Microglial depletion abolishes ischemic preconditioning in white matter.

Ischemic preconditioning (IPC) is a phenomenon whereby a brief, non-injurious ischemic exposure enhances tolerance to a subsequent ischemic challenge. The mechanism of IPC has mainly been studied in rodent stroke models where gray matter (GM) constitutes about 85% of the cerebrum. In humans, white matter (WM) is 50% of cerebral volume and is a critical component of stroke damage. We developed a novel CNS WM IPC model using the mouse optic nerve (MON) and identified the involved immune signaling pathways. Here we tested the hypothesis that microglia are necessary for WM IPC. Microglia were depleted by treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. MONs were exposed to transient ischemia in vivo, acutely isolated 72 h later, and subjected to oxygen-glucose deprivation (OGD) to simulate a severe ischemic injury (i.e., stroke). Functional and structural axonal recovery was assessed by recording compound action potentials (CAPs) and by microscopy using quantitative stereology. Microglia depletion eliminated IPC-mediated protection. In control mice, CAP recovery was improved in preconditioned MONs compared with non-preconditioned MONs, however, in PLX5622-treated mice, we observed no difference in CAP recovery between preconditioned and non-preconditioned MONs. Microgliadepletion also abolished IPC protective effects on axonal integrity and survival of mature (APC+ ) oligodendrocytes after OGD. IPC-mediated protection was independent of retinal injury suggesting it results from mechanistic processes intrinsic to ischemia-exposed WM. We conclude that preconditioned microglia are critical for IPC in WM. The "preconditioned microglia" phenotype might protect against other CNS pathologies and is a neurotherapeutic horizon worth exploring.

A Subpopulation of Microglia Generated in the Adult Mouse Brain Originates from Prominin-1-Expressing Progenitors.

Microglia maintain brain health and play important roles in disease and injury. Despite the known ability of microglia to proliferate, the precise nature of the population or populations capable of generating new microglia in the adult brain remains controversial. We identified Prominin-1 (Prom1; also known as CD133) as a putative cell surface marker of committed brain myeloid progenitor cells. We demonstrate that Prom1-expressing cells isolated from mixed cortical cultures will generate new microglia in vitro To determine whether Prom1-expressing cells generate new microglia in vivo, we used tamoxifen inducible fate mapping in male and female mice. Induction of Cre recombinase activity at 10 weeks in Prom1-expressing cells leads to the expression of TdTomato in all Prom1-expressing progenitors and newly generated daughter cells. We observed a population of new TdTomato-expressing microglia at 6 months of age that increased in size at 9 months. When microglia proliferation was induced using a transient ischemia/reperfusion paradigm, little proliferation from the Prom1-expressing progenitors was observed with the majority of new microglia derived from Prom1-negative cells. Together, these findings reveal that Prom1-expressing myeloid progenitor cells contribute to the generation of new microglia both in vitro and in vivo Furthermore, these findings demonstrate the existence of an undifferentiated myeloid progenitor population in the adult mouse brain that expresses Prom1. We conclude that Prom1-expressing myeloid progenitors contribute to new microglia genesis in the uninjured brain but not in response to ischemia/reperfusion.SIGNIFICANCE STATEMENT Microglia, the innate immune cells of the CNS, can divide to slowly generate new microglia throughout life. Newly generated microglia may influence inflammatory responses to injury or neurodegeneration. However, the origins of the new microglia in the brain have been controversial. Our research demonstrates that some newly born microglia in a healthy brain are derived from cells that express the stem cell marker Prominin-1. This is the first time Prominin-1 cells are shown to generate microglia.

Unipolar (Dendritic) Brush Cells Are Morphologically Complex and Require Tbr2 for Differentiation and Migration.

Previous studies demonstrated specific expression of transcription factor Tbr2 in unipolar brush cells (UBCs) of the cerebellum during development and adulthood. To further study UBCs and the role of Tbr2 in their development we examined UBC morphology in transgenic mouse lines (reporter and lineage tracer) and also examined the effects of Tbr2 deficiency in Tbr2 (MGI: Eomes) conditional knock-out (cKO) mice. In Tbr2 reporter and lineage tracer cerebellum, UBCs exhibited more complex morphologies than previously reported including multiple dendrites, bifurcating dendrites, and up to four dendritic brushes. We propose that "dendritic brush cells" (DBCs) may be a more apt nomenclature. In Tbr2 cKO cerebellum, mature UBCs were completely absent. Migration of UBC precursors from rhombic lip to cerebellar cortex and other nuclei was impaired in Tbr2 cKO mice. Our results indicate that UBC migration and differentiation are sensitive to Tbr2 deficiency. To investigate whether UBCs develop similarly in humans as in rodents, we studied Tbr2 expression in mid-gestational human cerebellum. Remarkably, Tbr2+ UBC precursors migrate along the same pathways in humans as in rodent cerebellum and disperse to create the same "fountain-like" appearance characteristic of UBCs exiting the rhombic lip.

