Degree Of Microglial Activation Research Articles

Quercetin Protects Against Global Cerebral ischemia‒reperfusion Injury by Inhibiting Microglial Activation and Polarization.

This study aims to investigate the protective effect of quercetin against global cerebral ischemia‒reperfusion (GCI/R) injury in rats and elucidate the underlying mechanism. A GCI/R injury rat model was established using a four-vessel occlusion (4-VO) method. An oxygen-glucose deprivation/reoxygenation (OGD/R) injury model was induced in BV2 cells. The extent of injury was assessed by evaluating neurological deficit scores (NDS) and brain water content and conducting behavioral tests. Pathomorphological changes in the prefrontal cortex were examined. Additionally, the study measured the levels of inflammatory cytokines, the degree of microglial activation and polarization, and the protein expression of Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor inducing interferon-β (TRIF). Quercetin pretreatment significantly ameliorated neurological impairment, improved learning and memory abilities, and reduced anxiety in rats subjected to GCI/R injury. Furthermore, quercetin administration effectively mitigated neuronal injury and brain edema. Notably, it suppressed microglial activation and hindered polarization toward the M1 phenotype. Simultaneously, quercetin downregulated the expression of TLR4 and TRIF proteins and attenuated the release of IL-1β and TNF-α. This study highlights the novel therapeutic potential of quercetin in alleviating GCI/R injury. Quercetin demonstrates its neuroprotective effects by inhibiting neuroinflammation and microglial activation while impeding their transformation into the M1 phenotype through modulation of the TLR4/TRIF pathway.

The role of FUT8‐catalyzed core fucosylation in Alzheimer’s amyloid‐β oligomer‐induced activation of human microglia

AbstractBackgroundA previous RNA‐seq study of human Alzheimer’s disease (AD) brain samples revealed significant upregulation of FUT8 coding for α1‐6 fucosyltransferase in parahippocampal gyrus, suggesting a role of FUT8 and the core fucosylation it catalyzes in early AD. Despite the well‐recognized roles of core fucosylation in immune regulation, its significance in microglial function, especially in neurodegenerative conditions such as AD, has rarely been studied.MethodHuman microglia derived from induced pluripotent stem cells (iMG) were used as the model system. To simulate microglia in the early stage of AD, we stimulated iMG derived from a subject of APOE3/3 genotype with amyloid‐β oligomer (AβO, 3 μM). The degree of microglia activation was determined by cytokine induction and MAPK phosphorylation. Lectins were used in flow cytometry, cytochemical staining, and lectin blotting to determine the changes in core fucosylation. Fucosylation was also examined by MS/MS‐based N‐glycan analysis. FUT8 expression in brain or cell samples was determined by quantitative PCR and Western blotting. FUT8 inhibition was induced by siRNA‐mediated gene silencing. The transcriptional regulation of FUT8 was investigated by ChIP‐PCR using p53 antibodies.ResultHuman iMG responded to AβO with a pattern of pro‐inflammatory activation within 24 hours. Using two lectins specifically recognizing α1,6 core fucose, AAL and LCA, in flow cytometry and fluorescence staining, we showed that such an activation was associated with increased core fucosylation. This observation will be further verified by N‐glycomic analysis. Inhibition of fucosylation by two inhibitors, 2FF and 6‐Alk‐Fuc, as well as FUT8 knockdown by specific siRNAs suppressed AβO‐induced iMG activation. AβO‐activated iMG also showed increased protein levels of FUT8 and p53. Because p53 was previously shown to be a direct transcriptional activator of FUT8 in tumor cells, we will test whether p53 is responsible for FUT8 upregulation by ChIP‐PCR. The in vivo relevance of our results is supported by qPCR results showing increased levels of FUT8 transcript in human temporal cortex, 5xFAD mouse cortex, and microglia isolated from 5xFAD mice.ConclusionOur data suggests that FUT8‐catalyzed α1‐6 core fucosylation is required for AβO‐induced microglia activation and may play a role in the pathogenesis of AD.

Synthetic torpor triggers a regulated mechanism in the rat brain, favoring the reversibility of Tau protein hyperphosphorylation.

