Joshua Crapser

All Publications

• Restoring hippocampal glucose metabolism rescues cognition across Alzheimer's disease pathologies. Science (New York, N.Y.) Minhas, P. S., Jones, J. R., Latif-Hernandez, A., Sugiura, Y., Durairaj, A. S., Wang, Q., Mhatre, S. D., Uenaka, T., Crapser, J., Conley, T., Ennerfelt, H., Jung, Y. J., Liu, L., Prasad, P., Jenkins, B. C., Ay, Y. A., Matrongolo, M., Goodman, R., Newmeyer, T., Heard, K., Kang, A., Wilson, E. N., Yang, T., Ullian, E. M., Serrano, G. E., Beach, T. G., Wernig, M., Rabinowitz, J. D., Suematsu, M., Longo, F. M., McReynolds, M. R., Gage, F. H., Andreasson, K. I. 2024; 385 (6711): eabm6131

  Abstract

  Impaired cerebral glucose metabolism is a pathologic feature of Alzheimer's disease (AD), with recent proteomic studies highlighting disrupted glial metabolism in AD. We report that inhibition of indoleamine-2,3-dioxygenase 1 (IDO1), which metabolizes tryptophan to kynurenine (KYN), rescues hippocampal memory function in mouse preclinical models of AD by restoring astrocyte metabolism. Activation of astrocytic IDO1 by amyloid beta and tau oligomers increases KYN and suppresses glycolysis in an aryl hydrocarbon receptor-dependent manner. In amyloid and tau models, IDO1 inhibition improves hippocampal glucose metabolism and rescues hippocampal long-term potentiation in a monocarboxylate transporter-dependent manner. In astrocytic and neuronal cocultures from AD subjects, IDO1 inhibition improved astrocytic production of lactate and uptake by neurons. Thus, IDO1 inhibitors presently developed for cancer might be repurposed for treatment of AD.

  View details for DOI 10.1126/science.abm6131

  View details for PubMedID 39172838

• Degenerate mapping of environmental location presages deficits in object-location encoding and memory in the 5xFAD mouse model for Alzheimer's disease. Neurobiology of disease Zhang, H., Chen, L., Johnston, K. G., Crapser, J., Green, K. N., Ha, N. M., Tenner, A. J., Holmes, T. C., Nitz, D. A., Xu, X. 2022: 105939

  Abstract

  A key challenge in developing diagnosis and treatments for Alzheimer's disease (AD) is to detect abnormal network activity at as early a stage as possible. To date, behavioral and neurophysiological investigations in AD model mice have yet to conduct a longitudinal assessment of cellular pathology, memory deficits, and neurophysiological correlates of neuronal activity. We therefore examined the temporal relationships between pathology, neuronal activities and spatial representation of environments, as well as object location memory deficits across multiple stages of development in the 5XFAD mice model and compared these results to those observed in wild-type mice. We performed longitudinal in vivo calcium imaging with miniscope on hippocampal CA1 neurons in behaving mice. We find that 5xFAD mice show amyloid plaque accumulation, depressed neuronal calcium activity during immobile states, and degenerate and unreliable hippocampal neuron spatial tuning to environmental location at early stages by 4 months of age while their object location memory (OLM) is comparable to WT mice. By 8 months of age, 5xFAD mice show deficits of OLM, which are accompanied by progressive degradation of spatial encoding and, eventually, impaired CA1 neural tuning to object-location pairings. Furthermore, depressed neuronal activity and unreliable spatial encoding at early stage are correlated with impaired performance in OLM at 8-month-old. Our results indicate the close connection between impaired hippocampal tuning to object-location and the presence of OLM deficits. The results also highlight that depressed baseline firing rates in hippocampal neurons during immobile states and unreliable spatial representation precede object memory deficits and predict memory deficits at older age, suggesting potential early opportunities for AD detecting.

  View details for DOI 10.1016/j.nbd.2022.105939

  View details for PubMedID 36462718

• Microglia as hackers of the matrix: sculpting synapses and the extracellular space. Cellular & molecular immunology Crapser, J. D., Arreola, M. A., Tsourmas, K. I., Green, K. N. 2021; 18 (11): 2472-2488

  Abstract

  Microglia shape the synaptic environment in health and disease, but synapses do not exist in a vacuum. Instead, pre- and postsynaptic terminals are surrounded by extracellular matrix (ECM), which together with glia comprise the four elements of the contemporary tetrapartite synapse model. While research in this area is still just beginning, accumulating evidence points toward a novel role for microglia in regulating the ECM during normal brain homeostasis, and such processes may, in turn, become dysfunctional in disease. As it relates to synapses, microglia are reported to modify the perisynaptic matrix, which is the diffuse matrix that surrounds dendritic and axonal terminals, as well as perineuronal nets (PNNs), specialized reticular formations of compact ECM that enwrap neuronal subsets and stabilize proximal synapses. The interconnected relationship between synapses and the ECM in which they are embedded suggests that alterations in one structure necessarily affect the dynamics of the other, and microglia may need to sculpt the matrix to modify the synapses within. Here, we provide an overview of the microglial regulation of synapses, perisynaptic matrix, and PNNs, propose candidate mechanisms by which these structures may be modified, and present the implications of such modifications in normal brain homeostasis and in disease.

