Microglial Marker Research Articles

Quantitative susceptibility mapping is more sensitive and specific than phase imaging in detecting chronic active multiple sclerosis lesion rims: pathological validation

Abstract Quantitative susceptibility mapping and phase imaging are used to identify multiple sclerosis lesions with paramagnetic rims that slowly expand over time, and are associated with earlier progression to disability, decreased brain volume, and increased frequency of clinical relapse. However, the presence of iron-laden microglia/macrophages at the lesion rim and demyelination within the lesion both contribute to phase and quantitative susceptibility mapping images. Therefore, simultaneous pathological validation is needed to assess accuracies in identifying iron-positive lesions. MRI was performed on fifteen multiple sclerosis brain slabs; thirty-two lesions of interest were processed for myelin, iron, and microglial markers. Three experienced readers classified lesions as rim positive or negative on quantitative susceptibility mapping and phase; these classifications were compared with Perls’ stain as the gold standard. All ten of the quantitative susceptibility mapping-positive lesions had iron-positive rims on histology. Of the sixteen phase-positive lesions, only ten had iron-positive rims on histology. Using Perls’ stain as the ground truth, the positive predictive value was 100% for quantitative susceptibility mapping and 63% for phase; the negative predictive value was 95% for quantitative susceptibility mapping and 94% for phase. Post-mortem imaging results demonstrate that quantitative susceptibility mapping is a more reliable indicator of an iron-positive rim compared to phase imaging.

Dietary blueberry before and/or after exposure to HZE particle radiation attenuates neuroinflammation, oxidative stress, glial cell activation, and memory deficits in rats.

Acute neuroinflammatory and oxidative-stress (OS)-inducing stressors, such as high energy and charge (HZE) particle irradiation, produce accelerated aging in the brain. Anti-inflammatory and antioxidant foods, such as blueberries (BB), attenuate neuronal and cognitive deficits when administered to rodents before or both before and after HZE particle exposure. However, the effects of post-stressor treatments are unknown and may be important to repair initial damage and prevent progressive neurodegeneration. This study assessed the differential efficacy and mechanistic targets of a BB-supplemented diet before and/or after HZE particle irradiation on neuroinflammation, OS, glial cell activation, and memory deficits. Two-month-old male Sprague-Dawley rats (n=120) consumed a 2% BB or control diet for 45 days. Rats were whole-body irradiated (150 cGy 56Fe) or were not irradiated, followed by a 45-day post-treatment interval in which they were fed a 2% BB or control diet. The novel object recognition test (NOR) was performed at the end of the post-treatment interval to evaluate memory. Biomarkers of neuroinflammation, OS, and glial cell activity were evaluated in the hippocampus and frontal cortex of rat brains following euthanasia. Statistical analyses included analysis of variance, t-tests, and Pearson correlations. Pre- and/or post-irradiation BB treatments were similarly effective at reducing 56Fe-induced recognition memory deficits on the NOR and the protein and/or mRNA expression of neuroinflammatory factors (tumor necrosis factor-ɑ [TNFɑ], inducible nitric oxide synthase [iNOS], cyclooxygenase-2 [COX-2], phosphorylated IκB-α [pIκB-ɑ]), one mediator of oxidative stress (NADPH oxidase [NOX2]), and markers for microglia and astrocyte activity (CD68 and glial fibrillary acidic protein [GFAP]) in the frontal cortex and hippocampus of rats 45 days post-irradiation (p < 0.05). Findings support the use of dietary post-treatments with antioxidant and anti-inflammatory properties to attenuate biochemical changes in the brain and memory deficits following acute neuroinflammatory/OS-inducing stressors, in addition to having protective benefits.

LCN2 Regulates Microglia Polarization Through the p38MAPK-PGC-1α-PPARγ Pathway to Alleviate Traumatic Brain Injury.

Traumatic brain injury (TBI) is a common traumatic event that imposes a significant burden on families and society. Lipocalin (LCN) is a class of multifunctional secreted lipoprotein molecules. This study aimed to explore the role and possible mechanism of LCN2 in TBI. A rat model of TBI was constructed and adeno-associated virus-coated shRNA-LCN2 was used to silence LCN2 expression. The modified neurological severity score (mNSS), learning and memory ability, pathological injury of brain tissue, number of neurons, and expression of neurotrophic factors were analyzed, and the expression of inflammatory factors, M1/M2 polarization of microglia, and p38MAPK-PGC-1α-PPARγ pathway after LCN2 silencing were further detected. Results found that LCN2 was highly expressed in the brain tissue of TBI rats, and there were obvious learning and cognitive impairments and pathological injury of brain tissue. After silencing LCN2, the mNSS was further increased, and the learning and cognitive ability was weakened. Similarly, silencing LCN2 increased the brain tissue water content, aggravated the histopathology degree, decreased the number of surviving neurons, and reduced the expression of neurotrophic factors in TBI model rats. In addition, the expression of M1 proinflammatory cytokines and polarization markers in microglia of TBI was increased, and the expression of M2 cytokines and markers was decreased after silencing LCN2. Silencing LCN2 also inhibited the activation of the p38MAPK-PGC-1α-PPARγ pathway. In conclusion, LCN2 was released by surviving neurons after TBI, and the increased LCN2 activated the p38MAPK-PGC-1α-PPARγ pathway, which promoted M2 polarization of microglia, and secreted neurotrophic factors, thereby alleviating secondary brain injury.