Introduction: Hyperphosphorylated Tau protein (PPTau) is the hallmark of tauopathic neurodegeneration. During "synthetic torpor" (ST), a transient hypothermic state which can be induced in rats by the local pharmacological inhibition of the Raphe Pallidus, a reversible brain Tau hyperphosphorylation occurs. The aim of the present study was to elucidate the - as yet unknown - molecular mechanisms underlying this process, at both a cellular and systemic level. Methods: Different phosphorylated forms of Tau and the main cellular factors involved in Tau phospho-regulation were assessed by western blot in the parietal cortex and hippocampus of rats induced in ST, at either the hypothermic nadir or after the recovery of euthermia. Pro- and anti-apoptotic markers, as well as different systemic factors which are involved in natural torpor, were also assessed. Finally, the degree of microglia activation was determined through morphometry. Results: Overall, the results show that ST triggers a regulated biochemical process which can dam PPTau formation and favor its reversibility starting, unexpectedly for a non-hibernator, from the hypothermic nadir. In particular, at the nadir, the glycogen synthase kinase-β was largely inhibited in both regions, the melatonin plasma levels were significantly increased and the antiapoptotic factor Akt was significantly activated in the hippocampus early after, while a transient neuroinflammation was observed during the recovery period. Discussion: Together, the present data suggest that ST can trigger a previously undescribed latent and regulated physiological process, that is able to cope with brain PPTau formation.

Differential Regulation of Microglial Activation in Response to Different Degree of Ischemia.

Microglia are primary immune cells within the brain and are rapidly activated after cerebral ischemia. The degree of microglial activation is closely associated with the severity of ischemia. However, it remains largely unclear how microglial activation is differentially regulated in response to a different degree of ischemia. In this study, we used a bilateral common carotid artery ligation (BCAL) model and induced different degrees of ischemia by varying the duration of ligation to investigate the microglial response in CX3CR1GFP/+ mice. Confocal microscopy, immunofluorescence staining, RNA sequencing, and qRT-PCR were used to evaluate the de-ramification, proliferation, and differential gene expression associated with microglial activation. Our results showed that 30 min of ischemia induced rapid de-ramification of microglia but did not have significant influence on the microglial density. In contrast, 60 min of ischemia led to a significant decrease in microglial density and more pronounced de-ramification of microglial processes. Importantly, 30 min of ischemia did not induce proliferation of microglia, but 60 min of ischemia led to a marked increase in the density of proliferative microglia. Further analysis utilized transcriptome sequencing showed that microglial activation is differentially regulated in response to different degrees of ischemia. A total of 1,097 genes were differentially regulated after 60 min of ischemia, but only 68 genes were differentially regulated after 30 min of ischemia. Pathway enrichment analysis showed that apoptosis, cell mitosis, immune receptor activity and inflammatory-related pathways were highly regulated after 60 min of ischemia compared to 30 min of ischemia. Multiple microglia-related genes such as Cxcl10, Tlr7, Cd86, Tnfrsf1a, Nfkbia, Tgfb1, Ccl2 and Il-6, were upregulated with prolonged ischemia. Pharmacological inhibition of CSF1 receptor demonstrated that CSF1R signaling pathway contributed to microglial proliferation. Together, these results suggest that the proliferation of microglia is gated by the duration of ischemia and microglia were differentially activated in responding to different degrees of ischemia.

Compatibility of intravitreally applied epidermal growth factor and amphiregulin

IntroductionTo examine the compatibility of intravitreally injected epidermal growth factor (EGF) and amphiregulin as EGF family member.MethodsFour rabbits (age: 4 months; body weight: 2.5 kg) received three intravitreal injections of EGF (100 ng) uniocularly in monthly intervals and underwent ocular photography, tonometry, biometry, and optical coherence tomography. After sacrificing the rabbits, the globes were histomorphometrically examined. In a second study part, eyes of 22 guinea pigs (age: 2–3 weeks) received two intravitreal administrations of amphiregulin (10 ng) or phosphate buffered solution (PBS) in 10-day interval, or were left untouched. Ten days after the second injection, the guinea pigs were sacrificed, the enucleated eyes underwent histological and immune-histological examinations.ResultsThe rabbit eyes with EGF injections versus the contralateral untouched eyes did not show significant differences in intraocular pressure (7.5 ± 2.4 mmHg vs. 6.8 ± 2.2 mmHg; P = 0.66), retinal thickness (158 ± 5 µm vs. 158 ± 3 µm; P = 1.0), cell counts in the retinal ganglion cell layer (3.3 ± 1.7 cells/150 µm vs. 3.0 ± 1.4 cells/150 µm; P = 0.83), inner nuclear layer (46.4 ± 23.2 cells/150 µm vs. 39.6 ± 6.4 cells/150 µm; P = 0.61), and outer nuclear layer (215 ± 108 cells/150 µm vs. 202 ± 47 cells/150 µm; P = 0.83), or any apoptotic retinal cells. The guinea pig eyes injected with amphiregulin versus eyes with PBS injections did not differ (P = 0.72) in the degree of microglial activation, and both groups did not differ from untouched eyes in number of apoptotic retinal cells and retinal gliosis.ConclusionsIntravitreal applications of EGF (100 ng) in rabbits nor intravitreal applications of amphiregulin (10 ng) in guinea pigs led to intraocular specific inflammation or any observed intraocular destructive effect. The findings support the notion of a compatibility of intraocular applied EGF and amphiregulin.