  View details for DOI 10.1038/s41423-021-00751-3

  View details for PubMedID 34413489

  View details for PubMedCentralID PMC8546068

• Subventricular zone/white matter microglia reconstitute the empty adult microglial niche in a dynamic wave. eLife Hohsfield, L. A., Najafi, A. R., Ghorbanian, Y., Soni, N., Crapser, J., Figueroa Velez, D. X., Jiang, S., Royer, S. E., Kim, S. J., Henningfield, C. M., Anderson, A., Gandhi, S. P., Mortazavi, A., Inlay, M. A., Green, K. N. 2021; 10

  Abstract

  Microglia, the brain's resident myeloid cells, play central roles in brain defense, homeostasis, and disease. Using a prolonged colony-stimulating factor 1 receptor inhibitor (CSF1Ri) approach, we report an unprecedented level of microglial depletion and establish a model system that achieves an empty microglial niche in the adult brain. We identify a myeloid cell that migrates from the subventricular zone and associated white matter areas. Following CSF1Ri, these amoeboid cells migrate radially and tangentially in a dynamic wave filling the brain in a distinct pattern, to replace the microglial-depleted brain. These repopulating cells are enriched in disease-associated microglia genes and exhibit similar phenotypic and transcriptional profiles to white-matter-associated microglia. Our findings shed light on the overlapping and distinct functional complexity and diversity of myeloid cells of the CNS and provide new insight into repopulating microglia function and dynamics in the mouse brain.

  View details for DOI 10.7554/eLife.66738

  View details for PubMedID 34423781

  View details for PubMedCentralID PMC8425950

• Microglial dyshomeostasis drives perineuronal net and synaptic loss in a CSF1R+/- mouse model of ALSP, which can be rescued via CSF1R inhibitors. Science advances Arreola, M. A., Soni, N., Crapser, J. D., Hohsfield, L. A., Elmore, M. R., Matheos, D. P., Wood, M. A., Swarup, V., Mortazavi, A., Green, K. N. 2021; 7 (35)

  Abstract

  Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia is an autosomal dominant neurodegenerative disease caused by mutations in colony-stimulating factor 1 receptor (CSF1R). We sought to identify the role of microglial CSF1R haploinsufficiency in mediating pathogenesis. Using an inducible Cx3cr1 CreERT2/+-Csf1r +/fl system, we found that postdevelopmental, microglia-specific Csf1r haploinsufficiency resulted in reduced expression of homeostatic microglial markers. This was associated with loss of presynaptic surrogates and the extracellular matrix (ECM) structure perineuronal nets. Similar phenotypes were observed in constitutive global Csf1r haploinsufficient mice and could be reversed/prevented by microglia elimination in adulthood. As microglial elimination is unlikely to be clinically feasible for extended durations, we treated adult CSF1R+/- mice at different disease stages with a microglia-modulating dose of the CSF1R inhibitor PLX5622, which prevented microglial dyshomeostasis along with synaptic- and ECM-related deficits. These data highlight microglial dyshomeostasis as a driver of pathogenesis and show that CSF1R inhibition can mitigate these phenotypes.

  View details for DOI 10.1126/sciadv.abg1601

  View details for PubMedID 34433559

  View details for PubMedCentralID PMC8386924

• To Kill a Microglia: A Case for CSF1R Inhibitors. Trends in immunology Green, K. N., Crapser, J. D., Hohsfield, L. A. 2020; 41 (9): 771-784

  Abstract

  Microglia, the brain's immune sentinels, have garnered much attention in recent years. Researchers have begun to identify the manifold roles that these cells play in the central nervous system (CNS), and this work has been greatly facilitated by microglial depletion paradigms. The varying degrees of spatiotemporal manipulation afforded by such techniques allow microglial ablation before, during, and/or following insult, injury, or disease. We review the major methods of microglial depletion, including toxin-based, genetic, and pharmacological approaches, which differ in key factors including depletion onset, duration, and off-target effects. We conclude that pharmacological CSF1R inhibitors afford the most extensive versatility in manipulating microglia, making them ideal candidates for future studies investigating microglial function in health and disease.

  View details for DOI 10.1016/j.it.2020.07.001

  View details for PubMedID 32792173

  View details for PubMedCentralID PMC7484341

• Microglia facilitate loss of perineuronal nets in the Alzheimer's disease brain. EBioMedicine Crapser, J. D., Spangenberg, E. E., Barahona, R. A., Arreola, M. A., Hohsfield, L. A., Green, K. N. 2020; 58: 102919