Temporal changes of spinal microglia in murine models of neuropathic pain: a scoping review.

Neuropathic pain (NP) is an ineffectively treated, debilitating chronic pain disorder that is associated with maladaptive changes in the central nervous system, particularly in the spinal cord. Murine models of NP looking at the mechanisms underlying these changes suggest an important role of microglia, the resident immune cells of the central nervous system, in various stages of disease progression. However, given the number of different NP models and the resource limitations that come with tracking longitudinal changes in NP animals, many studies fail to truly recapitulate the patterns that exist between pain conditions and temporal microglial changes. This review integrates how NP studies are being carried out in murine models and how microglia changes over time can affect pain behavior in order to inform better study design and highlight knowledge gaps in the field. 258 peer-reviewed, primary source articles looking at spinal microglia in murine models of NP were selected using Covidence. Trends in the type of mice, statistical tests, pain models, interventions, microglial markers and temporal pain behavior and microglia changes were recorded and analyzed. Studies were primarily conducted in inbred, young adult, male mice having peripheral nerve injury which highlights the lack of generalizability in the data currently being collected. Changes in microglia and pain behavior, which were both increased, were tested most commonly up to 2 weeks after pain initiation despite aberrant microglia activity also being recorded at later time points in NP conditions. Studies using treatments that decrease microglia show decreased pain behavior primarily at the 1- and 2-week time point with many studies not recording pain behavior despite the involvement of spinal microglia dysfunction in their development. These results show the need for not only studying spinal microglia dynamics in a variety of NP conditions at longer time points but also for better clinically relevant study design considerations.

Human brain microglia responses to amyloid beta and tau pathology in high pathology controls and across disease stages

AbstractBackgroundMicroglia responses to Aβ and tau pathology and the dysregulation of the microglial role in synaptic function may determine the onset and course of Alzheimer’s disease (AD). While significant work has been performed in mouse models, we still lack a complete understanding of physiological and pathological microglial states and functions in human AD brain.MethodFor immunoblotting of brain homogenates against multiple microglial markers, and flow cytometry (FC) analysis of synaptosomal fractions (SNAP25/CD47/Aβ(10G4)/phospho‐tau(AT8)), 49 cryopreserved human parietal cortex samples were categorized into four groups: low pathology control (LPC), high Aβ control (HAC), high pathology control (HPC), and AD. Selected microglia markers were assessed in the snRNAseq dataset (Rexach et al., 2023, bioRxiv) and validated by immunohistochemistry (IHC) analysis.ResultIn the LPC group, only 22.5% of Aβ‐positive (+)/p‐tau‐negative (‐) synaptosomes expressed CD47 on their surface, with a significant progressive increase in the percentage (%) of CD47+ events in the Aβ+/p‐tau‐ group across disease stages, reaching 53.4% in the AD group. Surprisingly, Aβ+/tau+ synaptosomes expressed the highest CD47 levels, suggesting protection from elimination. Higher levels of P2ry12 and CD206 were found in the LPC group compared to all other groups in brain homogenates. Interestingly, the levels of P2ry12, CD206, and Axl1 were significantly higher, and the level of Clec7a was significantly lower in the HPC group compared to the AD group. Linear regression modeling revealed a significant negative association between P2ry12 and synaptic tau pathology, as well as CD47 expression in p‐tau‐positive synaptosomes. Interestingly, in primary tauopathy microglia (but not AD microglia), IKFZ1‐regulated genes, including P2ry12, were upregulated according to snRNAseq and around p‐tau‐bearing neurons by IHC.ConclusionOur data suggest that in normal aging, synaptic Aβ accumulation leads to a diminishing of synaptic CD47 (‘don’t eat me’) signal, accelerating the clearance of malfunctioning Aβ but not p‐tau positive synapses. Upregulation of P2ry12 and CD206, accompanied by downregulation of Clec7a, may represent an early and potentially protective response to pre‐tangle tau pathology. Later in disease progression, P2ry12 reduction may drive a more reactive, phagocytic phenotype in response to tau pathology, leading to an increase in synaptic CD47 levels and synaptic pathology accumulation.