Comparisons of neuroinflammation, microglial activation, and degeneration of the locus coeruleus-norepinephrine system in APP/PS1 and aging mice

BackgroundThe role of microglia in Alzheimer’s disease (AD) pathogenesis is becoming increasingly important, as activation of these cell types likely contributes to both pathological and protective processes associated with all phases of the disease. During early AD pathogenesis, one of the first areas of degeneration is the locus coeruleus (LC), which provides broad innervation of the central nervous system and facilitates norepinephrine (NE) transmission. Though the LC-NE is likely to influence microglial dynamics, it is unclear how these systems change with AD compared to otherwise healthy aging.MethodsIn this study, we evaluated the dynamic changes of neuroinflammation and neurodegeneration in the LC-NE system in the brain and spinal cord of APP/PS1 mice and aged WT mice using immunofluorescence and ELISA.ResultsOur results demonstrated increased expression of inflammatory cytokines and microglial activation observed in the cortex, hippocampus, and spinal cord of APP/PS1 compared to WT mice. LC-NE neuron and fiber loss as well as reduced norepinephrine transporter (NET) expression was more evident in APP/PS1 mice, although NE levels were similar between 12-month-old APP/PS1 and WT mice. Notably, the degree of microglial activation, LC-NE nerve fiber loss, and NET reduction in the brain and spinal cord were more severe in 12-month-old APP/PS1 compared to 12- and 24-month-old WT mice.ConclusionThese results suggest that elevated neuroinflammation and microglial activation in the brain and spinal cord of APP/PS1 mice correlate with significant degeneration of the LC-NE system.

Mild Mechanic Stimulate on Acupoints Regulation of CGRP-Positive Cells and Microglia Morphology in Spinal Cord of Sciatic Nerve Injured Rats.

ObjectiveThe objective of this study is to observe the effects of mild mechanical stimulation on acupuncture points of spinal motor neurons and active substances of sciatic nerve injury in rats, and to explore the morphological basis for the recovery of motor function in rats with sciatic nerve injury, using mild acupuncture. Acupuncture in the local area of injury may cause further damage to the peripheral nerve injury. We believe that mild mechanical stimulation on the surface, using some specific acupuncture points can also have a positive effect on nerve repair. This method, called Chinese tuina, has existed for more than 2,000 years in China.MethodsThis study establishes a rat model using sciatic nerve crush injury. Rats received Chinese tuina in accordance with the principle of the three methods and three points, once a day, for 20 days. The rats’ status of hindlimb recovery was detected by a sciatic functional index. The labeled neuronal cell body was used to evaluate the fiber recovery after the rats’ sciatic nerve injury, using a neural tracing technique. Our team studied motor neuronal cell bodies, CGRP-positive cells, and the microglia of damaged sciatic nerves which were stained with fluorescent triple staining, adopting a confocal multi-layer scanning technique, and then the changes in neuronal activity distribution and expression, and changes of time and treatment were described, using the method of morphological description.ResultsSciatic nerve injury decreased the survival rate of motor neurons, affected CGRP-positive cells, and activated microglia in the ventral horn of the spinal cord. Compared with the model group, the survival of spinal ventral horn motor neurons was increased through tuina intervention. The swelling of CGRP-positive cells was alleviated, and the degree of microglia activation was less than that of the model group.ConclusionThis study used visual morphological findings to assess changes in neurons and active substances with time after injury of the peripheral nerve, and demonstrated that peripheral mild acupuncture intervention improved the capacity of neurofibrillary axoplasmic transport, regulated microglia activation, and significantly promoted the recovery of sciatic nerve injury.