  Abstract

  Microglia, the brain's principal immune cell, are increasingly implicated in Alzheimer's disease (AD), but the molecular interfaces through which these cells contribute to amyloid beta (Aβ)-related neurodegeneration are unclear. We recently identified microglial contributions to the homeostatic and disease-associated modulation of perineuronal nets (PNNs), extracellular matrix structures that enwrap and stabilize neuronal synapses, but whether PNNs are altered in AD remains controversial.Extensive histological analysis was performed on male and female 5xFAD mice at 4, 8, 12, and 18 months of age to assess plaque burden, microgliosis, and PNNs. Findings were validated in postmortem AD tissue. The role of neuroinflammation in PNN loss was investigated via LPS treatment, and the ability to prevent or rescue disease-related reductions in PNNs was assessed by treating 5xFAD and 3xTg-AD model mice with colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia.Utilizing the 5xFAD mouse model and human cortical tissue, we report that PNNs are extensively lost in AD in proportion to plaque burden. Activated microglia closely associate with and engulf damaged nets in the 5xFAD brain, and inclusions of PNN material are evident in mouse and human microglia, while aggrecan, a critical PNN component, deposits within human dense-core plaques. Disease-associated reductions in parvalbumin (PV)+ interneurons, frequently coated by PNNs, are preceded by PNN coverage and integrity impairments, and similar phenotypes are elicited in wild-type mice following microglial activation with LPS. Chronic pharmacological depletion of microglia prevents 5xFAD PNN loss, with similar results observed following depletion in aged 3xTg-AD mice, and this occurs despite plaque persistence.We conclude that phenotypically altered microglia facilitate plaque-dependent PNN loss in the AD brain.The NIH (NIA, NINDS) and the Alzheimer's Association.

  View details for DOI 10.1016/j.ebiom.2020.102919

  View details for PubMedID 32745992

  View details for PubMedCentralID PMC7399129

• Microglial depletion prevents extracellular matrix changes and striatal volume reduction in a model of Huntington's disease. Brain : a journal of neurology Crapser, J. D., Ochaba, J., Soni, N., Reidling, J. C., Thompson, L. M., Green, K. N. 2020; 143 (1): 266-288

  Abstract

  Huntington's disease is associated with a reactive microglial response and consequent inflammation. To address the role of these cells in disease pathogenesis, we depleted microglia from R6/2 mice, a rapidly progressing model of Huntington's disease marked by behavioural impairment, mutant huntingtin (mHTT) accumulation, and early death, through colony-stimulating factor 1 receptor inhibition (CSF1Ri) with pexidartinib (PLX3397) for the duration of disease. Although we observed an interferon gene signature in addition to downregulated neuritogenic and synaptic gene pathways with disease, overt inflammation was not evident by microglial morphology or cytokine transcript levels in R6/2 mice. Nonetheless, CSF1Ri-induced microglial elimination reduced or prevented disease-related grip strength and object recognition deficits, mHTT accumulation, astrogliosis, and striatal volume loss, the latter of which was not associated with reductions in cell number but with the extracellular accumulation of chondroitin sulphate proteoglycans (CSPGs)-a primary component of glial scars. A concurrent loss of proteoglycan-containing perineuronal nets was also evident in R6/2 mice, and microglial elimination not only prevented this but also strikingly increased perineuronal nets in the brains of naïve littermates, suggesting a new role for microglia as homeostatic regulators of perineuronal net formation and integrity.

  View details for DOI 10.1093/brain/awz363

  View details for PubMedID 31848580

  View details for PubMedCentralID PMC6935750

• Sustained microglial depletion with CSF1R inhibitor impairs parenchymal plaque development in an Alzheimer's disease model. Nature communications Spangenberg, E., Severson, P. L., Hohsfield, L. A., Crapser, J., Zhang, J., Burton, E. A., Zhang, Y., Spevak, W., Lin, J., Phan, N. Y., Habets, G., Rymar, A., Tsang, G., Walters, J., Nespi, M., Singh, P., Broome, S., Ibrahim, P., Zhang, C., Bollag, G., West, B. L., Green, K. N. 2019; 10 (1): 3758

  Abstract

  Many risk genes for the development of Alzheimer's disease (AD) are exclusively or highly expressed in myeloid cells. Microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for their survival. We designed and synthesized a highly selective brain-penetrant CSF1R inhibitor (PLX5622) allowing for extended and specific microglial elimination, preceding and during pathology development. We find that in the 5xFAD mouse model of AD, plaques fail to form in the parenchymal space following microglial depletion, except in areas containing surviving microglia. Instead, Aβ deposits in cortical blood vessels reminiscent of cerebral amyloid angiopathy. Altered gene expression in the 5xFAD hippocampus is also reversed by the absence of microglia. Transcriptional analyses of the residual plaque-forming microglia show they exhibit a disease-associated microglia profile. Collectively, we describe the structure, formulation, and efficacy of PLX5622, which allows for sustained microglial depletion and identify roles of microglia in initiating plaque pathogenesis.