Repeated exposure to high-dose nicotine induces prefrontal gray matter atrophy in adolescent male rats

Incidences of seizure after e-cigarette use in adolescents and young adults have been reported, raising the concern about the risk of nicotine overconsumption. Few previous studies have investigated the effects of nicotine at high doses on brain and behavior in adolescent animals. In this study, the effects of a 15-day repeated nicotine treatment at a daily dose of 2 mg/kg body weight were investigated in adolescent and adult male rats. Nicotine treatment abolished body weight gain in the adults, but did not affect the body weight significantly in the adolescents. Only the nicotine-treated adolescents showed significant changes in brain anatomy 1 day post-treatment, which manifested as a significant reduction of whole-brain gray matter (GM) volume, a further reduction of regional GM volume in the medial prefrontal cortex (mPFC) and altered GM volume covariations between the mPFC and a number of brain regions. The mPFC of nicotine-treated adolescent rats did not exhibit evident signs of neuronal degeneration and reactive astrocytosis, but showed a significantly decreased expression of presynaptic marker synaptophysin (SYN), along with a significantly increased oxidative stress and a significantly elevated expressions of microglial marker ionized calcium binding adaptor molecule 1 (IBA1). Together, these results suggested that repeated nicotine overdosing may shift regional redox, modulate microglia-mediated pruning, and give rise to structural/connectivity deficits in the mPFC of adolescent male rats.

Exploring the mechanism of Taohong Siwu Decoction in treating ischemic stroke injury via the circDnajc1/miR-27a-5p/C1qc signaling axis

BackgroundIschemic stroke (IS) is the most prevalent type of cerebrovascular disease. Taohong Siwu Decoction (THSWD) has been demonstrated to have neuroprotective benefits during Cerebral Ischemia-Reperfusion Injury (CIRI). Whether THSWD mitigates CIRI by modulating the circDnajc1/miR-27a-5p/C1qc signaling axis is not known. ObjectiveExamine how THSWD provides neuroprotective benefits in reducing the damage wrought by IS. MethodsWe employed middle cerebral artery occlusion/reperfusion (MCAO/R) rat model and oxygen glucose deprivation/re-oxygenation (OGD/R) in vitro model. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot (WB), and immunofluorescence analyses (IF) were utilized to detect microglial activation markers TMEM119, CD11b, and Iba1, inflammatory mediators CCL2, CCL6, IL1, IL6, IL10, and TNF-α, as well as circDnajc1, miR-27a-5p, C1qc, C3, and C5aR. ConclusionOur data suggest that THSWD can mitigate inflammatory response, inhibit microglial activation and neuron apoptosis, and exert neuroprotective effects by regulating the circDnajc1/miR-27a-5p/C1qc signaling axis.

Quercetin alleviates neonatal hypoxic-ischaemic brain injury by rebalancing microglial M1/M2 polarization through silent information regulator 1/ high mobility group box-1 signalling.

Neonatal hypoxic-ischaemic encephalopathy (HIE) remains one of the major causes of neonatal death and long-term neurological disability. Due to its complex pathogenesis, there are still many challenges in its treatment. In our previous studies, we found that quercetin can alleviate neurological dysfunction after hypoxic-ischaemic brain injury (HIBI) in neonatal mice. As demonstrated through in vitro experiments, quercetin can inhibit the activation of the TLR4/MyD88/NF-κB signalling pathway and the inflammatory response in the microglial cell line BV2 after oxygen-glucose deprivation. However, the in-depth mechanism still needs to be further elucidated. In the present study, 7day-old neonatal ICR mice or BV2 cells were treated with quercetin with or without the SIRT1 inhibitor EX527 via neurobehavioural, histopathological and molecular methods. In vivo experiments have shown that quercetin can significantly improve the performance of HI mice in behavioural tests, such as the Morris water maze, rotarod test and pole climbing test, and reduce HI insult-induced structural brain damage, cell apoptosis and hippocampal neuron loss. Quercetin also inhibited the immunofluorescence intensity of the microglial M1 marker CD16 + 32 and significantly downregulated the expression of the M1-related proteins iNOS, IL-1β and TNF-α. Moreover, quercetin increased the immunofluorescence intensity of the microglial M2 marker CD206 and significantly increased the expression of the M2-related proteins Arg-1 and IL-10. In addition, quercetin limits the nucleocytoplasmic translocation and release of microglial HMGB1 and further suppresses the activation of the downstream TLR4/MyD88/NF-κB signalling pathway. The above effects of quercetin are partially weakened by pretreatment with EX527. Similar results were found in in vitro experiments, and the mechanism further revealed that the rebalancing effect of quercetin on microglial polarization is achieved through the SIRT1-mediated reduction in HMGB1 acetylation levels. This study provides new and complementary insights into the neuroprotective effects of quercetin and a new direction for the treatment of neonatal HIE.

