage in these rats was assessed by inflammatory changes in comparison to sham-operated rats fed a standard diet. Results: Six weeks after IA induction (n = 30), irregular morphological changes, i.e., an enlarged vessel diameter and vascular wall, were observed in all of the left posterior cerebral arteries (Lt PCAs) prone to rupture. Approximately 20% of rats had ruptured IAs within 6 weeks. In brain harboring unruptured IAs at the PCA, the mRNA levels of RAGE and MR were higher, and that of ATP1α3 was lower than those in the sham-operated rats (p < 0.05, each). Immunohistochemically, elevated expression of RAGE and MR, and decreased expression of ATP1α3 were observed in the brain parenchyma adjacent to the Lt PCA, resulting in increased Iba-1 and S100B expression that reflected the inflammatory changes. There was no difference between the unruptured and ruptured aneurysm rat groups. Treatment with the MR antagonist esaxerenone abrogated these changes, and led to cerebral and vascular normalization and prolonged subarachnoid hemorrhage-free survival (p < 0.05). Conclusions: Regulation of the imbalance between the RAGE/MR pathway and ATP1α3 may help attenuate the damage in brain harboring IAs, and further studies are warranted to clarify the significance of the down-regulation of the MR/RAGE pathway and the up-regulation of ATP1α3 for attenuating the pathological changes in brain harboring IAs.
Background Medulloblastoma (MB) is the most common malignant brain tumor in children. Approximately one-third of MB patients remain incurable. Understanding the molecular mechanism of MB tumorigenesis is, therefore, critical for developing specific and effective treatment strategies. Our previous work demonstrated that astrocytes constitute the tumor microenvironment (TME) of MB and play an indispensable role in MB progression. However, the underlying mechanisms by which astrocytes are regulated and activated to promote MB remain elusive. Methods By taking advantage of Math1-Cre/Ptch1 loxp/loxp mice, which spontaneously develop MB, primary MB cells and astrocytes were isolated and then subjected to administration and coculture in vitro. Immunohistochemistry was utilized to determine the presence of C3a in MB sections. MB cell proliferation was evaluated by immunofluorescent staining. GFAP and cytokine expression levels in C3a-stimulated astrocytes were assessed by immunofluorescent staining, western blotting, q-PCR and ELISA. C3a receptor and TNF-α receptor expression was determined by PCR and immunofluorescent staining. p38 MAPK pathway activation was detected by western blotting. Transplanted MB mice were treated with a C3a receptor antagonist or TNF-α receptor antagonist to investigate their role in MB progression in vivo. Results We found that complement C3a, a fragment released from intact complement C3 following complement activation, was enriched in both human and murine MB tumor tissue, and its receptor was highly expressed on tumor-associated astrocytes (TAAs). We demonstrated that C3a activated astrocytes and promoted MB cell proliferation via the p38 MAPK pathway. Moreover, we discovered that C3a upregulated the production of proinflammatory cytokines, such as IL-6 and TNF-α in astrocytes. Application of the conditioned medium of C3a-stimulated astrocytes promoted MB cell proliferation, which was abolished by preincubation with a TNF-α receptor antagonist, indicating a TNF-α-dependent event. Indeed, we further demonstrated that administration of a selective C3a receptor or TNF-α receptor antagonist to mice subcutaneously transplanted with MB suppressed tumor progression in vivo. Conclusions C3a was released during MB development. C3a triggered astrocyte activation and TNF-α production via the p38 pathway, which promoted MB cell proliferation. Our findings revealed the novel role of C3a-mediated TNF-α production by astrocytes in MB progression. These findings imply that targeting C3a and TNF-α may represent a potential novel therapeutic approach for human MB.
Expression of Apelin decreased after spinal cord injury (SCI). A Spinal cord sections obtained from healthy uninjured rats (control) and SCI rats on days 1, 3, 7, 14 and 28 after SCI; Apelin protein levels were normalized to that of GAPDH after western blotting. B Gene expression of Apelin was quantified in total RNA isolated from spinal cord tissue using qRT-PCR with specific primers; GAPDH was used as a loading control for qRT-PCR. C Serum Apelin was assayed by ELISA after SCI. (*p < 0.05, **p < 0.01 vs. Sham group, #p < 0.05 14 d vs. 28 d, mean ± SD)
Changes of Apelin expression in spinal cord tissues during spinal cord injury. Immunofluorescence imaging showing co-localization of Apelin (green) with markers of specific cells types including NeuN (red, neurons), GFAP (red, astrocytes), and iba1 (red, microglia) in A normal spinal cord group and B 14 days after SCI; nuclei were counterstained with DAPI (blue). Co-localization appears yellow in the merged image. Scale bar = 60 µm
Identification of primary cultured neural stem cells (NSCs) and evaluation of their proliferation after administration of Apelin or its inhibitor ML221. A Morphology of NSCs under a light microscope, scale bar = 100 µm (left), 50 µm (middle) or 25 µm (right). B Identification of primary cultured NSCs, scale bar = 130 µm. C, D Multi-differentiation potential of NSCs was assessed by GFAP+ cells (green) and Olig2+ cells after 14 d, scale bar = 130 µm or 120 µm. Proliferation of NSCs under different dosages of Apelin and its inhibitor ML221 were determined by CCK8 assay at 24 h (E), 48 h (F), and 14 d (G) after SCI (*p < 0.05, **p < 0.01 vs. normal group, mean ± SD)