  View details for DOI 10.1038/s41467-019-11674-z

  View details for PubMedID 31434879

  View details for PubMedCentralID PMC6704256

• Evaluation of the Neuroprotective Effect of Sirt3 in Experimental Stroke. Translational stroke research Verma, R., Ritzel, R. M., Crapser, J., Friedler, B. D., McCullough, L. D. 2019; 10 (1): 57-66

  Abstract

  Sirtuins (Sirt) are a family of NAD+ dependent histone deacetylase (HDAC) proteins implicated in aging, cell cycle regulation, and metabolism. These proteins are involved in the epigenetic modification of neuromodulatory proteins after strokevia acetylation/deacetylation. The specific role of Sirt3, a mitochondrial sirtuin, in post-stroke injury has been relatively unexplored. Using male Sirt3 knockout (KO) mice and wild-type littermates (WT), we show that Sirt3 KO mice show significant neuroprotection at 3 days after ischemia/reperfusion (I/R) or stroke injury. The deacetylation activity of Sirt3, measured as the amount of reduced acetylated lysine, was increased after stroke. Stroke-induced increases in liver kinase 1 (LKB1) activity were also reduced in KO mice at 3 days after stroke. On further investigation, we found that the levels of Sirt1, another important member of the Sirtuin family, were increased in the brains of Sirt3 KO mice after stroke. To determine the translational relevance of these findings, we then tested the effects of pharmacological inhibition of Sirt3. We found no benefit of Sirt3 inhibition despite clear evidence of deacetylation. Overall, these data suggest that Sirt3 KO mice show neuroprotection by a compensatory rise in Sirt1 rather than the loss of Sirt3 after stroke. Further analysis reveals that the beneficial effects of Sirt1 might be mediated by a decrease in LKB1 activity after stroke. Finally, our data clearly demonstrate the importance of using both pharmacological and genetic methods in pre-clinical stroke studies.

  View details for DOI 10.1007/s12975-017-0603-x

  View details for PubMedID 29302794

• CD200-CD200R1 inhibitory signaling prevents spontaneous bacterial infection and promotes resolution of neuroinflammation and recovery after stroke. Journal of neuroinflammation Ritzel, R. M., Al Mamun, A. n., Crapser, J. n., Verma, R. n., Patel, A. R., Knight, B. E., Harris, N. n., Mancini, N. n., Roy-O'Reilly, M. n., Ganesh, B. P., Liu, F. n., McCullough, L. D. 2019; 16 (1): 40

  Abstract

  Ischemic stroke results in a robust inflammatory response within the central nervous system. As the immune-inhibitory CD200-CD200 receptor 1 (CD200R1) signaling axis is a known regulator of immune homeostasis, we hypothesized that it may play a role in post-stroke immune suppression after stroke.In this study, we investigated the role of CD200R1-mediated signaling in stroke using CD200 receptor 1-deficient mice. Mice were subjected to a 60-min middle cerebral artery occlusion and evaluated at days 3 and 7, representing the respective peak and early resolution stages of neuroinflammation in this model of ischemic stroke. Infarct size and behavioral deficits were assessed at both time points. Central and peripheral cellular immune responses were measured using flow cytometry. Bacterial colonization was determined in lung tissue homogenates both after acute stroke and in an LPS model of systemic inflammation.In wild-type (WT) animals, CD200R1 was expressed on infiltrating monocytes and lymphocytes after stroke but was absent on microglia. Early after ischemia (72 h), CD200R1-knockout (KO) mice had significantly poorer survival rates and an enhanced susceptibility to spontaneous bacterial colonization of the respiratory tract compared to wild-type (WT) controls, despite no difference in infarct or neurological deficits. While the CNS inflammation was resolved by day 7 post-stroke in WT mice, brain-resident microglia and monocyte activation persisted in CD200R1-KO mice, accompanied by a delayed, augmented lymphocyte response. At this time point, CD200R1-KO mice displayed greater weight loss, more severe neurological deficits, and impaired motor function compared to WT. Systemically, CD200R1-KO mice exhibited signs of persistent infection including lymphopenia, T cell activation and memory conversion, and narrowing of the TCR repertoire. These findings were confirmed in a second model of acute neuroinflammation induced by systemic endotoxin challenge.This study defines an essential role of CD200-CD200R1 signaling in stroke. Loss of CD200R1 led to high mortality, increased rates of post-stroke infection, and enhanced entry of peripheral leukocytes into the brain after ischemia, with no increase in infarct size. This suggests that the loss of CD200 receptor leads to enhanced peripheral inflammation that is triggered by brain injury.

  View details for DOI 10.1186/s12974-019-1426-3

  View details for PubMedID 30777093

  View details for PubMedCentralID PMC6378746

• Longitudinal Biochemical Assay Analysis of Mutant Huntingtin Exon 1 Protein in R6/2 Mice. Journal of Huntington's disease Morozko, E. L., Ochaba, J., Hernandez, S. J., Lau, A., Sanchez, I., Orellana, I., Kopan, L., Crapser, J., Duong, J. H., Overman, J., Yeung, S., Steffan, J. S., Reidling, J., Thompson, L. M. 2018; 7 (4): 321-335