Maresin-1 Ameliorates Sepsis-Induced Microglial Activation Through Modulation of the P38 MAPK Pathway.

Sepsis is a life-threatening disease characterized by a dysregulated immune response to infection, often leading to neuroinflammation. As a known immunomodulator, Maresin-1 (MaR1) may have potential applications in the treatment of sepsis-induced neuroinflammation, but its effects in this context are unknown. We used a mouse cecum ligation and puncture (CLP)-induced sepsis model and an in vitro lipopolysaccharide (LPS)-induced neuroinflammatory model of BV2 microglia. Expression of microglial cell markers (IBA1, CD11B, CD68, CD86 and CD206) and pro-inflammatory markers (iNOS and COX2) was assessed. The role of MaR1 in regulating the P38 MAPK pathway was explored using the P38 MAPK inhibitor SB203580. In the CLP model, an increased proportion of M1-type microglia was observed, and MaR1 was able to reverse it. However, the combination of SB203580 and MaR1 did not enhance the therapeutic effect compared to SB20580 alone. In vitro experiments, MaR1 inhibited LPS-induced P38 MAPK nuclear translocation and decreased the expression of pro-inflammatory markers such as iNOS and COX2. As with the animal results, no stacking effect could be obtained with the co-administration of SB203580 and MaR1. Our findings suggest that MaR1 attenuates sepsis-induced neuroinflammation mainly by inhibiting phosphorylation of P38 MAPK in microglial cells. This suggests that MaR1 may have a potential therapeutic role in the treatment of sepsis neuroinflammation.

Astragalus polysaccharide treatment relieves cerebral ischemia‒reperfusion injury by promoting M2 polarization of microglia by enhancing O-GlcNAcylation.

Cerebral ischemia‒reperfusion (I/R) injury seriously threatens the lives of patients. Astragalus polysaccharide (APS) is the main active ingredient of Astragalus membranaceus and has a wide range of pharmacological activities. Here, we aimed to explore the impacts of APS on cerebral I/R injury and its specific mechanisms. We established a cerebral I/R injury model using middle cerebral artery occlusion (MCAO)-treated rats and oxygen glucose deprivation/reoxygenation (OGD/R)-treated BV2 cells. The interleukin 1β (IL-1β), interleukin 6 (IL-6) and interleukin (IL-10) levels were determined using corresponding ELISA kits and RT‒qPCR. The levels of M1 microglial markers (INOS and CD16) and M2 microglial markers (Arg-1 and CD206) were measured by RT‒qPCR. The O-linked N-acetylglucosamine modification (O-GlcNAcylation), O-GlcNAc transfer (OGT) and O-GlcNAc glycosidase (OGA) protein levels were measured by Western blot.Our results showed that APS treatment decreased IL-1β (179.72 ± 9.08 vs. 81.33 ± 6.30) and IL-6 (445.56 ± 33.09 vs. 234.75 ± 27.62) levels and increased IL-10 (41.95 ± 4.18 vs. 86.40 ± 7.16) levels in OGD/R-treated BV2 cells (p < 0.001). In addition, APS promoted the M2 polarization of OGD/R-treated BV2 cells, manifested by an increase in Arg-1 (0.43 ± 0.04 vs. 0.76 ± 0.03) and CD206 (0.36 ± 0.03 vs. 0.65 ± 0.06) and a decrease in INOS (2.84 ± 0.39 vs. 1.56 ± 0.19) and CD16 (4.04 ± 0.36 vs. 1.88 ± 0.09) in OGD/R-treated BV2 cells (p < 0.001). Additionally, APS treatment increased the O-GlcNAcylation and OGT levels in OGD/R-treated BV2 cells, while OGT knockdown reversed the effect of APS in OGD/R-treated BV2 cells and MCAO-treated rats (p < 0.05).Our study demonstrated that APS alleviated cerebral I/R injury by promoting the M2 polarization of microglia by enhancing OGT-mediated O-GlcNAcylation.

