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.

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.

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.

Ischemic Preconditioning in White Matter: Magnitude and Mechanism.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon whereby brief ischemic exposure confers tolerance to a subsequent ischemic challenge. IPC has not been studied selectively in CNS white matter (WM), although stroke frequently involves WM. We determined whether IPC is present in WM and, if so, its mechanism. We delivered a brief in vivo preconditioning ischemic insult (unilateral common carotid artery ligation) to 12- to 14-week-old mice and determined WM ischemic vulnerability [oxygen-glucose deprivation (OGD)] 72 h later, using acutely isolated optic nerves (CNS WM tracts) from the preconditioned (ipsilateral) and control (contralateral) hemispheres. Functional and structural recovery was assessed by quantitative measurement of compound action potentials (CAPs) and immunofluorescent microscopy. Preconditioned mouse optic nerves (MONs) showed better functional recovery after OGD than the non-preconditioned MONs (31 ± 3 vs 17 ± 3% normalized CAP area, p < 0.01). Preconditioned MONs also showed improved axon integrity and reduced oligodendrocyte injury compared with non-preconditioned MONs. Toll-like receptor-4 (TLR4) and type 1 interferon receptor (IFNAR1), key receptors in innate immune response, are implicated in gray matter preconditioning. Strikingly, IPC-mediated WM protection was abolished in both TLR4(-/-) and IFNAR1(-/-) mice. In addition, IPC-mediated protection in WM was also abolished in IFNAR1(fl/fl) LysM(cre), but not in IFNAR1(fl/fl) control, mice. These findings demonstrated for the first time that IPC was robust in WM, the phenomenon being intrinsic to WM itself. Furthermore, WM IPC was dependent on innate immune cell signaling pathways. Finally, these data demonstrated that microglial-specific expression of IFNAR1 plays an indispensable role in WM IPC. SIGNIFICANCE STATEMENT: Ischemic preconditioning (IPC) has been studied predominantly in gray matter, but stroke in humans frequently involves white matter (WM) as well. Here we describe a novel, combined in vivo/ex vivo mouse model to determine whether IPC occurs in WM. It does. Using genetically altered mice, we identified two innate immune cell receptors, Toll-like receptor 4 and type 1 interferon receptor (IFNAR1), that are required for IPC-mediated protection in WM. Furthermore, using microglia-targeted IFNAR1 knockdown, we demonstrate that interferon signaling specifically in microglia is essential for this protection. The discovery of IPC as an intrinsic capability of WM is novel and important. This is also the first in vivo demonstration that cell-type-specific expression of an individual gene plays an indispensable role in IPC-mediated protection.

The broken branch: Darwin's evolutionary tree and the progress of medicine, the evolution of specialism and generalism.

The landscape of medical practice and health care has been transformed by specialization over the past Century. There has been an extraordinary acceleration in the proliferation of specialty practice, coincident with rapid growth in technological devices and their clinical applications during the past fifty years. Medicine and medical care are evolving rapidly, a process that has similarities to biologic evolution. Medical educators, policy makers, and practitioners might find a Darwinian overview of medicine and health care of interest.

Solidarity in medicine: the role of medical students and young doctors.

The descriptions of medical caring and of highly technical specialism and science utilize different vocabularies and language. Medical caring has an ancient glossary of words, gestures, and behavior which is rapidly being transformed by specialization and advances in the technology of communication. The technologic capabilities of intensive care have, for example, exceeded the human life span and forced redefinition of the meaning of being olive. There are risks for the contemporary profession as linguistic and technical evolution proceed at a faster pace than the evolution of human health and illth. The accentuation and acceleration of generational disparity between the young and the old diminishes the solidarity of the profession and the quality of care that it provides.

Data-driven design of a multiplexed, peptide-sensitized transistor to detect breath VOC markers of COVID-19.

Exhaled human breath contains a rich mixture of volatile organic compounds (VOCs) whose concentration can vary in response to disease or other stressors. Using simulated odorant-binding proteins (OBPs) and machine learning methods, we designed a multiplex of short VOC- and carbon-binding peptide probes that detect a characteristic "VOC fingerprint". Specifically, we target VOCs associated with COVID-19 in a compact, molecular sensor array that directly transduces vapor composition into multi-channel electrical signals. Rapidly synthesizable, chimeric VOC- and solid-binding peptides were derived from selected OBPs using multi-sequence alignment with protein database structures. Selective peptide binding to targeted VOCs and sensor surfaces was validated using surface plasmon resonance spectroscopy and quartz crystal microbalance. VOC sensing was demonstrated by peptide-sensitized, exposed-channel carbon nanotube transistors. The data-to-device pipeline enables the development of novel devices for non-invasive monitoring, diagnostics of diseases, and environmental exposure assessment.