  Abstract

  Biochemical analysis of mutant huntingtin (mHTT) aggregation species in HD mice is a common measure to track disease. A longitudinal and systematic study of how tissue processing affects detection of conformers has not yet been reported. Understanding the homeostatic flux of mHTT over time and under different processing conditions would aid in interpretation of pre-clinical assessments of disease interventions.Provide a systematic evaluation of tissue lysis methods and molecular and biochemical assays in parallel with behavioral readouts in R6/2 mice to establish a baseline for HTT exon1 protein accumulation.Established biochemical methods were used to process tissue from R6/2 mice of specific ages following behavior tasks. Aggregation states and accumulation of mHTT exon 1 protein were evaluated using multiple break and assay methods to determine potential conformational flux assay specificity in detection of mHTT species, and tissue specificity of conformers.Detection of mHTT exon 1 protein species varied based on biochemical processing and analysis providing a baseline for subsequent studies in R6/2 mice. Insoluble, high molecular weight species of mHTT exon 1 protein increased and tracked with onset of behavioral impairments in R6/2 mice using multiple assay methods.Conformational flux from soluble monomer to high molecular weight, insoluble species of mHTT exon 1 protein was generally consistent for multiple assay methods throughout R6/2 disease progression; however, the results support the use of multiple biochemical techniques to detect mHTT exon 1 protein species for preclinical assessments in HD mouse models expressing mHTT exon 1 protein.

  View details for DOI 10.3233/JHD-180329

  View details for PubMedID 30452420

  View details for PubMedCentralID PMC6294605

• A limited capacity for microglial repopulation in the adult brain. Glia Najafi, A. R., Crapser, J., Jiang, S., Ng, W., Mortazavi, A., West, B. L., Green, K. N. 2018; 66 (11): 2385-2396

  Abstract

  Microglia are the resident immune cell of the central nervous system (CNS), and serve to protect and maintain the local brain environment. Microglia are critically dependent on signaling through the colony-stimulating factor 1 receptor (CSF1R); administration of CSF1R inhibitors that cross the blood brain barrier (BBB) lead to the elimination of up to 99% of microglia, depending on CNS exposure and treatment duration. Once microglia are depleted, withdrawal of inhibitor stimulates repopulation of the entire CNS with new cells, conceivably enabling a therapeutic strategy for beneficial renewal of the entire microglial tissue. We have explored the kinetics and limits of this repopulation event and show that the rate of microglial repopulation is proportional to the extent of microglial depletion - greater depletion of microglia results in more rapid repopulation. Using a CSF1R inhibitor formulation that eliminates approximately 99% of microglia within 7 days, we subjected mice to multiple rounds of elimination (7 days' treatment) and repopulation (7 days' recovery) and found that the brain only has the capacity for a single complete repopulation event; subsequent elimination and CSF1R inhibitor withdrawal fail to repopulate the brain. However, if the recovery time between, or after, cycles is extended sufficiently then the brain can ultimately repopulate. These kinetic studies define the opportunities and possible limits of the remarkable renewal capacities of microglia.

  View details for DOI 10.1002/glia.23477

  View details for PubMedID 30370589

  View details for PubMedCentralID PMC6269202

• Aging alters the immunological response to ischemic stroke. Acta neuropathologica Ritzel, R. M., Lai, Y. J., Crapser, J. D., Patel, A. R., Schrecengost, A., Grenier, J. M., Mancini, N. S., Patrizz, A., Jellison, E. R., Morales-Scheihing, D., Venna, V. R., Kofler, J. K., Liu, F., Verma, R., McCullough, L. D. 2018; 136 (1): 89-110

  Abstract

  The peripheral immune system plays a critical role in aging and in the response to brain injury. Emerging data suggest inflammatory responses are exacerbated in older animals following ischemic stroke; however, our understanding of these age-related changes is poor. In this work, we demonstrate marked differences in the composition of circulating and infiltrating leukocytes recruited to the ischemic brain of old male mice after stroke compared to young male mice. Blood neutrophilia and neutrophil invasion into the brain were increased in aged animals. Relative to infiltrating monocyte populations, brain-invading neutrophils had reduced phagocytic potential, and produced higher levels of reactive oxygen species and extracellular matrix-degrading enzymes (i.e., MMP-9), which were further exacerbated with age. Hemorrhagic transformation was more pronounced in aged versus young mice relative to infarct size. High numbers of myeloperoxidase-positive neutrophils were found in postmortem human brain samples of old (> 71 years) acute ischemic stroke subjects compared to non-ischemic controls. Many of these neutrophils were found in the brain parenchyma. A large proportion of these neutrophils expressed MMP-9 and positively correlated with hemorrhage and hyperemia. MMP-9 expression and hemorrhagic transformation after stroke increased with age. These changes in the myeloid response to stroke with age led us to hypothesize that the bone marrow response to stroke is altered with age, which could be important for the development of effective therapies targeting the immune response. We generated heterochronic bone marrow chimeras as a tool to determine the contribution of peripheral immune senescence to age- and stroke-induced inflammation. Old hosts that received young bone marrow (i.e., Young → Old) had attenuation of age-related reductions in bFGF and VEGF and showed improved locomotor activity and gait dynamics compared to isochronic (Old → Old) controls. Microglia in young heterochronic mice (Old → Young) developed a senescent-like phenotype. After stroke, aged animals reconstituted with young marrow had reduced behavioral deficits compared to isochronic controls, and had significantly fewer brain-infiltrating neutrophils. Increased rates of hemorrhagic transformation were seen in young mice reconstituted with aged bone marrow. This work suggests that age alters the immunological response to stroke, and that this can be reversed by manipulation of the peripheral immune cells in the bone marrow.