Assessing the Effects of Cytoprotectants on Selective Neuronal Loss, Sensorimotor Deficit and Microglial Activation after Temporary Middle Cerebral Occlusion.

Although early reperfusion after stroke salvages the still-viable ischemic tissue, peri-infarct selective neuronal loss (SNL) can cause sensorimotor deficits (SMD). We designed a longitudinal protocol to assess the effects of cytoprotectants on SMD, microglial activation (MA) and SNL, and specifically tested whether the KCa3.1-blocker TRAM-34 would prevent SNL. Spontaneously hypertensive rats underwent 15 min middle-cerebral artery occlusion and were randomized into control or treatment group, which received TRAM-34 intraperitoneally for 4 weeks starting 12 h after reperfusion. SMD was assessed longitudinally using the sticky-label test. MA was quantified at day 14 using in vivo [11C]-PK111195 positron emission tomography (PET), and again across the same regions-of-interest template by immunofluorescence together with SNL at day 28. SMD recovered significantly faster in the treated group (p = 0.004). On PET, MA was present in 5/6 rats in each group, with no significant between-group difference. On immunofluorescence, both SNL and MA were present in 5/6 control rats and 4/6 TRAM-34 rats, with a non-significantly lower degree of MA but a significantly (p = 0.009) lower degree of SNL in the treated group. These findings document the utility of our longitudinal protocol and suggest that TRAM-34 reduces SNL and hastens behavioural recovery without marked MA blocking at the assessed time-points.

Activation of microglia by retroviral infection correlates with transient clearance of prions from the brain but does not change incubation time.

Prion diseases are fatal transmissible diseases, where conversion of the endogenous prion protein (PrPC ) into a misfolded isoform (PrPSc ) leads to neurodegeneration. Microglia, the immune cells of the brain, are activated in neurodegenerative disorders including prion diseases; however, their impact on prion disease pathophysiology is unclear with both beneficial PrPSc -clearing and detrimental potentially neurotoxic effects. Moreover, monocytes entering the brain from the periphery during disease course might add to disease pathophysiology. Here, the degree of microglia activation in the brain of prion infected mice with and without an additional intraperitoneal retrovirus infection was studied. Peripheral murine retrovirus infection leads to activation of parenchymal microglia without recruitment of monocytes. This activation correlated with transient clearance or delay in accumulation of infectious prions specifically from the brain at early time points in the diseases course. Microglia expression profiling showed upregulation of genes involved in protein degradation coinciding with prion clearance. This enforces a concept where microglia act beneficial in prion disease if adequately activated. Once microglia activation has ceased, prion disease reemerges leading to disease kinetics undistinguishable from the situation in prion-only infected mice. This might be caused by the loss of microglial homeostatic function at clinical prion disease.