The role of microglia in ischemic preconditioning.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a brief ischemic stimulus confers protection against a subsequent prolonged ischemic event. Initially thought to be due to mechanistic changes in neurons, our understanding of IPC has evolved to encompass a global reprogramming of the Central Nervous System (CNS) after transient ischemia/reperfusion that requires innate immune signaling pathways including Toll-like receptors (TLRs) and Type I interferons. Microglia are the CNS resident neuroimmune cells that express these key innate immune receptors. Studies suggest that microglia are required for IPC-mediated neuronal and axonal protection. Multiple paradigms targeting TLRs have converged on a distinctive Type I interferon response in microglia that is critical for preconditioning-mediated protection against ischemia. These pathways can be targeted through administration of TLR agonists, cytokines including interferon-ß, and pharmaceutical agents that induce preconditioning through cross-tolerance mechanisms. Transcriptomic analyses and single cell RNA studies point to specific gene expression signatures in microglia that functionally shift these mutable cells to an immunomodulatory or protective phenotype. Although there are technological challenges and gaps in knowledge to overcome, the targeting of specific molecular signaling pathways in microglia is a promising direction for development of novel and effective pharmacotherapies for stroke. Studies on preconditioning in animal models, including nonhuman primates, show promise as prophylactic preconditioning treatments for selected at risk patient populations. In addition, our growing understanding of the mechanisms of IPC-mediated protection is identifying novel cellular and molecular targets for therapeutic interventions that could apply broadly to both acute stroke and chronic vascular cognitive impairment patients.

Ischemic preconditioning induces cortical microglial proliferation and a transcriptomic program of robust cell cycle activation.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a subthreshold ischemic insult applied to the brain reduces damage caused by a subsequent more severe ischemic episode. Identifying key molecular and cellular mediators of IPC will provide critical information needed to develop novel therapies for stroke. Here we report that the transcriptomic response of acutely isolated preconditioned cortical microglia is dominated by marked upregulation of genes involved in cell cycle activation and cellular proliferation. Notably, this transcriptional response occurs in the absence of cortical infarction. We employed ex vivo flow cytometry, immunofluorescent microscopy, and quantitative stereology methods on brain tissue to evaluate microglia proliferation following IPC. Using cellular colocalization of microglial (Iba1) and proliferation (Ki67 and BrdU) markers, we observed a localized increase in the number of microglia and proliferating microglia within the preconditioned hemicortex at 72, but not 24, hours post-IPC. Our quantification demonstrated that the IPC-induced increase in total microglia was due entirely to proliferation. Furthermore, microglia in the preconditioned hemisphere had altered morphology and increased soma volumes, indicative of an activated phenotype. Using transgenic mouse models with either fractalkine receptor (CX3CR1)-haploinsufficiency or systemic type I interferon signaling loss, we determined that microglial proliferation after IPC is dependent on fractalkine signaling but independent of type I interferon signaling. These findings suggest there are multiple distinct targetable signaling pathways in microglia, including CX3CR1-dependent proliferation that may be involved in IPC-mediated protection.

Correction to: Neuroimmune Response in Ischemic Preconditioning.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon in which a brief period of cerebral ischemia confers transient tolerance to subsequent ischemic challenge. Research on IPC has implicated cellular, molecular, and systemic elements of the immune response in this phenomenon. Potent molecular mediators of IPC include innate immune signaling pathways such as Toll-like receptors and type 1 interferons. Brain ischemia results in release of pro- and anti-inflammatory cytokines and chemokines that orchestrate the neuroinflammatory response, resolution of inflammation, and transition to neurological recovery and regeneration. Cellular mediators of IPC include microglia, the resident central nervous system immune cells, astrocytes, and neurons. All of these cell types engage in cross-talk with each other using a multitude of signaling pathways that modulate activation/suppression of each of the other cell types in response to ischemia. As the postischemic neuroimmune response evolves over time there is a shift in function toward provision of trophic support and neuroprotection. Peripheral immune cells infiltrate the central nervous system en masse after stroke and are largely detrimental, with a few subtypes having beneficial, protective effects, though the role of these immune cells in IPC is largely unknown. The role of neural progenitor cells in IPC-mediated neuroprotection is another active area of investigation as is the role of microglial proliferation in this setting. A mechanistic understanding of these molecular and cellular mediators of IPC may not only facilitate more effective direct application of IPC to specific clinical scenarios, but also, more broadly, reveal novel targets for therapeutic intervention in stroke.

