Elimination Of Microglia Research Articles

Loss of C1q alters the auditory brainstem response.

Neural circuits in the auditory brainstem compute interaural time and intensity differences used to determine the locations of sound sources. These circuits display features that are specialized for these functions. The projection from the ventral cochlear nucleus (VCN) to the medial nucleus of the trapezoid (MNTB) body travels along highly myelinated fibers and terminates in the calyx of Held. This monoinnervating synapse emerges during development as multiple inputs are eliminated. We previously demonstrated that elimination of microglia with a colony stimulating factor-1 inhibitor results in impaired synaptic pruning so that multiple calyceal terminals reside on principal cells of MNTB. This inhibitor also resulted in impaired auditory brainstem responses (ABRs), with elevated thresholds and increased peak latencies. Loss of the microglial fractalkine receptor, CX3CR1, decreased peak latencies in the ABR. The mechanisms underlying these effects are not known. One prominent microglial signaling pathway involved in synaptic pruning and plasticity during development and aging is the C1q-initiated compliment cascade. Here we investigated the classical complement pathway initiator, C1q, in auditory brainstem maturation. We found that C1q expression is detected in the MNTB by the first postnatal week. C1q levels increased with age and were detected within microglia and surrounding the soma of MNTB principal neurons. Loss of C1q did not affect microglia-dependent calyceal pruning. Excitatory and inhibitory synaptic markers in the MNTB and LSO were not altered with C1q deletion. ABRs showed that C1q KO mice had normal hearing thresholds but shortened peak latencies. Altogether this study uncovers the developmental time frame of C1q expression in the sound localization pathway and shows a subtle functional consequence of C1q knockdown.

Transient CSF1R Inhibition in the Early Stage of AD Retains Homeostatic Microglia with Reduced Inflammasomes and Increased autophagy in the 5XFAD Model

AbstractBackgroundChronic neuroinflammation is one of the most conspicuous features of Alzheimer’s disease (AD). While microglia play a vital role in phagocytosing amyloid beta (Aβ) and restraining plaque extension, disease‐associated microglia are known to perpetuate neuroinflammation. Previous studies have shown that ∼80% microglia elimination through a month‐long colony‐stimulating factor 1 receptor (CSF1R) inhibition in the advanced stage of AD in 5X Familial AD (5XFAD) mice does not reduce Aβ levels or plaques but restrains synapse loss and neurodegeneration. On the other hand, prolonged CSF1R inhibition has been shown to prevent parenchymal plaque development. However, the immediate effects of transient CSF1R inhibition in the early stage of AD on residual microglial morphology and their metabolic fitness are unknown.MethodWe investigated the impact of transient CSF1R inhibition in three‐month‐old 5XFAD mice, a stage at which neuroinflammation has commenced, but Aβ plaques were minimal. Microglia from the hippocampus and the somatosensory cortex (SSC) of 5XFAD mice receiving a diet containing either the CSF1R inhibitor PLX5622 or a standard diet for ten days were examined for morphology, markers of NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3) inflammasome complex, autophagy, and mechanistic target of rapamycin (mTOR) signaling.ResultTen days of CSF1R inhibition resulted in 67‐70% depletion of microglia in the hippocampus and SSC. The residual microglia displayed a noninflammatory phenotype, typified by highly branched and ramified processes, reduced incidence of NLRP3 inflammasome complexes, and increased autophagy. Interestingly, Aβ plaque load was reduced, and mTOR signaling was unaltered in microglia but reduced in neurons. However, transient CSF1R inhibition did not impact astrocyte hypertrophy or neurogenesis in the hippocampus.ConclusionThe results showed that transient CSF1R inhibition in the early stage of AD promotes the retention of homeostatic microglia with reduced inflammasomes and better metabolic fitness but with no changes in mTOR signaling, which can restrain the perpetuation of neuroinflammation and improve phagocytosis of Aβ by microglia.

