Kristian Doyle

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• Doyle, K. P., & Buckwalter, M. S. (2014).

  A Mouse Model of Permanent Focal Ischemia: Distal Middle Cerebral Artery Occlusion

  . In Methods in Molecular Biology. doi:10.1007/978-1-4939-0320-7_9
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  Here we provide a standardized protocol for performing distal middle cerebral artery occlusion (DMCAO) in mice. DMCAO is a method of inducing permanent focal ischemia that is commonly used as a rodent stroke model. To perform DMCAO a temporal craniotomy is performed, and the middle cerebral artery (MCA) is permanently ligated at a point downstream of the lenticulostriate branches. The size of the lesion produced by this surgery is strain dependent. In C57BL/6J mice, DMCAO produces an infarct predominantly restricted to the barrel region of the somatosensory cortex, but in BALB/cJ mice, DMCAO generates a much larger lesion that incorporates more of the somatosensory cortex and part of the M1 region of the motor cortex. The larger lesion produced by DMCAO in BALB/cJ mice produces a clearer sensorimotor deficit, which is useful for investigating recovery from stroke. We also describe how to modify DMCAO in C57BL/6J mice with the application of hypoxia to generate a lesion and sensorimotor deficit that are similar in size to those produced by DMCAO alone in BALB/cJ mice. This is extremely useful for stroke experiments that require a robust sensorimotor deficit in transgenic mice created on a C57BL/6J background.

Journals/Publications

• Hashemi, E., Narain Srivastava, I., Aguirre, A., Tilahan Yoseph, E., Kaushal, E., Awani, A., Kyu Ryu, J., Akassoglou, K., Talebian, S., Chu, P., Pisani, L., Musolino, P., Steinman, L., Doyle, K., Robinson, W. H., Sharpe, O., Cayrol, R., Orchard, P., Lund, T., , Vogel, H., et al. (2023). A novel mouse model of cerebral adrenoleukodystrophy highlights NLRP3 activity in lesion pathogenesis. bioRxiv : the preprint server for biology.
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  We sought to create and characterize a mouse model of the inflammatory, cerebral demyelinating phenotype of X-linked adrenoleukodystrophy (ALD) that would facilitate the study of disease pathogenesis and therapy development. We also sought to cross-validate potential therapeutic targets such as fibrin, oxidative stress, and the NLRP3 inflammasome, in post-mortem human and murine brain tissues.
• Loppi, S. H., Tavera-Garcia, M. A., Becktel, D. A., Maiyo, B. K., Johnson, K. E., Nguyen, T. V., Schnellmann, R. G., & Doyle, K. P. (2023). Increased fatty acid metabolism and decreased glycolysis are hallmarks of metabolic reprogramming within microglia in degenerating white matter during recovery from experimental stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 271678X231157298.
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  The goal of this study was to evaluate changes in metabolic homeostasis during the first 12 weeks of recovery in a distal middle cerebral artery occlusion mouse model of stroke. To achieve this goal, we compared the brain metabolomes of ipsilateral and contralateral hemispheres from aged male mice up to 12 weeks after stroke to that of age-matched naïve and sham mice. There were 707 biochemicals detected in each sample by liquid chromatography-mass spectroscopy (LC-MS). Mitochondrial fatty acid β-oxidation, indicated by acyl carnitine levels, was increased in stroked tissue at 1 day and 4 weeks following stroke. Glucose and several glycolytic intermediates were elevated in the ipsilateral hemisphere for 12 weeks compared to the aged naïve controls, but pyruvate was decreased. Additionally, itaconate, a glycolysis inhibitor associated with activation of anti-inflammatory mechanisms in myeloid cells, was higher in the same comparisons. Spatial transcriptomics and RNA in situ hybridization localized these alterations to microglia within the area of axonal degeneration. These results indicate that chronic metabolic differences exist between stroked and control brains, including alterations in fatty acid metabolism and glycolysis within microglia in areas of degenerating white matter for at least 12 weeks after stroke.
• Loppi, S. H., Tavera-Garcia, M. A., Scholpa, N. E., Maiyo, B. K., Becktel, D. A., Morrison, H. W., Schnellmann, R. G., & Doyle, K. P. (2023). Boosting Mitochondrial Biogenesis Diminishes Foam Cell Formation in the Post-Stroke Brain. International journal of molecular sciences, 24(23).
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  Following ischemic stroke, the degradation of myelin and other cellular membranes surpasses the lipid-processing capabilities of resident microglia and infiltrating macrophages. This imbalance leads to foam cell formation in the infarct and areas of secondary neurodegeneration, instigating sustained inflammation and furthering neurological damage. Given that mitochondria are the primary sites of fatty acid metabolism, augmenting mitochondrial biogenesis (MB) may enhance lipid processing, curtailing foam cell formation and post-stroke chronic inflammation. Previous studies have shown that the pharmacological activation of the β2-adrenergic receptor (β2-AR) stimulates MB. Consequently, our study sought to discern the effects of intensified β2-AR signaling on MB, the processing of brain lipid debris, and neurological outcome using a mouse stroke model. To achieve this goal, aged mice were treated with formoterol, a long-acting β2-AR agonist, daily for two and eight weeks following stroke. Formoterol increased MB in the infarct region, modified fatty acid metabolism, and reduced foam cell formation. However, it did not reduce markers of post-stroke neurodegeneration or improve recovery. Although our findings indicate that enhancing MB in myeloid cells can aid in the processing of brain lipid debris after stroke, it is important to note that boosting MB alone may not be sufficient to significantly impact stroke recovery.