The microglial translocator protein (TSPO) in Alzheimer’s disease reflects a phagocytic phenotype

Translocator protein (TSPO) is a mitochondrial protein expressed by microglia, ligands for which are used as a marker of neuroinflammation in PET studies of Alzheimer’s disease (AD). We previously showed increasing TSPO load in the cerebral cortex with AD progression, consistent with TSPO PET scan findings. Here, we aim to characterise the microglial phenotype associated with TSPO expression to aid interpretation of the signal generated by TSPO ligands in patients. Human post-mortem sections of temporal lobe (TL) and cerebellum (Cb) from cases classified by Braak group (0–II, III–IV, V–VI; each n = 10) were fluorescently double labelled for TSPO and microglial markers: Iba1, HLA-DR, CD68, MSR-A and CD64. Quantification was performed on scanned images using QuPath software to assess the microglial phenotype of TSPO. Qualitative analysis was also performed for TSPO with GFAP (astrocytes), CD31 (endothelial cells) and CD163 (perivascular macrophages) to characterise the cellular profile of TSPO. The percentage of CD68+TSPO+ double-labelled cells was significantly higher than for other microglial markers in both brain regions and in all Braak stages, followed by MSR-A+TSPO+ microglia. Iba1+TSPO+ cells were more numerous in the cerebellum than the temporal lobe, while CD64+TSPO+ cells were more numerous in the temporal lobe. No differences were observed for the other microglial markers. TSPO expression was also detected in endothelial cells, but not detected in astrocytes nor in perivascular macrophages. Our data suggest that TSPO is mainly related to a phagocytic profile of microglia (CD68+) in human AD, potentially highlighting the ongoing neurodegeneration.

The impact of inactivation of the GH/IGF axis during aging on healthspan.

Several mouse lines with congenital growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis disruption have shown improved health and extended lifespan. The current study investigated how inactivating this axis, specifically during aging, impacts the healthspan. We used a tamoxifen-inducible global GH receptor (GHR) knockout mouse model starting at 12 months and followed the mice until 24 months of age (iGHRKO12-24 mice). We found sex- and tissue-specific effects, with some being pro-aging and others anti-aging. Measuring an array of cytokines in serum revealed that inactivation of the GH/IGF-1 axis at 12 months did not affect systemic inflammation during aging. On the other hand, hypothalamic inflammation was significantly reduced in iGHRKO12-24 mice, evidenced by GFAP+ (glial fibrillary acidic protein, a marker of astrocytes) and Iba-1+ (a marker for microglia). Liver RNAseq analysis indicated feminization of the male transcriptome, with significant changes in the expression of monooxygenase, sulfotransferase, and solute-carrier-transporter gene clusters. Finally, we found impaired bone morphology, more pronounced in male iGHRKO12-24 mice and correlated with GH/IGF-1 inactivation onset age. We conclude that inhibiting the GH/IGF-1 axis during aging only partially preserves the beneficial healthspan effects observed with congenital GH deficiency.

Morphology of Cortical Microglia in the Hyperacute Phase of Subarachnoid Hemorrhage.

Hemorrhagic stroke is the deadliest type of stroke. Cellular and molecular biomarkers are important for understanding the pathophysiology of stroke. Microglia are among the most promising biological markers. However, the morphological and physiological characteristics of microglia, as well as the structural and functional aspects of their interactions with neurons and other cells, are largely unknown. Due to the large number of different morphological phenotypes and very limited information on microglial changes in subarachnoid hemorrhage (SAH), we performed this study aimed at identifying the features of the distribution of various microglial phenotypes in the layers of the cerebral cortex in the hyperacute phase of non-traumatic SAH. We studied the distribution of various microglial phenotypes in the layers of the cerebral cortex of SAH non-survivors with a control group (coronary heart disease and sudden cardiac death were the underlying causes of death). An immunohistochemical study using antibodies to iba-1 (a marker of microglia) revealed changes in the morphological phenotypes of microglia in the cerebral cortex after subarachnoid hemorrhage. Significant differences between the groups indicate a rapid microglial response to injury. The findings indicate that there are quantitative and phenotypic changes in microglia in the cerebral cortex during early SAH in the human cortex.

Huang-Lian-Jie-Du decoction alleviates cognitive impairment in high-fat diet-induced obese mice via Trem2/Dap12/Syk pathway