Group 2 innate lymphoid cells suppress neuroinflammation and brain injury following intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) mobilizes circulating leukocytes that contribute to neuroinflammation and neural injury. However, little is known about the endogenous regulatory immune mechanisms to restrict neuroinflammation following ICH. We examined the role of group 2 innate lymphoid cells (ILC2) that are a specialized subset of innate immune modulators in a mouse model of ICH. We found accumulation of ILC2 in the brain following acute ICH and a concomitant increase of ILC2 within the peripheral lymph nodes. Depletion of ILC2 exacerbated neurodeficits and brain edema after ICH in male and female mice. This aggravated ICH injury was accompanied by augmented microglia activity and leukocyte infiltration. In contrast, expansion of ILC2 using IL-33 led to reduced ICH injury, microglia activity and leukocyte infiltration. Notably, elimination of microglia using a colony stimulating factor 1 receptor inhibitor diminished the exacerbation of ICH injury induced by depletion of ILC2. Brain-infiltrating ILC2 had upregulation of IL-13 after ICH. Results from in vitro assays revealed that ILC2 suppressed thrombin-induced inflammatory activity in microglia-like BV2 cells. Thus, our findings demonstrate that ILC2 suppress neuroinflammation and acute ICH injury.

Microglia activation mediates circadian rhythm disruption-induced cognitive impairment in mice

Alzheimer's disease (AD) is the leading cause of dementia and there are no effective treatments for this disease currently. Circadian rhythm disruption (CRD) is a hallmark of modern society that appears to be on the rise. It is well reported that AD is associated with disrupted circadian functioning and CRD can impair cognitive function. However, the cellular mechanisms underlying CRD-associated cognitive decline remain elusive. In this study, we investigated whether microglia are involved in CRD-induced cognitive decline. We established experimental ‘jet lag’ (phase delay of the light/dark cycles)-induced CRD mouse model and observed significant impairment of spatial learning and memory function in these mice. In the brain, CRD resulted in neuroinflammation, which was characterized by microglia activation and increased pro-inflammatory cytokine production, impairments in neurogenesis and reduction of synaptic proteins in the hippocampus. Interestingly, elimination of microglia with the colony stimulating factor-1 receptor inhibitor PLX3397 prevented CRD-induced neuroinflammation, cognitive decline, impairment of neurogenesis and loss of synaptic proteins. These findings collectively suggest that microglia activation plays a key role in CRD-induced cognitive deficit most likely through neuroinflammation-mediated impairments in adult neurogenesis and synapses.

Interleukin-17A modulates retinal inflammation by regulating microglial activation via the p38 MAPK pathway in experimental glaucoma neuropathy.

As a prototypical member of the IL-17 family, interleukin-17A (IL-17A) has received increasing attentions for its potent proinflammatory role as well as potential to be a key therapeutic target in human autoimmune inflammatory diseases; however, its roles in other pathological scenarios like neuroinflammations are not fully elucidated yet but appear essentially correlating and promising. Glaucoma is the leading cause of irreversible blindness with complicated pathogenesis still to be understood, where neuroinflammation was reported to be critically involved in its both initiation and progression. Whether IL-17A takes part in the pathogenesis of glaucoma through interfering neuroinflammation due to its potent proinflammatory effect is still unknown. In the present study, we investigated the role of IL-17A in the pathological process of glaucoma neuropathy as well as its relationship with the predominant immune inflammation mediator microglia in retina, trying to elucidate the underlying mechanisms from the view of inflammation modulation. In our study, RNA sequencing was performed for the retinas of chronic ocular hypertension (COH) and control mice. Western blot, RT-PCR, immunofluorescence, and ELISA were used to evaluate the microglial activation and proinflammatory cytokines release at conditioned levels of IL-17A, along with assessment of optic nerve integrity including retinal ganglion cells (RGCs) counting, axonal neurofilament quantification, and flash visual-evoked potential (F-VEP) examination. And the possibly involved signaling pathways were screened out to go through further validation in scenarios with conditioned IL-17A. Subsequently, IL-17A was found to be significantly upregulated in COH retina. Furthermore, suppression of IL-17A effectively diminished the loss of RGCs, improved axonal quality, and F-VEP performance in COH mice. Mechanistically, IL-17A promoted microglial activation and proinflammatory cytokines release along with enhanced phenotypic conversion of activated microglia to M2-type in early stage and to M1-type in late stage in glaucomatous retinas. Microglia elimination decreased the proinflammatory factors secretion, enhanced the RGCs survival and axonal quality mediated by IL-17A. Furthermore, IL-17A-induced the overactivation of microglia in glaucomatous condition was alleviated after blocking the p38 MAPK pathway. Taken together, IL-17A is involved in the regulation of retinal immune response and RGCs cell death in experimental glaucoma by essentially promoting retinal microglial activation via p38 MAPK signaling pathway. IL-17A dynamically regulates the phenotypic conversion of retinal microglia in experimental glaucoma partly depending on the duration of elevated intraocular pressure. Suppression of IL-17A contributes to alleviate glaucoma neuropathy and exhibits promising potential as an innovative target for therapeutic strategy in glaucoma.