Chimeric Peptide-Based Biomolecular Constructs for Versatile Nucleic Acid Biosensing.

Nucleic acid biomarkers hold great potential as key indicators for the diagnosis and monitoring of diseases. Herein we design and implement bifunctional chimeric biomolecules composed of a solid-binding peptide (SBP) domain that specifically adsorbs onto solid sensor surfaces and a peptide nucleic acid (PNA) moiety that facilitates anchoring of antisense oligonucleotide (ASO) probes for the detection of nucleic acid targets. A gold-binding peptide, AuBP1, previously selected by directed evolution to specifically bind to gold, served as the basis for immobilizing nucleic acid probes onto gold substrates. Using surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) analyses, we demonstrate the sequential biomolecular assembly of the heterofunctional solid-binding peptide-antisense oligomer (SBP-ASO) construct onto a sensor surface and the subsequent detection of DNA in an aqueous environment. The effect of steric hindrance on optimal probe assembly is observed, establishing that less packing density results in greater target capture efficacy. In addition, an adsorbed layer of chimeric solid-binding peptide-peptide nucleic acid (SBP-PNA) undergoes viscoelastic changes at the solid-liquid interface upon probe immobilization and DNA target capture, whereby the rigid biofunctional layer becomes more flexible. The dual nature of the chimeric construct is highly amenable to a variety of platforms allowing for both specific recognition and probe immobilization on the sensor surface, while the modular design of the solid-binding peptide-antisense oligonucleotide provides facile functionalization of a wide diversity of solid substrates.

RNAi reveals doublecortin is required for radial migration in rat neocortex.

Mutations in the doublecortin gene (DCX) in humans cause malformation of the cerebral neocortex. Paradoxically, genetic deletion of Dcx in mice does not cause neocortical malformation. We used electroporation of plasmids encoding short hairpin RNA to create interference (RNAi) of DCX protein in utero, and we show that DCX is required for radial migration in developing rat neocortex. RNAi of DCX causes both cell-autonomous and non-cell autonomous disruptions in radial migration, and creates two disruptions in neocortical development. First, many neurons prematurely stop migrating to form subcortical band heterotopias within the intermediate zone and then white matter. Second, many neurons migrate into inappropriate neocortical lamina within normotopic cortex. In utero RNAi can therefore be effectively used to study the specific cellular roles of DCX in neocortical development and to produce an animal model of double cortex syndrome.

Anthropometric assessment of nutritional status among highland Kashmiri children: Reevaluating the assumption of female nutritional disadvantage.

As part of a larger project investigating the health of rural villagers in Kashmir, India anthropometric data were collected from 132 residents of the highland village of Basmina. Here data for 70 children (53% of the total sample) less than 10 years old are examined and compared to United States reference data and presented as a percentage of the median and Z-scores to describe nutritional status differences by age and sex. Overall, these children maintain indices of weight/age, stature/age, and arm circumference (AC)/age at almost -2.0 Z-scores or more below reference medians. Weight/height and triceps skinfold/age approximate -1.4 and -1.3 Z-scores below the median, respectively. These data along with clinical observations suggest that the Basmina sample is low normal to mildly malnourished. However, preschool children show evidence of moderate and severe malnutrition. The entire child sample shows a higher prevalence of malnutrition when compared to other populations living under similar conditions. The results are contrary to the belief that Indian girls are at a nutritional disadvantage when compared to boys. In fact, preschool girls maintain higher, though not significant, percentages of weight/age, weight/height, and AC/age. Girls between 5 and 10 years old also maintain a higher triceps/age index. On the other hand, boys between 5 and 10 years old maintain a higher stature/age index, but the difference is not statistically significant.

Functional polymorphisms in toll-like receptor 4 are associated with worse outcome in acute ischemic stroke patients.

Toll-like receptor-4 (TLR4) is important in neuroinflammation. Single nucleotide polymorphisms (SNPs) in TLR4, including 1063 A/G [Asp299Gly] and 1363 C/T [Thr399Ile], are associated with altered immune responses but their effect on acute ischemic stroke (AIS) outcome is unknown. We collected demographic, clinical, laboratory, radiologic, and genotype data on 113 AIS patients and performed multivariate analyses to assess associations between TLR4 SNP haplotype and either neurological outcome, infection, or inflammatory markers. In adjusted analyses, TLR4 SNPs were associated with worse outcome as well as increases in circulating leukocytes, C-reactive protein, and interleukin-1 receptor antagonist. In AIS, variations in TLR4 may influence neurological outcome (for video abstract, please see Supplemental digital content 1 file, http://links.lww.com/WNR/A274).