• Zbesko, J. C., Stokes, J., Becktel, D. A., & Doyle, K. P. (2023). Targeting foam cell formation to improve recovery from ischemic stroke. Neurobiology of disease, 106130.
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  Inflammation is a crucial part of the healing process after an ischemic stroke and is required to restore tissue homeostasis. However, the inflammatory response to stroke also worsens neurodegeneration and creates a tissue environment that is unfavorable to regeneration for several months, thereby postponing recovery. In animal models, inflammation can also contribute to the development of delayed cognitive deficits. Myeloid cells that take on a foamy appearance are one of the most prominent immune cell types within chronic stroke infarcts. Emerging evidence suggests that they form as a result of mechanisms of myelin lipid clearance becoming overwhelmed, and that they are a key driver of the chronic inflammatory response to stroke. Therefore, targeting lipid accumulation in foam cells may be a promising strategy for improving recovery. The aim of this review is to provide an overview of current knowledge regarding inflammation and foam cell formation in the brain in the weeks and months following ischemic stroke and identify targets that may be amenable to therapeutic intervention.
• Dwyer, L. J., Stowe, A. M., Doyle, K., Popovich, P., Engler-Chiurazzi, E., LeGuern, C., Buckwalter, M. S., Poznansky, M. C., & Sîrbulescu, R. F. (2022). The 2022 FASEB virtual Catalyst Conference on B Cells in Injury and Regeneration, March 30, 2022. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(8), e22459.
• Ledford, J. G., Polverino, F., Brooks, H. L., Doyle, K. P., Boitano, S., Pederson, W. P., Ellerman, L. M., Sandoval, E. C., & Frye, J. B. (2022).

  Development of a Novel Mouse Model of Menopause-associated Asthma

  . American Journal of Respiratory Cell and Molecular Biology, 67(5), 605-609. doi:10.1165/rcmb.2022-0181le
• Pederson, W. P., Ellerman, L. M., Sandoval, E. C., Boitano, S., Frye, J. B., Doyle, K. P., Brooks, H. L., Polverino, F., & Ledford, J. G. (2022). Development of a Novel Mouse Model of Menopause-associated Asthma. American journal of respiratory cell and molecular biology, 67(5), 605-609.
• Schnellmann, R. G., Doyle, K. P., Zbesko, J. C., Yang, T., Tavera-garcia, M. A., Schnellmann, R. G., Nguyen, T. V., Morrison, H. W., Longo, F. M., Gonzalez, S., Garcia, F. G., Frye, J. B., Doyle, K. P., Crumpacker, R. H., Calderon, K. E., & Becktel, D. A. (2022). Post-Stroke Administration of the p75 Neurotrophin Receptor Modulator, LM11A-31, Attenuates Chronic Changes in Brain Metabolism, Increases Neurotransmitter Levels, and Improves Recovery.. The Journal of pharmacology and experimental therapeutics, 380(2), 126-141. doi:10.1124/jpet.121.000711
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  The aim of this study was to test whether poststroke oral administration of a small molecule p75 neurotrophin receptor (p75NTR) modulator (LM11A-31) can augment neuronal survival and improve recovery in a mouse model of stroke. Mice were administered LM11A-31 for up to 12 weeks, beginning 1 week after stroke. Metabolomic analysis revealed that after 2 weeks of daily treatment, mice that received LM11A-31 were distinct from vehicle-treated mice by principal component analysis and had higher levels of serotonin, acetylcholine, and dopamine in their ipsilateral hemisphere. LM11A-31 treatment also improved redox homeostasis by restoring reduced glutathione. It also offset a stroke-induced reduction in glycolysis by increasing acetyl-CoA. There was no effect on cytokine levels in the infarct. At 13 weeks after stroke, adaptive immune cell infiltration in the infarct was unchanged in LM11A-31-treated mice, indicating that LM11A-31 does not alter the chronic inflammatory response to stroke at the site of the infarct. However, LM11A-31-treated mice had less brain atrophy, neurodegeneration, tau pathology, and microglial activation in other regions of the ipsilateral hemisphere. These findings correlated with improved recovery of motor function on a ladder test, improved sensorimotor and cognitive abilities on a nest construction test, and less impulsivity in an open field test. These data support small molecule modulation of the p75NTR for preserving neuronal health and function during stroke recovery. SIGNIFICANCE STATEMENT: The findings from this study introduce the p75 neurotrophin receptor as a novel small molecule target for promotion of stroke recovery. Given that LM11A-31 is in clinical trials as a potential therapy for Alzheimer's disease, it could be considered as a candidate for assessment in stroke or vascular dementia studies.