BackgroundCognitive impairment induced by a high-fat diet (HFD) is common, but its mechanism is largely unknown. Huang-Lian-Jie-Du (HLJD) decoction is a classical and powerful prescription in China. It consists of four medicinal plants and is widely used in traditional Chinese medicines (TCM). Studies have shown that HLJD decoction is effective in treating obesity, depression, and so on. However, the therapeutic mechanism of HLJD is still poorly understood. PurposeOur study aimed to explore whether inflammatory factors and Trem2/Dap12/Syk pathway are involved in this process and whether HLJD treatment can repair cognitive impairment in HFD-induced obesity. MethodsTo obtain the obese mice, male mice were treated with HFD (60 Kcal% fat) for 16 weeks. After an additional eight weeks, HLJD decoction was administered orally at doses of 4 and 8 g/kg daily for eight weeks. The mice were then subjected to four behavior tests. Aβ42, total Tau, inflammatory-related, and microglial dysregulation-related markers expression were measured. Molecular docking analysis was also conducted to predict the interaction of the chemical constituents of HLJD with human TREM2, DAP12, and SYK. HLJD at doses of 12.5, 25, and 50 µg/mL or limonin at concentrations of 12.5, 25, and 50 µM were used to treat BV2 cells for 24 h. CCK8 assay and Trem2, Dap12, Syk, and p-Syk expression were measured. ResultsOur study revealed that cognitive impairment was evident in mice treated with HFD, indicating the impact of obesity on cognitive function. The expression of Aβ42 and total Tau in the hippocampus (HIP) was significantly higher in obese (HFD-V) mice compared to normal control (NC-V) mice. The Il6, Il1b, and Il10 mRNA expression levels were also markedly increased in the HIP of obese mice. Furthermore, Trem2, Dap12, p-Syk, and Iba1 expression were elevated in the HIP of obese mice. Importantly, HLJD treatment was found to repair cognitive impairment and lower the protein expression of Aβ42, Tau, Trem2, Dap12, p-Syk, and the expression of Il6, Il1b, and Il10 mRNA in HIP of HFD-V mice. The increased expression of Trem2, Dap12, p-Syk, and Iba1 in HIP after HFD consumption could be reduced after receiving HLJD decoction. The compound Limonin showed a well-predicted binding energy with TREM2, DAP12, and SYK. BV2 cells with HLJD or limonin detected the mRNA expressions of Trem2/Dap12. HLJD at 25 and 50 µg/mL decreased Trem2, Dap12, and p-Syk protein levels in BV2 cells. ConclusionThese results reveal that HLJD treatment could alleviate cognitive impairment in HFD-induced obese mice by controlling the activation of the Trem2/Dap12 pathway and reducing Syk phosphorylation in HIP microglia. HLJD and limonin suppressed Trem2/Dap12/Syk signaling pathway in BV2 cells. HLJD therapy might represent a novel treatment for patients with cognitive impairment induced by obesity.

TMIC-71. ELUCIDATING IMMUNE MICROENVIRONMENTAL DISTINCTIONS BETWEEN IDH-MUTANT ASTROCYTOMA AND OLIGODENDROGLIOMA USING SINGLE-CELL TRANSCRIPTIONAL PROFILING

Abstract Despite exhibiting longer overall survival that IDH-wt counterparts, IDH-mutant gliomas remain uniformly deadly and impact patients in the prime of adult life. The immune microenvironment of IDH-mutant gliomas remains incompletely characterized, particularly with regard to distinctions between IDH-mutant astrocytoma and IDH-mutant, 1p/19q codeleted oligodendroglioma. To address this knowledge gap, we subjected dissociated tumor suspensions derived from 7 astrocytoma and 7 oligodendroglioma clinical samples to flow cytometry-based sorting for the pan-immune marker, CD45, and the microglial marker, P2RY12. This process yielded both microglial and non-microglial immune cell populations, which were then analyzed by scRNA sequencing. This workflow revealed a single large cluster of microglia, characterized by expression of Galectin-1 and lysosomal Lipase A, which was present at a significantly higher level in oligodendroglioma. While the biological significance of this cluster is unclear, gene set enrichment analysis exploring phenotypic differences between microglia in astrocytoma and oligodendroglioma revealed significant increases in expression of gene sets related to antigen presentation in astrocytoma. Parallel analysis of the non-microglial immune fraction showed an increase in the expression of pro-inflammatory gene sets, such as genes related to cytokine secretion, within the CD8 T-cell compartment of astrocytomas. We also observed upregulation of the expression of inflammatory gene sets within the dendritic cells of astrocytomas. Taken together, these changes suggest that the immune compartment of astrocytoma is more active and inflamed, and targeting the immune system in these tumors may represent a potential therapeutic avenue.