(251) Opioids and microglia: a murine-derived microglia stable cell line study

The development of opioid hyperalgesia following long-term opioid use is a critical impediment in treating chronic pain. Activated microglia has been identified as a critical mediator in the development of opioid hyperalgesia. The mechanism by which opioids activate microglia remains unknown. To explore this question, we used a stable cell line of mouse-derived microglial cells, which allowed us to isolate the direct effect of opioids on microglia in the absence of neurons. The BV-2 microglial cell line, derived from mouse microglial cells, was cultured in vitro. Cells were exposed to 10uM morphine for 48 hours and the degree of microglial activation was assayed with immunohistochemistry using an antibody against IBA-1 and western blot measuring BDNF expression. Standard qPCR was run to probe the expression of BDNF and opioid receptor mRNA in this cell line before and after exposure to opioids. Glial inhibitors (+) naloxone and minocycline were co-administered with morphine and the effect on microglial activation and BDNF expression was measured. BV-2 cells cultured with morphine for 48 hours caused significant increase in BDNF expression. This was blocked by co-treatment with minocycline or (+) naloxone, the opioid inactive enantiomer of naloxone. Kappa and mu opioid mRNA was detected in resting BV-2 cells, with mu opioid receptor expression increasing after morphine treatment. This study found that microglia express opioid receptors and suggest opioids are able to directly activate microglia in vitro. (+) naloxone blocked microglial activation, suggesting (+) naloxone inhibits microglial activation via a non-opioid target. Studies elucidating the molecular target of (+) naloxone in this cell line are ongoing. The development of opioid hyperalgesia following long-term opioid use is a critical impediment in treating chronic pain. Activated microglia has been identified as a critical mediator in the development of opioid hyperalgesia. The mechanism by which opioids activate microglia remains unknown. To explore this question, we used a stable cell line of mouse-derived microglial cells, which allowed us to isolate the direct effect of opioids on microglia in the absence of neurons. The BV-2 microglial cell line, derived from mouse microglial cells, was cultured in vitro. Cells were exposed to 10uM morphine for 48 hours and the degree of microglial activation was assayed with immunohistochemistry using an antibody against IBA-1 and western blot measuring BDNF expression. Standard qPCR was run to probe the expression of BDNF and opioid receptor mRNA in this cell line before and after exposure to opioids. Glial inhibitors (+) naloxone and minocycline were co-administered with morphine and the effect on microglial activation and BDNF expression was measured. BV-2 cells cultured with morphine for 48 hours caused significant increase in BDNF expression. This was blocked by co-treatment with minocycline or (+) naloxone, the opioid inactive enantiomer of naloxone. Kappa and mu opioid mRNA was detected in resting BV-2 cells, with mu opioid receptor expression increasing after morphine treatment. This study found that microglia express opioid receptors and suggest opioids are able to directly activate microglia in vitro. (+) naloxone blocked microglial activation, suggesting (+) naloxone inhibits microglial activation via a non-opioid target. Studies elucidating the molecular target of (+) naloxone in this cell line are ongoing.

Can MS lesion stages be distinguished with MRI? A postmortem MRI and histopathology study

In multiple sclerosis (MS), a histopathological distinction is made between different stages of white matter (WM) lesions. These lesions are characterized as preactive, active, chronic active or chronic inactive, depending on the degree of microglia activation and degree of demyelination. The different lesions are not distinguishable on conventional magnetic resonance imaging (MRI) scans at standard clinical field strengths, but might be distinguished using more advanced, quantitative MRI methods, such as T1 relaxation time (T1-RT) mapping. To investigate this, postmortem brain material from 20 MS patients was investigated, using both T1-RT MRI at 1.5T and histopathology. The brain material contained a total of 9 preactive, 18 active, 30 chronic active and 14 chronic inactive lesions, as well as 38 areas of normal appearing WM (NAWM). Our results show that, at 1.5T, T1-RT qMRI can only distinguish between categories NAWM/preactive, active and chronic WM lesions. Advanced imaging at standard field strengths, such as conventional imaging measures, is therefore insufficient to differentiate the WM lesions in MS, and higher field strengths may be required to achieve better pathological differentiation of these lesions.

A death receptor 6-amyloid precursor protein pathway regulates synapse density in the mature CNS but does not contribute to Alzheimer's disease-related pathophysiology in murine models.

Recent studies implicate death receptor 6 (DR6) in an amyloid precursor protein (APP)-dependent pathway regulating developmental axon pruning, and in a pruning pathway operating during plastic rearrangements in adult brain. DR6 has also been suggested to mediate toxicity in vitro of Aβ peptides derived from APP. Given the link between APP, Aβ, and Alzheimer's disease (AD), these findings have raised the possibility that DR6 contributes to aspects of neurodegeneration in AD. To test this possibility, we have used mouse models to characterize potential function(s) of DR6 in the adult CNS and in AD-related pathophysiology. We show that DR6 is broadly expressed within the adult CNS and regulates the density of excitatory synaptic connections onto pyramidal neurons in a genetic pathway with APP. DR6 knock-out also gives rise to behavioral abnormalities, some of which are similar to those previously documented in APP knock-out animals. However, in two distinct APP transgenic models of AD, we did not observe any alteration in the formation of amyloid plaques, gliosis, synaptic loss, or cognitive behavioral deficits with genetic deletion of DR6, though we did observe a transient reduction in the degree of microglial activation in one model. Our results support the view that DR6 functions with APP to modulate synaptic density in the adult CNS, but do not provide evidence for a role of DR6 in the pathophysiology of AD.

A novel technique for morphometric quantification of subarachnoid hemorrhage-induced microglia activation.