  View details for DOI 10.1007/s00401-018-1859-2

  View details for PubMedID 29752550

  View details for PubMedCentralID PMC6015099

• Multiparity improves outcomes after cerebral ischemia in female mice despite features of increased metabovascular risk. Proceedings of the National Academy of Sciences of the United States of America Ritzel, R. M., Patel, A. R., Spychala, M., Verma, R., Crapser, J., Koellhoffer, E. C., Schrecengost, A., Jellison, E. R., Zhu, L., Venna, V. R., McCullough, L. D. 2017; 114 (28): E5673-E5682

  Abstract

  Females show a varying degree of ischemic sensitivity throughout their lifespan, which is not fully explained by hormonal or genetic factors. Epidemiological data suggest that sex-specific life experiences such as pregnancy increase stroke risk. This work evaluated the role of parity on stroke outcome. Age-matched virgin (i.e., nulliparous) and multiparous mice were subjected to 60 min of reversible middle cerebral artery occlusion and evaluated for infarct volume, behavioral recovery, and inflammation. Using an established mating paradigm, fetal microchimeric cells present in maternal mice were also tracked after parturition and stroke. Parity was associated with sedentary behavior, weight gain, and higher triglyceride and cholesterol levels. The multiparous brain exhibited features of immune suppression, with dampened baseline microglial activity. After acute stroke, multiparous mice had smaller infarcts, less glial activation, and less behavioral impairment in the critical recovery window of 72 h. Behavioral recovery was significantly better in multiparous females compared with nulliparous mice 1 mo after stroke. This recovery was accompanied by an increase in poststroke angiogenesis that was correlated with improved performance on sensorimotor and cognitive tests. Multiparous mice had higher levels of VEGF, both at baseline and after stroke. GFP+ fetal cells were detected in the blood and migrated to areas of tissue injury where they adopted endothelial morphology 30 d after injury. Reproductive experience has profound and complex effects on neurovascular health and disease. Inclusion of female mice with reproductive experience in preclinical studies may better reflect the life-long patterning of ischemic stroke risk in women.

  View details for DOI 10.1073/pnas.1607002114

  View details for PubMedID 28645895

  View details for PubMedCentralID PMC5514696

• Early retinal inflammatory biomarkers in the middle cerebral artery occlusion model of ischemic stroke. Molecular vision Ritzel, R. M., Pan, S. J., Verma, R., Wizeman, J., Crapser, J., Patel, A. R., Lieberman, R., Mohan, R., McCullough, L. D. 2016; 22: 575-88

  Abstract

  The transient middle cerebral artery occlusion (MCAO) model of stroke is one of the most commonly used models to study focal cerebral ischemia. This procedure also results in the simultaneous occlusion of the ophthalmic artery that supplies the retina. Retinal cell death is seen days after reperfusion and leads to functional deficits; however, the mechanism responsible for this injury has not been investigated. Given that the eye may have a unique ocular immune response to an ischemic challenge, this study examined the inflammatory response to retinal ischemia in the MCAO model.Young male C57B/6 mice were subjected to 90-min transient MCAO and were euthanized at several time points up to 7 days. Transcription of inflammatory cytokines was measured with quantitative real-time PCR, and immune cell activation (e.g., phagocytosis) and migration were assessed with ophthalmoscopy and flow cytometry.Observation of the affected eye revealed symptoms consistent with Horner's syndrome. Light ophthalmoscopy confirmed the reduced blood flow of the retinal arteries during occlusion. CX3CR1-GFP reporter mice were then employed to evaluate the extent of the ocular microglia and monocyte activation. A significant increase in green fluorescent protein (GFP)-positive macrophages was seen throughout the ischemic area compared to the sham and contralateral control eyes. RT-PCR revealed enhanced expression of the monocyte chemotactic molecule CCL2 early after reperfusion followed by a delayed increase in the proinflammatory cytokine TNF-α. Further analysis of peripheral leukocyte recruitment by flow cytometry determined that monocytes and neutrophils were the predominant immune cells to infiltrate at 72 h. A transient reduction in retinal microglia numbers was also observed, demonstrating the ischemic sensitivity of these cells. Blood-eye barrier permeability to small and large tracer molecules was increased by 72 h. Retinal microglia exhibited enhanced phagocytic activity following MCAO; however, infiltrating myeloid cells were significantly more efficient at phagocytizing material at all time points. Immune homeostasis in the affected eye was largely restored by 7 days.This work demonstrates that there is a robust inflammatory response in the eye following MCAO, which may contribute to a worsening of retinal injury and visual impairment. These results mirror what has been observed in the brain after MCAO, suggesting a conserved inflammatory signaling response to ischemia in the central nervous system. Imaging of the eye may therefore serve as a useful non-invasive prognostic indicator of brain injury after MCAO. Future studies are needed to determine whether this inflammatory response is a potential target for therapeutic manipulation in retinal ischemia.