TMIC-70. CORRELATION OF LLT-1 AND NLRC4 INFLAMMASOME AND ITS EFFECT ON GLIOBLASTOMA PROGNOSIS

Abstract PURPOSE LLT-1 is a well-known ligand for the natural killer (NK) cell inhibitory receptor NKRP1A. Here, we examined NLRC4 inflammasome components and LLT-1 expression in glioblastoma (GBM) tissues to elucidate potential associations and interactions between these factors. METHODS Nine GBM tissues were collected for experiments and RNA sequencing, while fifty-two tumor core tissues were collected for RNA sequencing. Expression of LLT-1 and other proteins was assessed by immunofluorescence. Computational analyses utilized RNA-seq data from 296 and 52 patients from the Chinese Glioma Genome Atlas and CHA medical records, respectively, using survival, non-negative matrix factorization clustering, Gene Ontology enrichment, and protein-protein interaction analyses. Receptor-ligand interactions between tumor and immune cells were confirmed by single cell RNA sequencing analysis. RESULTS In GBM tissues, LLT-1 was predominantly colocalized with Glial fibrillary acidic protein (GFAP) -expressing astrocytes, but not with microglial markers like Iba-1. Additionally, LLT-1 and activated NLRC4 inflammasomes were co-expressed mainly in intratumoral astrocytes, suggesting an association between LLT-1, NLRC4, and glioma malignancy. High LLT-1 expression correlates with poor prognosis, particularly in the mesenchymal subtype, and is associated with TNF and NOD-like receptor signaling pathway enrichment, indicating a potential role in tumor inflammation and progression. At the single-cell level, mesenchymal-like malignant cells were highly enriched in the TNF, NLR, and IL-1 signaling pathways compared to other malignant cell types. CONCLUSION We revealed an association between NLRC4 inflammasome activity and LLT-1 expression, suggesting a novel regulatory pathway involving TNF, inflammasomes, and IL-1, potentially offering new anti-glioma approaches by enhancing NK cell-based treatments.

Evaluation of the Efficacy of a Full-Spectrum Low-THC Cannabis Plant Extract Using In Vitro Models of Inflammation and Excitotoxicity.

Evidence has accumulated that Cannabis-derived compounds have the potential to treat neuroinflammatory changes present in neurodevelopmental conditions such as autism spectrum disorder. However, research is needed on the specific brain health benefits of strains of whole Cannabis extract that are ready for commercial production. Here, we explore the anti-inflammatory and neuroprotective effects of NTI-164, a genetically unique high-cannabidiol (CBD), low-Δ9-tetrahydrocannabinol extract, and also CBD alone on BV-2 microglia and SHSY-5Y neurons. Inflammation-induced up-regulation of microglial inflammatory markers was significantly attenuated by NTI-164, but not by CBD. NTI-164 promoted undifferentiated neuron proliferation and differentiated neuron survival under excitotoxic conditions. These effects suggest the potential for NTI-164 as a treatment for neuropathologies.

TMOD-28. MODELING MICROENVIRONMENTAL DYNAMICS: A NOVEL 3D GLIOMA-NEURON FUSION MODEL

Abstract Gliomas engage in bidirectional interactions with their microenvironment, wherein neuronal activity influences glioma proliferation and gliomas induce neuronal hyperexcitability. Although various models elucidate this complex interplay, a preference for a three-dimensional (3D) cell culture model has emerged to mimic intra-tumoral heterogeneity and microenvironmental interactions influencing glioma invasion, resistance, and recurrence. This study introduces an in vitro, self-assembled, scaffold-free, neuron-glioma spheroid fusion model to investigate the electrophysiological and functional interplay between glioma and its microenvironment. Primary mouse cortical neurons (MCN) were isolated from prenatal brain tissues and were cultured at 500,000 cells/spheroid on ultra-low attachment plates. Glioma organoids (WHO Grade 2-4) were developed from primary patient-derived tissue samples and were allowed to self-assemble for at least two weeks. After spontaneous spiking activity was detected in MCN spheroids, they were combined with glioma organoids in culture and allowed to fuse. A multielectrode array (MEA) was used to characterize the electrophysiological properties of the fusion model. Structural and functional characterizations were performed using immunofluorescence staining of proliferation, microglial, astrocytic, synaptic, and tumor markers. Additionally, the electrophysiological effects of the dual-IDH-mutant inhibitor, Vorasidenib, were assessed in WHO grade 2-4 IDH-mutant gliomas. Electrophysiological analysis of glioma-neuron co-cultures using MEA demonstrated a significantly increased network synchrony and firing rate in the fusion model when compared with the neuron-only condition, consistent with 2D models in the past. In correlation, the fusion model also demonstrated a significant increase in the Ki67 proliferation index across WHO grade 2-4 gliomas when compared to the glioma organoid-only condition. Vorasidenib treatment decreased neuronal firing rate and synchrony. Together, these results offer a high-throughput 3D model for recapitulating the tumor-brain microenvironment for both low and high-grade gliomas. Future studies will focus on the use of this model for drug screening and the exploration of malignant transformation in gliomas.