Neuronal-microglial liver X receptor β activating decrease neuroinflammation and chronic stress-induced depression-related behavior in mice

Depression is accompanied by excessive neuroinflammation. Liver X receptor β (LXRβ) has been reported as a newly emerging target that exerts systemic and organic inflammation modulation. However, the modulatory mechanism in alleviating neuroinflammation are far from being revealed. In the current study, depression-related behaviors in mice were induced by chronic unpredictable mild stress (CUMS) and corticosterone (CORT) drinking. Mice received either TO901317, PLX-5622 and intra- bilateral basolateral amygdale (BLA) injection of rAAV9-hSyn-hM3D(Gq)-eGFP to activate LXRβ, eliminate microglia and pharmacogenetic activate neurons in BLA, respectively, followed by behavioral tests. Microglial pro-inflammatory and pro-phagocytic activation, as well as nuclear factor-κB (NF-κB) signaling pathway, NLRP3 inflammasome activation and interleukin-1β (IL-1β) release in BLA were investigated. Moreover, pro-inflammatory activation of BV2 cells-induced by CORT with or without TO901317 was detected. Neuroinflammation indicated by IL-1β release was measured in a co-culture system of HT22-primary microglia with or without TO901317. Our results indicated that chronic stress induced depression-related behaviors, which were accompanied with microglial pro-inflammatory and pro-phagocytic activation, as well as NF-κB signaling pathway and NLRP3 inflammasome activation in BLA. Accordingly, pharmacological activation of LXRβ inhibited microglial pro-inflammatory and pro-phagocytic activation, as well as NF-κB signaling pathway and NLRP3 inflammasome activation, and IL-1β release both in vivo and in vitro. Finally, both elimination of microglia and pharmacogenetic activation of neurons in BLA protected mice from chronic stress-induced depression-related behavior. Collectively, pharmacological activation of neuronal-microglial LXRβ alleviates depression-related behavior by modulating excessive neuroinflammation via inhibiting NF-κB signaling pathway and NLRP3 inflammasome activation.

Significant Sex Differences in the Efficacy of the CSF1R Inhibitor-PLX5622 on Rat Brain Microglia Elimination.

Microglia play pivotal roles in central nervous system development, homeostasis, responses to trauma, and neurodegenerative and neuropsychiatric disorders with significant sex-bias in their symptoms and prevalence. Survival of the microglia in adult brains depends on the expression of the colony-stimulating factor 1 receptor (CSF1R). The inhibition of CSF1R by brain-permeant PLX5622 in the chow eliminates, within 5–10 days, ~90% of the microglia in female and male mice, thereby enabling the investigation of the roles of the microglia in health and pathological mice models. Because of a prevailing “impression” that PLX5622 is ineffective in rats, it has hardly been used in studies of adult rats. Here, we report that effective microglia elimination by PLX5622-chow in rats is highly sex-dependent. Our observations provide missing information for the limited use and interpretation of PLX5622 in biomedical studies of the microglia in rat models. The sex differences that are too often overlooked must be carefully considered and clearly emphasized.

Effects of CSF1R inhibitor PLX3397 on reinforcement learning ability

PLX3397 is an orally administrable CSF1 receptor inhibitor that is expected to be a therapeutic agent for cell tumors such as tendon synovial giant cell tumors. Administration of high doses of PLX3397 is known to eliminate microglia selectively and may affect neuronal functions via the loss of microglia. The elimination of microglia has been known to affect exploratory tasks involving aversive stimuli. However, the effect of the microglial elimination on the ability of reward-based reinforcement learning has been unknown. In this study, C57BL6 mice were administrated with PLX3397 for three weeks and were tested with a 5-armed bandit task (5-ABT). 5-ABT is an exploratory operant conditioning task in which each of the five choices has a different reward probability. The task contains three subtasks: one in which all choices had a reward probability of 30% (ALL), a subtask in which only one choice had a reward probability of 50% and the rest had a reward probability of 0% (BIT), and a subtask in which the correct choice in BIT was reversed and four options had a reward probability of 30% (REV). As a result, there was no significant difference in the entropy of choice (exploration pattern) in the subtask ALL between the PLX3397-treated group and the control group. There was no significant difference between the groups in the number of steps required for the BIT and REV subtasks to exceed 50% correct. We also estimated the learning rates of the mice by fitting the behavioral sequences to a Q-learning model and found no significant differences between the groups. These results suggest that microglial elimination has no significant impact on reinforcement learning ability.