Subarachnoid hemorrhage (SAH) is a neurologic catastrophe and poor outcome is typically attributed to vasospasm; however, there is also evidence that SAH causes a pro-inflammatory state and these two phenomena may be interrelated. SAH causes activation of microglia, but the time course and degree of microglial activation after SAH and its link to poor patient outcome and vasospasm remains unknown. Transgenic mice expressing eGFP under the control of the CX3CR1 locus, in which microglia are endogenously fluorescent, were randomly assigned to control or SAH groups. Immunohistochemistry for CD-68 and CD-31 was performed at different time points after SAH. Using confocal microscopy and MatLab software, we have developed a novel technique to detect and quantify the stages of microglial activation and return to quiescence using an automated computerized morphometric analysis. We detected a statistically significant decrease in microglial process complexity 2 and 7 days following SAH. In addition, we detected a statistically significant increase in microglial domain volume 1 day following SAH; however, microglial domain volume returned to baseline by 2 days. Most techniques for microglia assessment are qualitative, not quantitative, and are therefore inadequate to address the effects of anti-inflammatory drug treatment or other therapies after SAH. Using novel image analysis techniques we were able to reproducibly quantify activation of microglia following SAH, which will improve our ability to study the biology of microglial activation, and may ultimately improve management of disease progression and response to therapies directed at microglial activation.

Full and partial peroxisome proliferation-activated receptor-gamma agonists, but not delta agonist, rescue of dopaminergic neurons in the 6-OHDA Parkinsonian model is associated with inhibition of microglial activation and MMP expression

BackgroundPeroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor that has been shown to have anti-inflammatory and matrix metalloproteinase (MMP) inhibitor properties. PPARγ agonists have been shown to have neuroprotective effects in various neurodegeneration models where inflammation is implicated, including models of Parkinson's disease. However, no studies have looked at the effects of partial PPARγ agonists. Experimental approachThe neuroprotective effects of the PPARγ full agonist, pioglitazone (20mg/kg), partial PPARγ agonist GW855266X (15mg/kg) and PPAR-δ full agonist GW610742X (10mg/kg) were investigated in the 6-hydroxydopamine (6-OHDA) model of Parkinson's disease when administered prior to or post 6-OHDA lesioning. The integrity of the nigrostriatal system was assessed by assessing the numbers dopaminergic neurons in the substantia nigra (SN) and by assessing striatal dopamine content. The degree of microglia activation in the SN was also immunohistochemistry assessed utilizing the marker OX-6 for activated microglia and CD-68 a marker for phagocytic microglia. Additionally we performed immunocytochemistry for MMP3 in the SN. Finally, we investigated whether a period of drug withdrawal for a further 7days affected the neuroprotection produced by the PPARγ agonists. Key resultsBoth Pioglitazone and GW855266X protected against 6-OHDA induced loss of dopaminergic neurons in the substantia nigra and depletion of striatal dopamine when administered orally twice daily for either 1) 7day prior to and 7days post lesioning or 2) for 7days starting 2days post lesioning when neurons will be severely traumatized. 6-OHDA lesioning was associated with an increase in microglia activation and in numbers of MMP-3 immunoreactive cells which was attenuated by pioglitazone and GW855266X. Neuroprotective effects were not replicated using the PPARδ agonist GW610742X. Subsequent withdrawal of both Pioglitazone and GW855266X, for a further 7days negated any neuroprotective effect suggesting that long-term administration may be required to attenuate the inflammatory response. Conclusions and implicationsFor the first time a partial PPAR-γ agonist has been shown to be neuroprotectory when administered post lesioning in a Parkinsonian model. Effects may be via the inhibition of microglial and MMP activation and support further research.

Meningeal inflammation is widespread and linked to cortical pathology in multiple sclerosis

Meningeal inflammation in the form of ectopic lymphoid-like structures has been suggested to play a prominent role in the development of cerebral cortical grey matter pathology in multiple sclerosis. The aim of this study was to analyse the incidence and distribution of B cell follicle-like structures in an extensive collection of cases with secondary progressive multiple sclerosis with a wide age range and to determine their relationship to diffuse meningeal inflammation, white matter perivascular infiltrates and microglial activation. One hundred and twenty three cases with secondary progressive multiple sclerosis were examined for the presence of meningeal and perivascular immune cell infiltrates in tissue blocks and/or whole coronal macrosections encompassing a wide array of brain areas. Large, dense, B cell-rich lymphocytic aggregates were screened for the presence of follicular dendritic cells, proliferating B cells and plasma cells. Ectopic B cell follicle-like structures were found, with variable frequency, in 49 cases (40%) and were distributed throughout the forebrain, where they were most frequently located in the deep sulci of the temporal, cingulate, insula and frontal cortex. Subpial grey matter demyelinated lesions were located both adjacent to, and some distance from such structures. The presence of B cell follicle-like structures was associated with an accompanying quantitative increase in diffuse meningeal inflammation that correlated with the degree of microglial activation and grey matter cortical demyelination. The median age of disease onset, time to disease progression, time to wheelchair dependence and age at death all differed significantly in these cases when compared with those without B cell follicle-like structures. Our findings suggest that meningeal infiltrates may play a contributory role in the underlying subpial grey matter pathology and accelerated clinical course, which is exacerbated in a significant proportion of cases by the presence of B cell follicle-like structures.