Ischemia/Reperfusion Induces Interferon-Stimulated Gene Expression in Microglia.

Innate immune signaling is important in the pathophysiology of ischemia/reperfusion (stroke)-induced injury and recovery. Several lines of evidence support a central role for microglia in these processes. Recent work has identified Toll-like receptors (TLRs) and type I interferon (IFN) signaling in both ischemia/reperfusion-induced brain injury and ischemic preconditioning-mediated neuroprotection. To determine the effects of "ischemia/reperfusion-like" conditions on microglia, we performed genomic analyses on wild-type (WT) and TLR4-/- cultured microglia after sequential exposure to hypoxia/hypoglycemia and normoxia/normoglycemia (H/H-N/N). We observed increased expression of type 1 IFN-stimulated genes (ISGs) as the predominant transcriptomal feature of H/H-N/N-exposed WT, but not TLR4-/-, microglia. Microarray analysis on ex vivo sorted microglia from ipsilateral male mouse cortex after a transient in vivo ischemic pulse also demonstrated robust expression of ISGs. Type 1 IFNs, including the IFN-αs and IFN-ß, activate the interferon-α/ß receptor (IFNAR) complex. We confirmed both in vitro H/H-N/N- and in vivo ischemia/reperfusion-induced microglial ISG responses by quantitative real-time PCR and demonstrated that both were dependent on IFNAR1. We characterized the effects of hypoxia/hypoglycemia on phosphorylation of signal transducer and activator of transcription 1 (STAT1), release of type 1 IFNs, and surface expression of IFNAR1 in microglia. We demonstrated that IFN-ß induces dose-dependent secretion of ISG chemokines in cultured microglia and robust ISG expression in microglia both in vitro and in vivo Finally, we demonstrated that the microglial ISG chemokine responses to TLR4 agonists were dependent on TLR4 and IFNAR1. Together, these data suggest novel ischemia/reperfusion-induced pathways for both TLR4-dependent and -independent, IFNAR1-dependent, type 1 IFN signaling in microglia.SIGNIFICANCE STATEMENT Stroke is the fifth leading cause of death in the United States and is a leading cause of serious long-term disability worldwide. Innate immune responses are critical in stroke pathophysiology, and microglia are key cellular effectors in the CNS response to ischemia/reperfusion. Using a transcriptional analysis approach, we identified a robust interferon (IFN)-stimulated gene response within microglia exposed to ischemia/reperfusion in both in vitro and in vivo experimental paradigms. Using a number of complementary techniques, we have demonstrated that these responses are dependent on innate immune signaling components including Toll-like receptor-4 and type I IFNs. We have also elucidated several novel ischemia/reperfusion-induced microglial signaling mechanisms.

Behavior of Xeno-Transplanted Undifferentiated Human Induced Pluripotent Stem Cells Is Impacted by Microenvironment Without Evidence of Tumors.

Human pluripotent stem cells (hPSC) have great clinical potential through the use of their differentiated progeny, a population in which there is some concern over risks of tumorigenicity or other unwanted cellular behavior due to residual hPSC. Preclinical studies using human stem cells are most often performed within a xenotransplant context. In this study, we sought to measure how undifferentiated hPSC behave following xenotransplant. We directly transplanted undifferentiated human induced pluripotent stem cells (hIPSC) and human embryonic stem cells (hESC) into the adult mouse brain ventricle and analyzed their fates. No tumors or precancerous lesions were present at more than one year after transplantation. This result differed with the tumorigenic capacity we observed after allotransplantation of mouse ESC into the mouse brain. A substantial population of cellular derivatives of undifferentiated hESC and hIPSC engrafted, survived, and migrated within the mouse brain parenchyma. Within brain structures, transplanted cell distribution followed a very specific pattern, suggesting the existence of distinct microenvironments that offer different degrees of permissibility for engraftment. Most of the transplanted hESC and hIPSC that developed into brain cells were NeuN+ neuronal cells, and no astrocytes were detected. Substantial cell and nuclear fusion occurred between host and transplanted cells, a phenomenon influenced by microenvironment. Overall, hIPSC appear to be largely functionally equivalent to hESC in vivo. Altogether, these data bring new insights into the behavior of stem cells without prior differentiation following xenotransplantation into the adult brain.