Exosomes activate hippocampal microglia in atrial fibrillation through long-distance heart–brain communication

BackgroundThere is growing evidence that atrial fibrillation (AF) is a risk factor for cognitive impairment (CI) and dementia in the presence or absence of stroke. The purpose of this study was to explore the mechanism of CI caused by AF.MethodsEighteen male canines were randomly divided into a sham group, a pacing group, and a pacing + GW4869 group. An experimental model of AF was established by rapid atrial pacing (450 beats/min) for 2 weeks, and the sham group received pacemaker implantation without atrial pacing. The GW4869 group received an intravenous GW4869 injection (0.3 mg/kg, once a day) during pacing. All canines were locally injected with Ad-CD63-RFP in epicardial adipose tissue (EAT) to trace the exosomes. Ultracentrifugation was employed to isolate EAT-derived exosomes, followed by RNA sequencing and quantitative real-time PCR (qRT-PCR) to assess RNA in both exosomes and hippocampal tissue. The miRanda database was used to predict the targeting relationships between miRNA and mRNA, which were further validated by luciferase reporter assays. Western blot analysis was conducted to detect exosomal markers (CD63, CD81, TSG101) in EAT exosomes, while immunofluorescence was used to detect Ad-CD63-RFP signals in both EAT and hippocampal tissues, as well as microglial activation marker IBA-1. To further explore the effects of exosomes on microglial cells, in vitro experiments using brain microvascular endothelial cells (bEnd3) and microglial cells (BV2) were conducted. IBA-1 expression and RNA levels in BV2 cells were analyzed by immunofluorescence and qRT-PCR, respectively.ResultsAfter 14 days of pacing of the canine atrium, compared to the sham group, both the pacing and GW4869 groups exhibited an increased number of AF inductions, along with prolonged AF duration. The fluorescence intensity of Ad-CD63-RFP and the microglial activation marker IBA-1 were markedly greater in the hippocampus. RNA sequencing showed that the differentially expressed gene cfa-miR-22e in EAT exosomes was upregulated, and its target gene IL33 was downregulated in the hippocampus. qRT-PCR showed that the levels of cfa-miR-22e were increased in both EAT exosomes and the hippocampus, while the expression of IL-33, a target of cfa-miR-22e, was decreased in the hippocampus. The administration of GW4869 abolished these effects. The in vitro results from bEnd3 and BV2 cell experiments were consistent with the conclusions drawn from the in vivo studies.ConclusionOur study indicated that the exosomes secreted by EAT in canines with AF can penetrate the BBB and activate microglia in the hippocampus through the cfa-miR-22e/IL33 signalling pathway.

A Defined Diet Combined with Sonicated Inoculum Provides a High Incidence, Moderate Severity Form of Experimental Autoimmune Encephalomyelitis (EAE).

Myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-peptide induced experimental autoimmune encephalomyelitis (EAE) is a model for inflammation of the brain and spinal cord. However, its severity and incidence vary within and between laboratories. Severe scores can lead to premature termination and are both unnecessary for readouts and detrimental to animal welfare. Ideally, the model would have high incidence, moderate severity, and low interindividual variability to fulfill the "Refine" aspect of the 3R concept. Nevertheless, most efforts to increase incidence also increase the severity. When the effects of potential therapies are tested, moderate severity is sufficient to detect useful drug effects as long as variation is low. Low variation can also reduce group sizes, which supports the "Reduce" aspect of 3R approaches in disease modeling. We set out to reduce variation and control severity by assessing the effects of mouse age, dietary fiber, antigen emulsion, and the dose of MOG and pertussis toxin on incidence, variability, and severity in the MOG-EAE model. We compared 14- and 33-week-old female C57BL/6 mice and varied the diet and inoculum in two studies. We measured disease signs in vivo as well as gene expression in the brain and spinal cord and histology by immunofluorescence. Ordinary one-way ANOVA was used for multiple comparisons. The most reliable induction conditions were with a low-fermentative/fiber diet (AIN 93M) combined with a sonicated emulsion of the MOG35-55-peptide. High-dose pertussis toxin increased EAE severity and incidence in 14-week-old mice (25% survival) while being more moderate in mature mice (100% survival). Varying all parameters suggests that duration of prefeeding defined diet, emulsion quality, and mouse maturity were factors that increase uniformity of response allowing incidence to reach 100% without excess severity. Microglia and astrocyte-associated markers were upregulated proportionally to score consistent with known EAE pathology. A defined fiber/high-sugar diet with sonicated inoculum provides for a moderate severity, high incidence, and less variable EAE. The resulting uniformity in animal response and associated cytokine patterns, and the strong link to a defined diet, suggest that this may be a more clinically translatable protocol for the induction of EAE. This is consistent with reported effects of low-fermentable diets on immune modulation in human patients with autoimmune diseases.