• Zbesko, J. C., Schnellmann, R. G., Nguyen, T. V., Doyle, K. P., Zbesko, J. C., Wu, H. J., Tavera-garcia, M. A., Schnellmann, R. G., Nguyen, T. V., Li, A., Hayes, M., Grover, J. W., Garcia, F. G., Frye, J. B., Doyle, K. P., Chung, A. G., Calderon, K., & Becktel, D. A. (2022). Repeated Administration of 2-Hydroxypropyl-β-Cyclodextrin (HPβCD) Attenuates the Chronic Inflammatory Response to Experimental Stroke.. The Journal of neuroscience : the official journal of the Society for Neuroscience, 42(2), 325-348. doi:10.1523/jneurosci.0933-21.2021
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  Globally, more than 67 million people are living with the effects of ischemic stroke. Importantly, many stroke survivors develop a chronic inflammatory response that may contribute to cognitive impairment, a common and debilitating sequela of stroke that is insufficiently studied and currently untreatable. 2-Hydroxypropyl-β-cyclodextrin (HPβCD) is an FDA-approved cyclic oligosaccharide that can solubilize and entrap lipophilic substances. The goal of the present study was to determine whether the repeated administration of HPβCD curtails the chronic inflammatory response to stroke by reducing lipid accumulation within stroke infarcts in a distal middle cerebral artery occlusion mouse model of stroke. To achieve this goal, we subcutaneously injected young adult and aged male mice with vehicle or HPβCD 3 times per week, with treatment beginning 1 week after stroke. We evaluated mice at 7 weeks following stroke using immunostaining, RNA sequencing, lipidomic, and behavioral analyses. Chronic stroke infarct and peri-infarct regions of HPβCD-treated mice were characterized by an upregulation of genes involved in lipid metabolism and a downregulation of genes involved in innate and adaptive immunity, reactive astrogliosis, and chemotaxis. Correspondingly, HPβCD reduced the accumulation of lipid droplets, T lymphocytes, B lymphocytes, and plasma cells in stroke infarcts. Repeated administration of HPβCD also preserved NeuN immunoreactivity in the striatum and thalamus and c-Fos immunoreactivity in hippocampal regions. Additionally, HPβCD improved recovery through the protection of hippocampal-dependent spatial working memory and reduction of impulsivity. These results indicate that systemic HPβCD treatment following stroke attenuates chronic inflammation and secondary neurodegeneration and prevents poststroke cognitive decline.SIGNIFICANCE STATEMENT Dementia is a common and debilitating sequela of stroke. Currently, there are no available treatments for poststroke dementia. Our study shows that lipid metabolism is disrupted in chronic stroke infarcts, which causes an accumulation of uncleared lipid debris and correlates with a chronic inflammatory response. To our knowledge, these substantial changes in lipid homeostasis have not been previously recognized or investigated in the context of ischemic stroke. We also provide a proof of principle that solubilizing and entrapping lipophilic substances using HPβCD could be an effective strategy for treating chronic inflammation after stroke and other CNS injuries. We propose that using HPβCD for the prevention of poststroke dementia could improve recovery and increase long-term quality of life in stroke sufferers.
• Becktel, D. A., Zbesko, J. C., Frye, J. B., Chung, A. G., Hayes, M., Calderon, K., Grover, J. W., Li, A., Garcia, F. G., Tavera-Garcia, M. A., Schnellmann, R. G., Wu, H. J., Nguyen, T. V., & Doyle, K. P. (2021). Repeated administration of 2-hydroxypropyl-β-cyclodextrin (HPβCD) attenuates the chronic inflammatory response to experimental stroke. The Journal of neuroscience : the official journal of the Society for Neuroscience.
  More info

  Globally, more than 67 million people are living with the effects of ischemic stroke. Importantly, many stroke survivors develop a chronic inflammatory response that may contribute to cognitive impairment, a common and debilitating sequela of stroke that is insufficiently studied and currently untreatable. 2-hydroxypropyl-β-cyclodextrin (HPβCD) is an FDA-approved cyclic oligosaccharide that can solubilize and entrap lipophilic substances. The goal of the present study was to determine whether the repeated administration of HPβCD curtails the chronic inflammatory response to stroke by reducing lipid accumulation within stroke infarcts in a distal middle cerebral artery occlusion mouse model of stroke. To achieve this goal, we subcutaneously injected young adult and aged male mice with vehicle or HPβCD three times per week, with treatment beginning one week after stroke. We evaluated mice at 7 weeks following stroke using immunostaining, RNA sequencing, lipidomic, and behavioral analyses. Chronic stroke infarct and peri-infarct regions of HPβCD-treated mice were characterized by an upregulation of genes involved in lipid metabolism and a downregulation of genes involved in innate and adaptive immunity, reactive astrogliosis, and chemotaxis. Correspondingly, HPβCD reduced the accumulation of lipid droplets, T lymphocytes, B lymphocytes, and plasma cells in stroke infarcts. Repeated administration of HPβCD also preserved NeuN immunoreactivity in the striatum and thalamus and c-Fos immunoreactivity in hippocampal regions. Additionally, HPβCD improved recovery through the protection of hippocampal-dependent spatial working memory and reduction of impulsivity. These results indicate that systemic HPβCD treatment following stroke attenuates chronic inflammation and secondary neurodegeneration and prevents post-stroke cognitive decline.Dementia is a common and debilitating sequela of stroke. Currently, there are no available treatments for post-stroke dementia. Our study shows that lipid metabolism is disrupted in chronic stroke infarcts, which causes an accumulation of uncleared lipid debris and correlates with a chronic inflammatory response. To our knowledge, these substantial changes in lipid homeostasis have not been previously recognized or investigated in the context of ischemic stroke. We also provide a proof of principle that solubilizing and entrapping lipophilic substances using HPβCD could be an effective strategy for treating chronic inflammation after stroke and other CNS injuries. We propose that using HPβCD for the prevention of post-stroke dementia could improve recovery and increase long-term quality of life in stroke sufferers.