Thrombin induces release of proinflammatory chemokines interleukin-8 and interferon-γ-induced protein-10 from cultured human fetal astrocytes.

Thrombin is a multifunctional serine proteinase that induces a variety of responses from neural cells by cleavage of proteinase-activated receptors (PARs) including PAR1 and PAR4. Thrombin/PAR signaling has been implicated in the neuroinflammatory response that occurs in the brain following stroke and other central nervous system pathologies. The neuroinflammatory response involves astrocytes and results in induction of proinflammatory chemokines including interleukin-8 (IL-8 or CXCL8) and interferon-γ-induced protein-10 (IP-10 or CXCL10) in these cells. Astroctyes are known to express PARs, however the effect of thrombin on astrocytic chemokine secretion is unknown. Here we characterize the ability of thrombin to induce proliferation/metabolic activity and chemokine secretion in primary human fetal astrocytes. Thrombin induces dose-dependent astrocyte proliferation as well as release of both IL-8 and IP-10, but not IL-6 or the chemokine regulated and normal T cell expressed and secreted (RANTES). The chemokine responses were mimicked by PAR1, but not PAR4, activating peptides. Our data indicate that astrocytic chemokine release is part of the neuroinflammatory response triggered by the exposure of the central nervous system to thrombin.

Darwin's earthquake.

Charles Darwin experienced a major earthquake in the Concepción-Valdivia region of Chile 175 years ago, in February 1835. His observations dramatically illustrated the geologic principles of James Hutton and Charles Lyell which maintained that the surface of the earth was subject to alterations by natural events, such as earthquakes, volcanoes, and the erosive action of wind and water, operating over very long periods of time. Changes in the land created new environments and fostered adaptations in life forms that could lead to the formation of new species. Without the demonstration of the accumulation of multiple crustal events over time in Chile, the biologic implications of the specific species of birds and tortoises found in the Galapagos Islands and the formulation of the concept of natural selection might have remained dormant.

A letter from the United States: the fox in our backyard--science, serendipity and surprise.

The story of how Charles Darwin composed The Origin of Species, published in November of 1859, has been told many times during the bicentennial of Darwin s birth and the sesquicentennial of the publication of the book. It is a history well known to biologists and historians of science. The heated debate that accompanied the demonstration of natural selection as a mechanism of speciation and continues to the present is surprising. Human beings do not welcome surprise: "the emotion aroused by something unexpected." The history of science and human intellect, however, illustrate the creative stimulus of surprise and serendipity in the development of human knowledge and the evolution of culture. The lives of Homo sapiens would not change if our intellect was unable or unwilling to respond to the unexpected and to make connections between surprising and commonplace events. The rich diversity of South American life was surprising to the European travelers of the 18th and 19th centuries: surprising by its beauty and profusion, but also by its similarities to the creatures of Europe and Africa. Darwin s curiosity sought and welcomed surprise).

Typhoid fever during pregnancy: case report and review.

Typhoid fever, like many gastroenteric infections, is considered a particular risk during pregnancy because of reduced peristaltic activity in the gastrointestinal and biliary tracts and increased prevalence of biliary 'sludge' and concretions. Antibiotic resistance among salmonellae makes the choice of antibiotics for initial treatment of infection difficult before cultures and sensitivities are reported. Because of the potential risks of some antimicrobial agents for pregnancy, the selection of antibiotic therapy is further complicated.

A novel pathogenic pathway of immune activation detectable before clinical onset in Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by both neurological and systemic abnormalities. We examined the peripheral immune system and found widespread evidence of innate immune activation detectable in plasma throughout the course of HD. Interleukin 6 levels were increased in HD gene carriers with a mean of 16 years before the predicted onset of clinical symptoms. To our knowledge, this is the earliest plasma abnormality identified in HD. Monocytes from HD subjects expressed mutant huntingtin and were pathologically hyperactive in response to stimulation, suggesting that the mutant protein triggers a cell-autonomous immune activation. A similar pattern was seen in macrophages and microglia from HD mouse models, and the cerebrospinal fluid and striatum of HD patients exhibited abnormal immune activation, suggesting that immune dysfunction plays a role in brain pathology. Collectively, our data suggest parallel central nervous system and peripheral pathogenic pathways of immune activation in HD.