Myosin 1f-mediated activation of microglia contributes to the photoreceptor degeneration in a mouse model of retinal detachment

Photoreceptor death and neurodegeneration is the leading cause of irreversible vision loss. The inflammatory response of microglia plays an important role in the process of neurodegeneration. In this study, we chose retinal detachment as the model of photoreceptor degeneration. We found Myosin 1f was upregulated after retinal detachment, and it was specifically expressed in microglia. Deficiency of myosin 1f protected against photoreceptor apoptosis by inhibiting microglia activation. The elimination of microglia can abolish the protective effect of myosin 1f deficiency. After stimulation by LPS, microglia with myosin 1f deficiency showed downregulation of the MAPK and AKT pathways. Our results demonstrated that myosin 1f plays a crucial role in microglia-induced neuroinflammation after retinal injury and photoreceptor degeneration by regulating two classic inflammatory pathways and thereby decreasing the expression of inflammatory cytokines. Knockout of myosin 1f reduces the intensity of the immune response and prevents cell death of photoreceptor, suggesting that myosin 1f can be inhibited to prevent a decline in visual acuity after retinal detachment.

Microglia Depletion-Induced Remodeling of Extracellular Matrix and Excitatory Synapses in the Hippocampus of Adult Mice

The extracellular matrix (ECM) plays a key role in synaptogenesis and the regulation of synaptic functions in the central nervous system. Recent studies revealed that in addition to dopaminergic and serotoninergic neuromodulatory systems, microglia also contribute to the regulation of ECM remodeling. In the present work, we investigated the physiological role of microglia in the remodeling of perineuronal nets (PNNs), predominantly associated with parvalbumin-immunopositive (PV+) interneurons, and the perisynaptic ECM around pyramidal neurons in the hippocampus. Adult mice were treated with PLX3397 (pexidartinib), as the inhibitor of colony-stimulating factor 1 receptor (CSF1-R), to deplete microglia. Then, confocal analysis of the ECM and synapses was performed. Although the elimination of microglia did not alter the overall number or intensity of PNNs in the CA1 region of the hippocampus, it decreased the size of PNN holes and elevated the expression of the surrounding ECM. In the neuropil area in the CA1 str. radiatum, the depletion of microglia increased the expression of perisynaptic ECM proteoglycan brevican, which was accompanied by the elevated expression of presynaptic marker vGluT1 and the increased density of dendritic spines. Thus, microglia regulate the homeostasis of pre- and postsynaptic excitatory terminals and the surrounding perisynaptic ECM as well as the fine structure of PNNs enveloping perisomatic—predominantly GABAergic—synapses.

CX3CR1 mutation alters synaptic and astrocytic protein expression, topographic gradients, and response latencies in the auditory brainstem.

The precise and specialized circuitry in the auditory brainstem develops through adaptations of cellular and molecular signaling. We previously showed that elimination of microglia during development impairs synaptic pruning that leads to maturation of the calyx of Held, a large encapsulating synapse that terminates on neurons of the medial nucleus of the trapezoid body (MNTB). Microglia depletion also led to a decrease in glial fibrillary acidic protein (GFAP), a marker for mature astrocytes. Here, we investigated the role of signaling through the fractalkine receptor (CX3CR1), which is expressed by microglia and mediates communication with neurons. CX3CR1-/- and wild-type mice were studied before and after hearing onset and at 9 weeks of age. Levels of GFAP were significantly increased in the MNTB in mutants at 9 weeks. Pruning was unaffected at the calyx of Held, but we found an increase in expression of glycinergic synaptic marker in mutant mice at P14, suggesting an effect on maturation of inhibitory inputs. We observed disrupted tonotopic gradients of neuron and calyx size in MNTB in mutant mice. Auditory brainstem recording (ABR) revealed that CX3CR1-/- mice had normal thresholds and amplitudes but decreased latencies and interpeak latencies, particularly for the highest frequencies. These results demonstrate that disruption of fractalkine signaling has a significant effect on auditory brainstem development. Our findings highlight the importance of neuron-microglia-astrocyte communication in pruning of inhibitory synapses and establishment of tonotopic gradients early in postnatal development.