• Hoyer-Kimura, C., Konhilas, J. P., Mansour, H. M., Polt, R., Doyle, K. P., Billheimer, D., & Hay, M. (2021). Neurofilament light: a possible prognostic biomarker for treatment of vascular contributions to cognitive impairment and dementia. Journal of neuroinflammation, 18(1), 236.
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  Decreased cerebral blood flow and systemic inflammation during heart failure (HF) increase the risk for vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer disease-related dementias (ADRD). We previously demonstrated that PNA5, a novel glycosylated angiotensin 1-7 (Ang-(1-7)) Mas receptor (MasR) agonist peptide, is an effective therapy to rescue cognitive impairment in our preclinical model of VCID. Neurofilament light (NfL) protein concentration is correlated with cognitive impairment and elevated in neurodegenerative diseases, hypoxic brain injury, and cardiac disease. The goal of the present study was to determine (1) if treatment with Ang-(1-7)/MasR agonists can rescue cognitive impairment and decrease VCID-induced increases in NfL levels as compared to HF-saline treated mice and, (2) if NfL levels correlate with measures of cognitive function and brain cytokines in our VCID model.
• Nguyen, T. V., Crumpacker, R. H., Calderon, K. E., Garcia, F. G., Zbesko, J. C., Frye, J. B., Gonzalez, S., Becktel, D. A., Yang, T., Tavera-Garcia, M. A., Morrison, H. W., Schnellmann, R. G., Longo, F. M., & Doyle, K. P. (2021). Post-stroke administration of the p75 neurotrophin receptor modulator, LM11A-31, attenuates chronic changes in brain metabolism, increases neurotransmitter levels, and improves recovery. The Journal of pharmacology and experimental therapeutics.
  More info

  The aim of this study was to test whether post-stroke oral administration of a small molecule p75 neurotrophin receptor(p75) modulator (LM11A-31) can augment neuronal survival and improve recovery in a mouse model of stroke. Mice were administered LM11A-31 for up to 12 weeks, beginning 1 week after stroke. Metabolomic analysis revealed that after 2 weeks of daily treatment, mice that received LM11A-31 were distinct from vehicle treated mice by principal component analysis and had higher levels of serotonin, acetylcholine, and dopamine in their ipsilateral hemisphere. LM11A-31 treatment also improved redox homeostasis by restoring reduced glutathione. It also offset a stroke induced reduction in glycolysis by increasing acetyl-CoA. There was no effect on cytokine levels in the infarct. At 13 weeks following stroke, adaptive immune cell infiltration in the infarct was unchanged in LM11A-31 treated mice, indicating that LM11A-31 does not alter the chronic inflammatory response to stroke at the site of the infarct. However, LM11A-31 treated mice had less brain atrophy, neurodegeneration, tau pathology, and microglial activation in other regions of the ipsilateral hemisphere. These findings correlated with improved recovery of motor function on a ladder test, improved sensorimotor and cognitive abilities on a nest construction test, and less impulsivity in an open field test. These data support small molecule modulation of the p75 neurotrophin receptor for preserving neuronal health and function during stroke recovery. The findings from this study introduce the p75 neurotrophin receptor as a novel small molecule target for promotion of stroke recovery. Given that LM11A-31 is in clinical trials as a potential therapy for Alzheimer's disease, it could be considered as a candidate for assessment in stroke or vascular dementia studies.
• Zbesko, J. C., Stokes, J., Nguyen, T. V., Gerardo, D. K., Frye, J. B., Doyle, K. P., Calderon, K., Bhattacharya, D., & Becktel, D. A. (2021). IgA natural antibodies are produced following T-cell independent B-cell activation following stroke.. Brain, behavior, and immunity, 91, 578-586. doi:10.1016/j.bbi.2020.09.014
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  Up to 30% of stroke patients experience cognitive decline within one year of their stroke. There are currently no FDA-approved drugs that can prevent post-stroke cognitive decline, in part due to a poor understanding of the mechanisms involved. We have previously demonstrated that a B-lymphocyte response to stroke, marked by IgA + cells, can cause delayed cognitive dysfunction in mice and that a similar adaptive immune response occurs in the brains of some human stroke patients that suffer from vascular dementia. The stimuli which trigger B-lymphocyte activation following stroke, and their target antigens, are still unknown. Therefore, to learn more about the mechanisms by which B-lymphocytes become activated following stroke we first characterized the temporal kinetics of the B-lymphocyte, T-lymphocyte, and plasma cell (PC) response to stroke in the brain by immunohistochemistry (IHC). We discovered that B-lymphocyte, T-lymphocyte, and plasma cell infiltration within the infarct progressively increases between 2 and 7 weeks after stroke. We then compared the B-lymphocyte response to stroke in WT, MHCII-/-, CD4-/-, and MyD88-/- mice to determine if B-lymphocytes mature into IgA + PCs through a T-lymphocyte and MyD88 dependent mechanism. Our data from a combination of IHC and flow cytometry indicate that following stroke, a population of IgA + PCs develops independently of CD4 + helper T-lymphocytes and MyD88 signaling. Subsequent sequencing of immunoglobulin genes of individual IgA + PCs present within the infarct identified a novel population of natural antibodies with few somatic mutations in complementarity-determining regions. These findings indicate that a population of IgA + PCs develops in the infarct following stroke by B-lymphocytes interacting with one or more thymus independent type 2 (TI-2) antigens, and that they produce IgA natural antibodies.