Emodin-induced microglial apoptosis is associated with TRB3 induction

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a natural anthraquinone compound isolated from the rhizome of rhubarb, has been reported to treat brain injury after intracerebral hemorrhage. Treatment of neurons with emodin is able to decrease glutamate excitotoxicity, modulate calcium homeostasis, and induce Bcl-2 expression. However, the effects of emodin on the brain-resident innate immune cells are unclear. In the present study, the mouse microglial cell line, BV-2, was selected to investigate the effects of emodin on microglial activation and apoptosis. Cell viability and apoptosis were sequentially measured with the CellTiter-Glo Luminescent Cell Viability Assay, YOPRO-1 and Caspase-Glo 3/7 Assay Systems. The degree of microglial activation was evaluated using quantitative RT-PCR to measure expression of inflammatory markers. Treatment of BV-2 cells with emodin caused caspase-mediated apoptosis in a dose-dependent manner, and emodin augmented LPS-induced microglial apoptosis to repress inflammatory activation. In response to emodin treatment, reactive oxygen species (ROS) production was increased, and TRB3 was markedly activated. siRNA knockdown of TRB3 attenuated emodin-induced microglial apoptosis. Ectopic overexpression of TRB3 decreased cell viability and was associated with dysregulation of the prosurvival Akt/FOXO3 pathway. These results demonstrate that emodin induces BV-2 cell apoptosis through TRB3 and consequently eliminates inflammatory microglia. Our findings provide a novel molecular basis through which emodin exerts neuroprotective effects, treating brain injury after intracerebral hemorrhage.

Inhibition of the Striatal Specific Phosphodiesterase PDE10A Ameliorates Striatal and Cortical Pathology in R6/2 Mouse Model of Huntington's Disease

BackgroundHuntington's disease is a devastating neurodegenerative condition for which there is no therapy to slow disease progression. The particular vulnerability of striatal medium spiny neurons to Huntington's pathology is hypothesized to result from transcriptional dysregulation within the cAMP and CREB signaling cascades in these neurons. To test this hypothesis, and a potential therapeutic approach, we investigated whether inhibition of the striatal-specific cyclic nucleotide phosphodiesterase PDE10A would alleviate neurological deficits and brain pathology in a highly utilized model system, the R6/2 mouse.Methodology/Principal FindingsR6/2 mice were treated with the highly selective PDE10A inhibitor TP-10 from 4 weeks of age until euthanasia. TP-10 treatment significantly reduced and delayed the development of the hind paw clasping response during tail suspension, deficits in rotarod performance, and decrease in locomotor activity in an open field. Treatment prolonged time to loss of righting reflex. These effects of PDE10A inhibition on neurological function were reflected in a significant amelioration in brain pathology, including reduction in striatal and cortical cell loss, the formation of striatal neuronal intranuclear inclusions, and the degree of microglial activation that occurs in response to the mutant huntingtin-induced brain damage. Striatal and cortical levels of phosphorylated CREB and BDNF were significantly elevated.Conclusions/SignificanceOur findings provide experimental support for targeting the cAMP and CREB signaling pathways and more broadly transcriptional dysregulation as a therapeutic approach to Huntington's disease. It is noteworthy that PDE10A inhibition in the R6/2 mice reduces striatal pathology, consistent with the localization of the enzyme in medium spiny neurons, and also cortical pathology and the formation of neuronal nuclear inclusions. These latter findings suggest that striatal pathology may be a primary driver of these secondary pathological events. More significantly, our studies point directly to an accessible new therapeutic approach to slow Huntington's disease progression, namely, PDE10A inhibition. There is considerable activity throughout the pharmaceutical industry to develop PDE10A inhibitors for the treatment of basal ganglia disorders. The present results strongly support the investigation of PDE10A inhibitors as a much needed new treatment approach to Huntington's disease.