  View details for PubMedID 27293375

  View details for PubMedCentralID PMC4893077

• Ischemic stroke induces gut permeability and enhances bacterial translocation leading to sepsis in aged mice. Aging Crapser, J., Ritzel, R., Verma, R., Venna, V. R., Liu, F., Chauhan, A., Koellhoffer, E., Patel, A., Ricker, A., Maas, K., Graf, J., McCullough, L. D. 2016; 8 (5): 1049-63

  Abstract

  Aging is an important risk factor for post-stroke infection, which accounts for a large proportion of stroke-associated mortality. Despite this, studies evaluating post-stroke infection rates in aged animal models are limited. In addition, few studies have assessed gut microbes as a potential source of infection following stroke. Therefore we investigated the effects of age and the role of bacterial translocation from the gut in post-stroke infection in young (8-12 weeks) and aged (18-20 months) C57Bl/6 male mice following transient middle cerebral artery occlusion (MCAO) or sham surgery. Gut permeability was examined and peripheral organs were assessed for the presence of gut-derived bacteria following stroke. Furthermore, sickness parameters and components of innate and adaptive immunity were examined. We found that while stroke induced gut permeability and bacterial translocation in both young and aged mice, only young mice were able to resolve infection. Bacterial species seeding peripheral organs also differed between young (Escherichia) and aged (Enterobacter) mice. Consequently, aged mice developed a septic response marked by persistent and exacerbated hypothermia, weight loss, and immune dysfunction compared to young mice following stroke.

  View details for DOI 10.18632/aging.100952

  View details for PubMedID 27115295

  View details for PubMedCentralID PMC4931853

• Reversal of the Detrimental Effects of Post-Stroke Social Isolation by Pair-Housing is Mediated by Activation of BDNF-MAPK/ERK in Aged Mice. Scientific reports Verma, R., Harris, N. M., Friedler, B. D., Crapser, J., Patel, A. R., Venna, V., McCullough, L. D. 2016; 6: 25176

  Abstract

  Social isolation (SI) increases stroke-related mortality and morbidity in clinical populations. The detrimental effects of SI have been successfully modeled in the laboratory using young animals. Mechanistically, the negative effects of SI in young animals are primarily mediated by an enhanced inflammatory response to injury and a reduction in neurotrophic factors. However, the response to brain injury differs considerably in the aged. Given that SI is more prevalent in aged populations, we hypothesized that isolation, even when initiated after stroke, would delay recovery in aged mice. We found that aged isolated male mice had significantly increased infarct volume, neurological deficits, and serum IL-6 levels three days after stroke compared to pair housed (PH) mice. Using RT(2) Profiler PCR Array and real-time quantitative PCR we found several important synaptic plasticity genes were differentially expressed in post-stroke SI mice. Furthermore, paired mice showed improved memory and neurobehavioral recovery four weeks after injury. Mechanistic and histological studies showed that the beneficial effects of pair housing are partially mediated by BDNF via downstream MAPK/ERK signaling and restoration of axonal basic myelin protein levels.

  View details for DOI 10.1038/srep25176

  View details for PubMedID 27125783

  View details for PubMedCentralID PMC4850427

• Age-Associated Resident Memory CD8 T Cells in the Central Nervous System Are Primed To Potentiate Inflammation after Ischemic Brain Injury. Journal of immunology (Baltimore, Md. : 1950) Ritzel, R. M., Crapser, J., Patel, A. R., Verma, R., Grenier, J. M., Chauhan, A., Jellison, E. R., McCullough, L. D. 2016; 196 (8): 3318-30

  Abstract

  Aging is associated with an increase in basal inflammation in the CNS and an overall decline in cognitive function and poorer recovery following injury. Growing evidence suggests that leukocyte recruitment to the CNS is also increased with normal aging, but, to date, no systematic evaluation of these age-associated leukocytes has been performed. In this work, the effect of aging on CNS leukocyte recruitment was examined. Aging was associated with more CD45(high) leukocytes, primarily composed of conventional CD8(+) T cells. These results were strain independent and seen in both sexes. Intravascular labeling and immunohistology revealed the presence of parenchymal CD8(+) T cells in several regions of the brain, including the choroid plexus and meninges. These cells had effector memory (CD44(+)CD62L(-)) and tissue-resident phenotypes and expressed markers associated with TCR activation. Analysis of TCRvβ repertoire usage suggested that entry into the CNS is most likely stochastic rather than Ag driven. Correlational analyses revealed a positive association between CD8 T cell numbers and decreased proinflammatory function of microglia. However, the effects of cerebral ischemia and ex vivo stimulation of these cells dramatically increased production of TNF, IFN-γ, and MCP-1/CCL2. Taken together, we identified a novel population of resident memory, immunosurveillant CD8 T cells that represent a hallmark of CNS aging and appear to modify microglia homeostasis under normal conditions, but are primed to potentiate inflammation and leukocyte recruitment following ischemic injury.