• Bartlett, M. J., Flores, A. J., Ye, T., Smidt, S. I., Dollish, H. K., Stancati, J. A., Farrell, D. C., Parent, K. L., Doyle, K. P., Besselsen, D. G., Heien, M. L., Cowen, S. L., Steece-Collier, K., Sherman, S. J., & Falk, T. (2020). Preclinical evidence in support of repurposing sub-anesthetic ketamine as a treatment for L-DOPA-induced dyskinesia. Experimental neurology, 333, 113413.
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  Parkinson's disease (PD) is the second most common neurodegenerative disease. Pharmacotherapy with L-DOPA remains the gold-standard therapy for PD, but is often limited by the development of the common side effect of L-DOPA-induced dyskinesia (LID), which can become debilitating. The only effective treatment for disabling dyskinesia is surgical therapy (neuromodulation or lesioning), therefore effective pharmacological treatment of LID is a critical unmet need. Here, we show that sub-anesthetic doses of ketamine attenuate the development of LID in a rodent model, while also having acute anti-parkinsonian activity. The long-term anti-dyskinetic effect is mediated by brain-derived neurotrophic factor-release in the striatum, followed by activation of ERK1/2 and mTOR pathway signaling. This ultimately leads to morphological changes in dendritic spines on striatal medium spiny neurons that correlate with the behavioral effects, specifically a reduction in the density of mushroom spines, a dendritic spine phenotype that shows a high correlation with LID. These molecular and cellular changes match those occurring in hippocampus and cortex after effective sub-anesthetic ketamine treatment in preclinical models of depression, and point to common mechanisms underlying the therapeutic efficacy of ketamine for these two disorders. These preclinical mechanistic studies complement current ongoing clinical testing of sub-anesthetic ketamine for the treatment of LID by our group, and provide further evidence in support of repurposing ketamine to treat individuals with PD. Given its clinically proven therapeutic benefit for both treatment-resistant depression and several pain states, very common co-morbidities in PD, sub-anesthetic ketamine could provide multiple therapeutic benefits for PD in the future.
• Doyle, K. P., & Buckwalter, M. S. (2020). Immunological mechanisms in poststroke dementia.. Current Opinion in Neurology, 33(1), 30-36. doi:10.1097/wco.0000000000000783
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  Purpose of review To review new evidence on links between poststroke dementia and inflammation. Recent findings Although there are still no treatments for poststroke dementia, recent evidence has improved our understanding that stroke increases the risk of incident dementia and worsens cognitive trajectory for at least a decade afterwards. Within approximately the first year dementia onset is associated with stroke severity and location, whereas later absolute risk is associated with more traditional dementia risk factors, such as age and imaging findings. The molecular mechanisms that underlie increased risk of incident dementia in stroke survivors remain unproven; however new data in both human and animal studies suggests links between cognitive decline and inflammation. These point to a model where chronic brain inflammation, provoked by inefficient clearance of myelin debris and a prolonged innate and adaptive immune response, causes poststroke dementia. These localized immune events in the brain may themselves be influenced by the peripheral immune state at key times after stroke. Summary This review recaps clinical evidence on poststroke dementia, new mechanistic links between the chronic inflammatory response to stroke and poststroke dementia, and proposes a model of immune-mediated neurodegeneration after stroke.
• Falk, T., Sherman, S. J., Steece-Collier, K., Cowen, S. L., Heien, M. L., Doyle, K., Besselsen, D. G., Parent, K. L., Farrell, D. C., Stancati, J. A., Dollish, H. K., Smidt, S. I., Ye, T., Flores, A. J., & Bartlett, M. J. (2020). Preclinical evidence in support of repurposing sub-anesthetic ketamine as a treatment for L-DOPA-induced dyskinesia.. Experimental Neurology, 333, 113413. doi:https://doi.org/10.1016/j.expneurol.2020.113413
  More info

  Parkinson's disease (PD) is the second most common neurodegenerative disease. Pharmacotherapy with L-DOPA remains the gold-standard therapy for PD, but is often limited by the development of the common side effect of L-DOPA-induced dyskinesia (LID), which can become debilitating. The only effective treatment for disabling dyskinesia is surgical therapy (neuromodulation or lesioning), therefore effective pharmacological treatment of LID is a critical unmet need. Here, we show that sub-anesthetic doses of ketamine attenuate the development of LID in a rodent model, while also having acute anti-parkinsonian activity. The long-term anti-dyskinetic effect is mediated by brain-derived neurotrophic factor-release in the striatum, followed by activation of ERK1/2 and mTOR pathway signaling. This ultimately leads to morphological changes in dendritic spines on striatal medium spiny neurons that correlate with the behavioral effects, specifically a reduction in the density of mushroom spines, a dendritic spine phenotype that shows a high correlation with LID. These molecular and cellular changes match those occurring in hippocampus and cortex after effective sub-anesthetic ketamine treatment in preclinical models of depression, and point to common mechanisms underlying the therapeutic efficacy of ketamine for these two disorders. These preclinical mechanistic studies complement current ongoing clinical testing of sub-anesthetic ketamine for the treatment of LID by our group, and provide further evidence in support of repurposing ketamine to treat individuals with PD. Given its clinically proven therapeutic benefit for both treatment-resistant depression and several pain states, very common co-morbidities in PD, sub-anesthetic ketamine could provide multiple therapeutic benefits for PD in the future.