New parameters for analysis of changes microglial morphology using stereology and histomorphometry

New parameters for analysis of changes microglial morphology using stereology and histomorphometryIn histological evaluation of experimental and pathological specimens, the degree of microglial activation is often evaluated on the basis of morphological features of the cells including the size and shape of the cells, the number and length of cell processes, as well as by increased expression of markers such as CD11b, CD45 (LCA) and Iba‐1. From stereological studies it is known that microglial activation is followed by proliferation of microglial cells and change in the morphology of the cells.To extend the list of morphological parameters that can be measured quantitatively for comparative studies, we combined digital image analysis and stereological methods for analysis of the morphology of microglia labeled by Iba‐1. In the molecular layer of fascia dentata of the hippocampus from 6 male C57Bl6‐mice the total number of microglial cells was 16 906 cells, the mean cell size was 151.3 µm3, and the average length of cell processes was 80.0 µm/cell. We found microglial cell bodies were ovoid in shape, as judged by the ratio of the shortest and longest diameter (ratio = 0.61) and that on average 5.65 cell processes were extending at the equatorial plane of the cell.

Triptolide protects against 1-methyl-4-phenyl pyridinium-induced dopaminergic neurotoxicity in rats: Implication for immunosuppressive therapy in Parkinson’s disease

Neuroinflammation with microglial activation has been implicated to have a strong association with the progressive dopaminergic neuronal loss in Parkinson's disease (PD). The present study was undertaken to evaluate the activation profile of microglia in 1-methyl-4-phenyl pyridinium (MPP+)-induced hemiparkinsonian rats. Triptolide, a potent immunosuppressant and microglia inhibitor, was then examined for its efficacy in protecting dopaminergic neurons from injury and ameliorating behavioral disabilities induced by MPP+. The rat model of PD was established by intranigral microinjection of MPP+. At baseline and on day 1, 3, 7, 14, 21 following MPP+ injection, the degree of microglial activation was examined by detecting the immunodensity of OX-42 (microglia marker) in the substantia nigra (SN). The number of viable dopaminergic neurons was determined by measuring tyrosine hydroxylase (TH) positive neurons in the SN. Behavioral performances were evaluated by counting the number of rotations induced by apomorphine, calculating scores of forelimb akinesia and vibrissae-elicited forelimb placing asymmetry. Intranigral injection of MPP+ resulted in robust activation of microglia, progressive depletion of dopaminergic neurons, and ongoing aggravation of behavioral disabilities in rats. Triptolide significantly inhibited microglial activation, partially prevented dopaminergic cells from death and improved behavioral performances. These data demonstrated for the first time a neuroprotective effect of triptolide on dopaminergic neurons in MPP+-induced hemiparkinsonian rats. The protective effect of triptolide may, at least partially, be related to the inhibition of MPP+-induced microglial activation. Our results lend strong support to the use of immunosuppressive agents in the management of PD.

Time- and activation-dependency of the protective effect of microglia on astrocytes exposed to peroxide-induced oxidative stress

Microglial as well as astroglial activation (gliosis) are common phenomena of neuropathological processes in the brain. Nevertheless, the role of glial cell communication during gliosis is still not completely understood. The data we present with this work are a part of a bigger study on microglial-astroglial cell communication. The purpose of this study was to examine the impact of microglial-astroglial cell interactions on the cytotoxic effect of hydrogen peroxide (H2O2) on astrocytes and to characterize the microglia-induced astroprotective effect in detail. Therefore, purified cell cultures of microglia and astroglia from neonatal rat brains were used. Astrocytes were pre-treated with conditioned medium from lipopolysaccharides (LPS)-activated microglia and then exposed to oxidative stress induced by H2O2 under varying conditions. Activated microglia significantly reduced the cytotoxic effect of H2O2. As well in the indirect co-culture model using conditioned medium of microglia as in a direct co-culture model with different portions of microglial cells on the astroglial monolayer the astroprotective effect clearly correlated with the degree of microglial activation. The protective effect on astrocytes was induced in less than 24 h and lasted more than 4 days. Taken together, our results indicate that microglial activation might play an important role in the modulation of the oxidative stress resistance of glial tissue during gliosis.