  View details for DOI 10.4049/jimmunol.1502021

  View details for PubMedID 26962232

  View details for PubMedCentralID PMC4868658

• Age- and location-related changes in microglial function. Neurobiology of aging Ritzel, R. M., Patel, A. R., Pan, S., Crapser, J., Hammond, M., Jellison, E., McCullough, L. D. 2015; 36 (6): 2153-63

  Abstract

  Inflammation in the central nervous system (CNS) is primarily regulated by microglia. No longer considered a homogenous population, microglia display a high degree of heterogeneity, immunological diversity and regional variability in function. Given their low rate of self-renewal, the microenvironment in which microglia reside may play an important role in microglial senescence. This study examines age-related changes in microglia in the brain and spinal cord. Using ex-vivo flow cytometry analyses, functional assays were performed to assess changes in microglial morphology, oxidative stress, cytokine production, and phagocytic activity with age in both the brain and spinal cord. The regional CNS environment had a significant effect on microglial activity with age. Blood-CNS barrier permeability was greater in the aging spinal cord compared with aging brain; this was associated with increased tissue cytokine levels. Aged microglia had deficits in phagocytosis at baseline and after stimulus-induced activation. The identification of age-specific, high scatter microglia together with the use of ex-vivo functional analyses provides the first functional characterization of senescent microglia. Age and regional-specificity of CNS disease should be taken into consideration when developing immune-modulatory treatments.

  View details for DOI 10.1016/j.neurobiolaging.2015.02.016

  View details for PubMedID 25816747

• Functional differences between microglia and monocytes after ischemic stroke. Journal of neuroinflammation Ritzel, R. M., Patel, A. R., Grenier, J. M., Crapser, J., Verma, R., Jellison, E. R., McCullough, L. D. 2015; 12: 106

  Abstract

  The brain's initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood-brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models.Using a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days.We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke.In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.

  View details for DOI 10.1186/s12974-015-0329-1

  View details for PubMedID 26022493

  View details for PubMedCentralID PMC4465481

• One is the deadliest number: the detrimental effects of social isolation on cerebrovascular diseases and cognition. Acta neuropathologica Friedler, B., Crapser, J., McCullough, L. 2015; 129 (4): 493-509

  Abstract

  The deleterious effects of chronic social isolation (SI) have been recognized for several decades. Isolation is a major source of psychosocial stress and is associated with an increased prevalence of vascular and neurological diseases. In addition, isolation exacerbates morbidity and mortality following acute injuries such as stroke or myocardial infarction. In contrast, affiliative social interactions can improve organismal function and health. The molecular mechanisms underlying these effects are unknown. Recently, results from large epidemiological trials and pre-clinical studies have revealed several potential mediators of the detrimental effects of isolation. At least three major biological systems have been implicated: the neuroendocrine (HPA) axis, the immune system, and the autonomic nervous system. This review summarizes studies examining the relationship between isolation and mortality and the pathophysiological mechanisms underlying SI. Cardiovascular, cerebrovascular, and neurological diseases including atherosclerosis, myocardial infarction, ischemic stroke and Alzheimer's disease are given special emphasis in the context of SI. Sex differences are highlighted and studies are separated into clinical and basic science for clarity.

  View details for DOI 10.1007/s00401-014-1377-9

  View details for PubMedID 25537401

  View details for PubMedCentralID PMC4369164

• Inhibition of mitochondrial p53 abolishes the detrimental effects of social isolation on ischemic brain injury. Stroke Venna, V. R., Verma, R., O'Keefe, L. M., Xu, Y., Crapser, J., Friedler, B., McCullough, L. D. 2014; 45 (10): 3101-4

  Abstract

  Social isolation (SI) increases stroke incidence and delays poststroke recovery. Women may be at greater risk from the negative consequences of SI, but few studies have examined both sexes in experimental models, and none have evaluated the effects of isolation initiated after stroke. The effects of poststroke SI in men and women were examined, and the role of mitochondrial P53 was evaluated.C57Bl6 mice were pair-housed (PH; male and ovariectomized female) for 2 weeks, subjected to stroke and then assigned to a housing condition (isolated or PH). The effects of housing on infarct volume and recovery were examined. Changes in Bcl-2 and mitochondrial p53 were assessed by Western blot. A mitochondrial p53 inhibitor (pifithrin-μ) was given to mice of both sexes.Compared with pair-housed mice, poststroke SI significantly increased infarct size in both sexes; SI mice also had worse neurological deficits. The detrimental effects of SI paralleled increases in mitochondrial p53 levels. Pharmacological inhibition of mitochondrial p53 using pifithrin-μ abolished the detrimental effects of SI and reduced cell death.Poststroke SI results in increased ischemic injury in both sexes. The effect of housing on infarct was more pronounced in women. Targeting the mitochondrial P53 pathway could minimize the detrimental effects of isolation after stroke.

  View details for DOI 10.1161/STROKEAHA.114.006553

  View details for PubMedID 25205311

  View details for PubMedCentralID PMC4192598