• Zbesko, J. C., Frye, J. B., Becktel, D. A., Gerardo, D. K., Stokes, J., Calderon, K., Nguyen, T. V., Bhattacharya, D., & Doyle, K. P. (2020). IgA natural antibodies are produced following T-cell independent B-cell activation following stroke. Brain, behavior, and immunity.
  More info

  Up to 30% of stroke patients experience cognitive decline within one year of their stroke. There are currently no FDA-approved drugs that can prevent post-stroke cognitive decline, in part due to a poor understanding of the mechanisms involved. We have previously demonstrated that a B-lymphocyte response to stroke, marked by IgA+ cells, can cause delayed cognitive dysfunction in mice and that a similar adaptive immune response occurs in the brains of some human stroke patients that suffer from vascular dementia. The stimuli which trigger B-lymphocyte activation following stroke, and their target antigens, are still unknown. Therefore, to learn more about the mechanisms by which B-lymphocytes become activated following stroke we first characterized the temporal kinetics of the B-lymphocyte, T-lymphocyte, and plasma cell (PC) response to stroke in the brain by immunohistochemistry (IHC). We discovered that B-lymphocyte, T-lymphocyte, and plasma cell infiltration within the infarct progressively increases between 2 and 7 weeks after stroke. We then compared the B-lymphocyte response to stroke in WT, MHCII, CD4, and MyD88 mice to determine if B-lymphocytes mature into IgA+ PCs through a T-lymphocyte and MyD88 dependent mechanism. Our data from a combination of IHC and flow cytometry indicate that following stroke, a population of IgA+ PCs develops independently of CD4+ helper T-lymphocytes and MyD88 signaling. Finally, sequencing of immunoglobulin genes of individual IgA+ PCs present within the infarct identified a novel population of natural antibodies with few somatic mutations in complementarity-determining regions. These findings indicate that a population of IgA+ PCs develops in the infarct following stroke by B-lymphocytes interacting with one or more thymus independent type 2 (TI-2) antigens, and that they produce IgA natural antibodies.
• Doyle, K. P., & Buckwalter, M. S. (2019). Immunological mechanisms in poststroke dementia. Current opinion in neurology.
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  To review new evidence on links between poststroke dementia and inflammation.
• Hay, M., Polt, R., Heien, M. L., Vanderah, T. W., Largen-Milnes, T. M., Rodgers, K. E., Falk, T., Bartlett, M. J., Doyle, K., & Konhilas, J. (2019). A Novel Angiotensin-(1-7)-glycosylated Mas Receptor Agonist for Treating Vascular Cognitive Impairment and Inflammation Related Memory Dysfunction. The Journal of pharmacology and experimental therapeutics.
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  A growing body of evidence indicates that decreased brain blood flow, increased reactive oxygen species production (ROS), and pro-inflammatory mechanisms accelerate the progression of neurodegenerative diseases such as vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer's disease and related dementias (ADRD). There is an urgent clinical need for safe and effective therapies for the treatment and prevention of cognitive impairment known to occur in patients with VCID and chronic inflammatory diseases such as heart failure (HF), hypertension and diabetes. This study used our mouse model of VCID/HF to test our novel glycosylated Angiotensin-(1-7) peptide, PNA5, as a therapy to treat VCID and investigate circulating inflammatory biomarkers that may be involved. We demonstrate that PNA5 has greater brain penetration as compared to the native Angiotensin-(1-7) peptide. Moreover, following treatment with 1.0/mg/kg subcutaneously for 21 days, PNA5 exhibits up to 10 days of sustained cognitive protective effects in our VCID/HF mice that last beyond the peptide half-life. PNA5 reversed object recognition impairment in VCID/HF mice and rescued spatial memory impairment. PNA5 activation of MasR results in a dose-dependent inhibition of ROS in human endothelial cells. Lastly, PNA5 treatment decreased VCID/HF induced activation of brain microglia/macrophages and inhibited circulating TNF-α, IL-7 and G-CSF serum levels while increasing the anti-inflammatory cytokine IL-10. These results suggest that PNA5 is an excellent candidate and "first-in-class" therapy for treating VCID and other inflammatory related brain diseases.
• Felix, K. M., Jaimez, I. A., Nguyen, T. V., Ma, H., Raslan, W. A., Klinger, C. N., Doyle, K. P., & Wu, H. J. (2018). Gut Microbiota Contributes to Resistance Against Pneumococcal Pneumonia in Immunodeficient Rag Mice. Frontiers in cellular and infection microbiology, 8, 118.