Specific depletion of resident microglia in the early stage of stroke reduces cerebral ischemic damage

BackgroundIschemia can induce rapid activation of microglia in the brain. As key immunocompetent cells, reactive microglia play an important role in pathological development of ischemic stroke. However, the role of activated microglia during the development of ischemia remains controversial. Thus, we aimed to investigate the function of reactive microglia in the early stage of ischemic stroke.MethodsA Rose Bengal photothrombosis model was applied to induce targeted ischemic stroke in mice. CX3CR1CreER:R26iDTR mice were used to specifically deplete resident microglia through intragastric administration of tamoxifen (Ta) and intraperitoneal injection of diphtheria toxin (DT). At day 3 after ischemic stroke, behavioral tests were performed. After that, mouse brains were collected for further histological analysis and detection of mRNA expression of inflammatory factors.ResultsThe results showed that specific depletion of microglia resulted in a significant decrease in ischemic infarct volume and improved performance in motor ability 3 days after stroke. Microglial depletion caused a remarkable reduction in the densities of degenerating neurons and inducible nitric oxide synthase positive (iNOS+) cells. Importantly, depleting microglia induced a significant increase in the mRNA expression level of anti-inflammatory factors TGF-β1, Arg1, IL-10, IL-4, and Ym1 as well as a significant decline of pro-inflammatory factors TNF-α, iNOS, and IL-1β 3 days after stroke.ConclusionsThese results suggest that activated microglia is an important modulator of the brain’s inflammatory response in stroke, contributing to neurological deficit and infarct expansion. Modulation of the inflammatory response through the elimination of microglia at a precise time point may be a promising therapeutic approach for the treatment of cerebral ischemia.

Neuroinflammatory changes of the normal brain tissue in cured mice following combined radiation and anti-PD-1 blockade therapy for glioma

The efficacy of combining radiation therapy with immune checkpoint inhibitor blockade to treat brain tumors is currently the subject of multiple investigations and holds significant therapeutic promise. However, the long-term effects of this combination therapy on the normal brain tissue are unknown. Here, we examined mice that were intracranially implanted with murine glioma cell line and became long-term survivors after treatment with a combination of 10 Gy cranial irradiation (RT) and anti-PD-1 checkpoint blockade (aPD-1). Post-mortem analysis of the cerebral hemisphere contralateral to tumor implantation showed complete abolishment of hippocampal neurogenesis, but neural stem cells were well preserved in subventricular zone. In addition, we observed a drastic reduction in the number of mature oligodendrocytes in the subcortical white matter. Importantly, this observation was evident specifically in the combined (RT + aPD-1) treatment group but not in the single treatment arm of either RT alone or aPD-1 alone. Elimination of microglia with a small molecule inhibitor of colony stimulated factor-1 receptor (PLX5622) prevented the loss of mature oligodendrocytes. These results identify for the first time a unique pattern of normal tissue changes in the brain secondary to combination treatment with radiotherapy and immunotherapy. The results also suggest a role for microglia as key mediators of the adverse treatment effect.

Loss of homeostatic microglia in rare neurological disorders: implications for cell transplantation