Microglial Interferon Signaling and White Matter.

Microglia, the resident immune cells of the CNS, are primary regulators of the neuroimmune response to injury. Type I interferons (IFNs), including the IFNαs and IFNß, are key cytokines in the innate immune system. Their activity is implicated in the regulation of microglial function both during development and in response to neuroinflammation, ischemia, and neurodegeneration. Data from numerous studies in multiple sclerosis (MS) and stroke suggest that type I IFNs can modulate the microglial phenotype, influence the overall neuroimmune milieu, regulate phagocytosis, and affect blood-brain barrier integrity. All of these IFN-induced effects result in numerous downstream consequences on white matter pathology and microglial reactivity. Dysregulation of IFN signaling in mouse models with genetic deficiency in ubiquitin specific protease 18 (USP18) leads to a severe neurological phenotype and neuropathological changes that include white matter microgliosis and pro-inflammatory gene expression in dystrophic microglia. A class of genetic disorders in humans, referred to as pseudo-TORCH syndrome (PTS) for the clinical resemblance to infection-induced TORCH syndrome, also show dysregulation of IFN signaling, which leads to severe neurological developmental disease. In these disorders, the excessive activation of IFN signaling during CNS development results in a destructive interferonopathy with similar induction of microglial dysfunction as seen in USP18 deficient mice. Other recent studies implicate "microgliopathies" more broadly in neurological disorders including Alzheimer's disease (AD) and MS, suggesting that microglia are a potential therapeutic target for disease prevention and/or treatment, with interferon signaling playing a key role in regulating the microglial phenotype.

Neuroimmune Response in Ischemic Preconditioning.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon in which a brief period of cerebral ischemia confers transient tolerance to subsequent ischemic challenge. Research on IPC has implicated cellular, molecular, and systemic elements of the immune response in this phenomenon. Potent molecular mediators of IPC include innate immune signaling pathways such as Toll-like receptors and type 1 interferons. Brain ischemia results in release of pro- and anti-inflammatory cytokines and chemokines that orchestrate the neuroinflammtory response, resolution of inflammation, and transition to neurological recovery and regeneration. Cellular mediators of IPC include microglia, the resident central nervous system immune cells, astrocytes, and neurons. All of these cell types engage in cross-talk with each other using a multitude of signaling pathways that modulate activation/suppression of each of the other cell types in response to ischemia. As the postischemic neuroimmune response evolves over time there is a shift in function toward provision of trophic support and neuroprotection. Peripheral immune cells infiltrate the central nervous system en masse after stroke and are largely detrimental, with a few subtypes having beneficial, protective effects, though the role of these immune cells in IPC is largely unknown. The role of neural progenitor cells in IPC-mediated neuroprotection is another active area of investigation as is the role of microglial proliferation in this setting. A mechanistic understanding of these molecular and cellular mediators of IPC may not only facilitate more effective direct application of IPC to specific clinical scenarios, but also, more broadly, reveal novel targets for therapeutic intervention in stroke.

Calibrated forceps model of spinal cord compression injury.

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Endogenous proliferation after spinal cord injury in animal models.

Spinal cord injury (SCI) results in motor and sensory deficits, the severity of which depends on the level and extent of the injury. Animal models for SCI research include transection, contusion, and compression mouse models. In this paper we will discuss the endogenous stem cell response to SCI in animal models. All SCI animal models experience a similar peak of cell proliferation three days after injury; however, each specific type of injury promotes a specific and distinct stem cell response. For example, the transection model results in a strong and localized initial increase of proliferation, while in contusion and compression models, the initial level of proliferation is lower but encompasses the entire rostrocaudal extent of the spinal cord. All injury types result in an increased ependymal proliferation, but only in contusion and compression models is there a significant level of proliferation in the lateral regions of the spinal cord. Finally, the fate of newly generated cells varies from a mainly oligodendrocyte fate in contusion and compression to a mostly astrocyte fate in the transection model. Here we will discuss the potential of endogenous stem/progenitor cell manipulation as a therapeutic tool to treat SCI.