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  causes infection-related mortality worldwide. Immunocompromised individuals, including young children, the elderly, and those with immunodeficiency, are especially vulnerable, yet little is known regarding related pathogenesis and protection in immunocompromised hosts. Recently, strong interest has emerged in the gut microbiota's impact on lung diseases, or the "gut-lung axis." However, the mechanisms of gut microbiota protection against gut-distal lung diseases like pneumonia remain unclear. We investigated the role of the gut commensal, segmented filamentous bacteria (SFB), against pneumococcal pneumonia in immunocompetent and immunocompromised mouse models. For the latter, we chose the Rag model, with adaptive immune deficiency. Immunocompetent adaptive protection against infection is based on antibodies against pneumococcal capsular polysaccharides, prototypical T cell independent-II (TI-II) antigens. Although SFB colonization enhanced TI-II antibodies in C57BL/6 mice, our data suggest that SFB did not further protect these immunocompetent animals. Indeed, basal B cell activity in hosts without SFB is sufficient for essential protection against . However, in immunocompromised Rag mice, we demonstrate a gut-lung axis of communication, as SFB influenced lung protection by regulating innate immunity. Neutrophil resolution is crucial to recovery, since an unchecked neutrophil response causes severe tissue damage. We found no early neutrophil recruitment differences between hosts with or without SFB; however, we observed a significant drop in lung neutrophils in the resolution phase of infection, which corresponded with lower CD47 expression, a molecule that inhibits phagocytosis of apoptotic cells, in SFB-colonized Rag mice. SFB promoted a shift in lung neutrophil phenotype from inflammatory neutrophils expressing high levels of CD18 and low levels of CD62L, to pro-resolution neutrophils with low CD18 and high CD62L. Blocking CD47 in SFB(-) mice increased pro-resolution neutrophils, suggesting CD47 down-regulation may be one neutrophil-modulating mechanism SFB utilizes. The SFB-induced lung neutrophil phenotype remained similar with heat-inactivated treatment, indicating these SFB-induced changes in neutrophil phenotype during the resolution phase are not simply secondary to better bacterial clearance in SFB(+) than SFB(-) mice. Together, these data demonstrate that the gut commensal SFB may provide much-needed protection in immunocompromised hosts in part by promoting neutrophil resolution post lung infection.
• Nguyen, T. V., Hayes, M., Zbesko, J. C., Frye, J. B., Congrove, N. R., Belichenko, N. P., McKay, B. S., Longo, F. M., & Doyle, K. P. (2018). Alzheimer's associated amyloid and tau deposition co-localizes with a homeostatic myelin repair pathway in two mouse models of post-stroke mixed dementia. Acta neuropathologica communications, 6(1), 100.
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  The goal of this study was to determine the chronic impact of stroke on the manifestation of Alzheimer's disease (AD) related pathology and behavioral impairments in mice. To accomplish this goal, we used two distinct models. First, we experimentally induced ischemic stroke in aged wildtype (wt) C57BL/6 mice to determine if stroke leads to the manifestation of AD-associated pathological β-amyloid (Aβ) and tau in aged versus young adult wt mice. Second, we utilized a transgenic (Tg) mouse model of AD (hAPP-SL) to determine if stroke leads to the worsening of pre-existing AD pathology, as well as the development of pathology in brain regions not typically expressed in AD Tg mice. In the wt mice, there was delayed motor recovery and an accelerated development of cognitive deficits in aged mice compared to young adult mice following stroke. This corresponded with increased brain atrophy, increased cholinergic degeneration, and a focal increase of Aβ in areas of axonal degeneration in the ipsilateral hemisphere of the aged animals. By contrast, in the hAPP-SL mice, we found that ischemia induced aggravated behavioral deficits in conjunction with a global increase in Aβ tau, and cholinergic pathology compared to hAPP-SL mice that underwent a sham stroke procedure. With regard to a potential mechanism, in both models, we found that the stroke-induced Aβ and tau deposits co-localized with increased levels of β-secretase 1 (BACE1), along with its substrate, neuregulin 1 (NGR1) type III, both of which are proteins integral for myelin repair. Based on these findings, we propose that the chronic sequelae of stroke may be ratcheting-up a myelin repair pathway, and that the consequent increase in BACE1 could be causing an inadvertent cleavage of its alternative substrate, AβPP, resulting in greater Aβ seeding and pathogenesis.
• Taylor, S., Mehina, E., White, E., Reeson, P., Yongblah, K., Doyle, K. P., & Brown, C. E. (2018). Suppressing Interferon-γ Stimulates Microglial Responses and Repair of Microbleeds in the Diabetic Brain. The Journal of neuroscience : the official journal of the Society for Neuroscience, 38(40), 8707-8722.
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  Microcirculatory damage is a common complication for those with vascular risk factors, such as diabetes. To resolve vascular insults, the brain's immune cells (microglia) must rapidly envelop the site of injury. Currently, it is unknown whether Type 1 diabetes, a condition associated with chronic immune system dysfunction, alters microglial responses to damage and what mechanisms are responsible. Using two-photon microscopy in adult male mice, we show that microglial envelopment of laser-induced cerebral microbleeds is diminished in a hyperglycemic mouse model of Type 1 diabetes, which could not be fully rescued with chronic insulin treatment. Microglia were important for vessel repair because reduced microglial accumulation in diabetic mice or near-complete depletion in healthy controls was associated with greater secondary leakage of the damaged vessel. Broadly suppressing inflammation with dexamethasone in diabetic mice but not healthy controls, significantly enhanced microglial responses to microbleeds and attenuated secondary vessel leakage. These enhancements were associated with changes in IFN-γ signaling because dexamethasone suppressed abnormally high levels of IFN-γ protein levels in brain and blood serum of diabetic mice. Further, blocking IFN-γ in diabetic mice with neutralizing antibodies restored normal microglial chemotaxic responses and purinoceptor gene expression, as well as mitigated secondary leakage. These results suggest that abnormal IFN-γ signaling disrupts microglial function in the diabetic brain, and that immunotherapies targeting IFN-γ can stimulate microglial repair of damaged vessels. Although Type 1 diabetes is an established risk factor for vascular complications, such as microbleeds, and is known to hinder wound healing in the body, no study has examined how diabetes impacts the brain's innate immune reparative response (involving cells called microglia) to vascular injury. Here we show that microglial responses to brain microbleeds were diminished in diabetic animals, which also exacerbated secondary leakage from damaged vessels. These impairments were related to abnormally high levels of the proinflammatory cytokine IFN-γ because reducing IFN-γ with immunosuppressant drugs or blocking antibodies helped restore normal microglial responses and repair of damaged vessels. These data highlight the use of IFN-γ modulating therapeutics to enhance vascular repair in at-risk populations.