Microglia originating from yolk sac exert various functions to maintain the homeostasis in the brain, and their functional breakdown appears to be involved in the pathophysiology of various neurological diseases. In this review article, loss of homeostatic microglia and new therapeutic approaches for rare neurological disorders are discussed. ASLP (adult-onset leukoencephalopathy with axonal spheroids and pigmented glia) known as a primary microgliopathy is an adult-onset leukoencephalopathy caused by CSF1R mutation. CSF1 receptor encoded by CSF1R plays an important role in the function of microglia. In brain of ALSP patients, homeostatic microglia are significantly reduced. The biallelic mutations for CSF1R cause childhood-onset severe phenotype and elimination of microglia from the brain parenchyma. Since microglia also almost disappear in CSF1R-deficient mice and rats, CSF1R deficiency and loss of microglia appear to be tightly associated across species. Based on the underlying mechanism of homeostatic microglia loss, novel approaches using cell transplantation of normal microglia-like cells have been attempted. Transplantation of wild-type bone marrow cells into Csf1r-/- mice results in replacement by donor-derived microglial-like cells in the recipient's brain. The concept of "microglial niche" may explain the rationale behind the microglial cell transplantation in disease condition(s). Hematopoietic stem cell transplantation (HSCT) has been attempted in 4 patients with ALSP. Beneficial effects by showing stabilization of the disease course have been observed. Although the effectiveness of HSCT for ALSP patients warrants further investigation, the approach of cell transplantation that replaces ruptured homeostatic microglia with normal microglia-like cells seems to be promising.

Microglia Elimination Increases Neural Circuit Connectivity and Activity in Adult Mouse Cortex.

Microglia have crucial roles in sculpting synapses and maintaining neural circuits during development. To test the hypothesis that microglia continue to regulate neural circuit connectivity in adult brain, we have investigated the effects of chronic microglial depletion, via CSF1R inhibition, on synaptic connectivity in the visual cortex in adult mice of both sexes. We find that the absence of microglia dramatically increases both excitatory and inhibitory synaptic connections to excitatory cortical neurons assessed with functional circuit mapping experiments in acutely prepared adult brain slices. Microglia depletion leads to increased densities and intensities of perineuronal nets. Furthermore, in vivo calcium imaging across large populations of visual cortical neurons reveals enhanced neural activities of both excitatory neurons and parvalbumin-expressing interneurons in the visual cortex following microglia depletion. These changes recover following adult microglia repopulation. In summary, our new results demonstrate a prominent role of microglia in sculpting neuronal circuit connectivity and regulating subsequent functional activity in adult cortex.SIGNIFICANCE STATEMENT Microglia are the primary immune cell of the brain, but recent evidence supports that microglia play an important role in synaptic sculpting during development. However, it remains unknown whether and how microglia regulate synaptic connectivity in adult brain. Our present work shows chronic microglia depletion in adult visual cortex induces robust increases in perineuronal nets, and enhances local excitatory and inhibitory circuit connectivity to excitatory neurons. Microglia depletion increases in vivo neural activities of both excitatory neurons and parvalbumin inhibitory neurons. Our new results reveal new potential avenues to modulate adult neural plasticity by microglia manipulation to better treat brain disorders, such as Alzheimer's disease.

MP07-19 ACYLOXYACYL HYDROLASE REGULATES MICROGLIAL PHENOTYPE IN A MOUSE MODEL OF INTERSTITIAL CYSTITIS

INTRODUCTION AND OBJECTIVE: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a devastating condition of chronic pelvic pain. We previously linked a locus encoding acyloxyacyl hydrolase (Aoah) with pain severity in a murine model of IC. AOAH-deficient mice develop pelvic allodynia and exhibit symptoms co-morbid with IC/BPS, such as altered voiding and a depressive phenotype. Microglia are the resident immune cells of the central nervous system (CNS), and studies have linked altered microglial morphology and activation to neuropathic pain and depression. It is also well established that microglial activation can be modulated by the gut microbiota, and AOAH dysbiosis has been reported in IC/BPS patients. Therefore, we examined the microglial phenotype in AOAH-deficient mice and the potential role of microglia in modulating pelvic pain. METHODS: To analyze changes in microglial morphology, brains from wild type (WT) C57BL/6 and AOAH-deficient mice were sectioned and stained for the microglial marker P2RY12. Following immunohistochemistry, z-stacks were acquired and skeletal analyses were performed using Image J. Activation of microglia was measured through Western blotting and a proteome profiler array of mouse cytokines using brain lysates. To address the functional role of microglia in pelvic pain, microglia were eliminated from the CNS of AOAH-deficient and WT mice by oral administration of chow containing PLX5622. Mice were exposed to mechanical stimulation of the pelvic area with von Frey filaments prior to and following microglial elimination. RESULTS: Our preliminary data revealed altered microglial morphology in AOAH-deficient mice compared to WT mice. AOAH-deficient mice exhibited hyper-ramified microglia, suggesting a surveillance phenotype, in the cortex and Barrington’s nucleus, an essential region for micturition. We also observed hyper-ramified microglia in the paraventricular nucleus, a regulator of stress and depression. Microglia were less ramified in the hippocampus, consistent with an activated phenotype. Due to the morphological changes, we expect to observe differences in cytokine release between WT and AOAH-deficient mice, reflecting these differential activation states. In addition, we expect that microglia elimination will alleviate pelvic allodynia and depression in AOAH-deficient mice. CONCLUSIONS: Overall, we observed that microglial phenotype is dependent on AOAH, suggesting microglia-specific receptors or cytokines may be considered as future therapeutic targets for treating IC/BPS. Source of Funding: NIDDK awards U01 DK082342 and R01 DK066112-06S1

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments.

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 °C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Microglia elimination caused prolonged increases in sleep following both peripheral and central inflammatory challenges in the mouse

Microglia elimination caused prolonged increases in sleep following both peripheral and central inflammatory challenges in the mouse

Attenuation of neuroinflammation reverses Adriamycin-induced cognitive impairments

Numerous clinical studies have established the debilitating neurocognitive side effects of chemotherapy in the treatment of breast cancer, often referred as chemobrain. We hypothesize that cognitive impairments are associated with elevated microglial inflammation in the brain. Thus, either elimination of microglia or restoration of microglial function could ameliorate cognitive dysfunction. Using a rodent model of chronic Adriamycin (ADR) treatment, a commonly used breast cancer chemotherapy, we evaluated two strategies to ameliorate chemobrain: 1) microglia depletion using the colony stimulating factor-1 receptor (CSF1R) inhibitor PLX5622 and 2) human induced pluripotent stem cell-derived microglia (iMG)-derived extracellular vesicle (EV) treatment. In strategy 1 mice received ADR once weekly for 4 weeks and were then administered CSF1R inhibitor (PLX5622) starting 72 h post-ADR treatment. ADR-treated animals given a normal diet exhibited significant behavioral deficits and increased microglial activation 4–6 weeks later. PLX5622-treated mice exhibited no ADR-related cognitive deficits and near complete depletion of IBA-1 and CD68+ microglia in the brain. Cytokine and RNA sequencing analysis for inflammation pathways validated these findings. In strategy 2, 1 week after the last ADR treatment, mice received retro-orbital vein injections of iMG-EV (once weekly for 4 weeks) and 1 week later, mice underwent behavior testing. ADR-treated mice receiving EV showed nearly complete restoration of cognitive function and significant reductions in microglial activation as compared to untreated ADR mice. Our data demonstrate that ADR treatment elevates CNS inflammation that is linked to cognitive dysfunction and that attenuation of neuroinflammation reverses the adverse neurocognitive effects of chemotherapy.

Elimination of microglia in mouse spinal cord alters the retrograde CNS plasticity observed following peripheral axon injury

Following the transection of peripherally located sympathetic preganglionic axons of the cervical sympathetic trunk (CST), transient retrograde neuronal and glial responses occur in the intermediolateral cell column (IML) of the spinal cord, the location of the parent neuronal cell bodies. The role of microglia in this central response to peripheral axon injury was examined in mice fed the PLX5622 diet containing colony-stimulating factor-1 receptor (CSF-1R) inhibitor for 28 days, which eliminated approximately 90% of spinal cord microglia. Microglia elimination did not impact baseline neurotransmitter expression in the IML neurons, and the typical neuronal plasticity observed following CST transection was unaffected. Oligodendrocyte precursor cells (OPCs) were significantly increased at one week post injury in the IML of mice fed the control diet, with no change in mature oligodendrocytes (OLs). Following microglia elimination, the baseline population of OPCs in the IML was increased, suggesting increased OPC proliferation. Injury in the microglia depleted mice resulted in no additional increase in OPCs. Though baseline astrocyte activation and GFAP protein expression were unaffected, microglia elimination led to increased activation and GFAP protein post injury when compared with mice fed the control diet. These results reveal that microglia regulate the baseline OPC population in the uninjured spinal cord and that activated microglia influence the activities of OL lineage cells as well as astrocytes. The regulatory roles of microglia observed in this study likely contribute to the long term survival of the IML neurons observed following the distal axon injury.