• Wu, H. J., Doyle, K., Nguyen, T., Felix, K. M., Jaimez, I., Ma, H., Raslan, W., & Klinger, C. (2018).

  Gut microbiota enhances neutrophil resolution in immunocompromised hosts to improve response to pneumococcal pneumonia.

  . The Journal of Immunology, 200(1_Supplement), 173.10-173.10. doi:10.4049/jimmunol.200.supp.173.10
• Zbesko, J. C., Nguyen, T. V., Yang, T., Frye, J. B., Hussain, O., Hayes, M., Chung, A., Day, W. A., Stepanovic, K., Krumberger, M., Mona, J., Longo, F. M., & Doyle, K. P. (2018). Glial scars are permeable to the neurotoxic environment of chronic stroke infarcts. Neurobiology of disease, 112, 63-78.
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  Following stroke, the damaged tissue undergoes liquefactive necrosis, a stage of infarct resolution that lasts for months although the exact length of time is currently unknown. One method of repair involves reactive astrocytes and microglia forming a glial scar to compartmentalize the area of liquefactive necrosis from the rest of the brain. The formation of the glial scar is a critical component of the healing response to stroke, as well as other central nervous system (CNS) injuries. The goal of this study was to evaluate the toxicity of the extracellular fluid present in areas of liquefactive necrosis and determine how effectively it is segregated from the remainder of the brain. To accomplish this goal, we used a mouse model of stroke in conjunction with an extracellular fluid toxicity assay, fluorescent and electron microscopy, immunostaining, tracer injections into the infarct, and multiplex immunoassays. We confirmed that the extracellular fluid present in areas of liquefactive necrosis following stroke is toxic to primary cortical and hippocampal neurons for at least 7 weeks following stroke, and discovered that although glial scars are robust physical and endocytic barriers, they are nevertheless permeable. We found that molecules present in the area of liquefactive necrosis can leak across the glial scar and are removed by a combination of paravascular clearance and microglial endocytosis in the adjacent tissue. Despite these mechanisms, there is delayed atrophy, cytotoxic edema, and neuron loss in regions adjacent to the infarct for weeks following stroke. These findings suggest that one mechanism of neurodegeneration following stroke is the failure of glial scars to impermeably segregate areas of liquefactive necrosis from surviving brain tissue.
• Branca, C., Ferreira, E., Nguyen, T. V., Doyle, K., Caccamo, A., & Oddo, S. (2017). Genetic reduction of Nrf2 exacerbates cognitive deficits in a mouse model of Alzheimer's disease. Human molecular genetics, 26(24), 4823-4835.
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  Aging is the major risk factor for several neurodegenerative diseases, including Alzheimer's disease (AD). However, the mechanisms by which aging contributes to neurodegeneration remain elusive. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor that regulates expression of a vast number of genes by binding to the antioxidant response element. Nrf2 levels decrease as a function of age, and reduced Nrf2 levels have been reported in postmortem human brains and animal models of AD. Nevertheless, it is still unknown whether Nrf2 plays a role in the cognitive deficits associated with AD. To address this question, we used a genetic approach to remove the Nrf2 gene from APP/PS1 mice, a widely used animal model of AD. We found that the lack of Nrf2 significantly exacerbates cognitive deficits in APP/PS1, without altering gross motor function. Specifically, we found an exacerbation of deficits in spatial learning and memory, as well as in working and associative memory. Different brain regions control these behavioral tests, indicating that the lack of Nrf2 has a global effect on brain function. The changes in cognition were linked to an increase in Aβ and interferon-gamma (IFNγ) levels, and microgliosis. The changes in IFNγ levels are noteworthy as previously published evidence indicates that IFNγ can increase microglia activation and induce Aβ production. Our data suggest a clear link between Nrf2 and AD-mediated cognitive decline and further strengthen the connection between Nrf2 and AD.
• Danilo, C. A., Constantopoulos, E., McKee, L. A., Chen, H., Regan, J. A., Lipovka, Y., Lahtinen, S., Stenman, L. K., Nguyen, T. V., Doyle, K. P., Slepian, M. J., Khalpey, Z. I., & Konhilas, J. P. (2017). Bifidobacterium animalis subsp. lactis 420 mitigates the pathological impact of myocardial infarction in the mouse. Beneficial microbes, 8(2), 257-269.
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  There is a growing appreciation that our microbial environment in the gut plays a critical role in the maintenance of health and the pathogenesis of disease. Probiotic, beneficial gut microbes, administration can directly attenuate cardiac injury and post-myocardial infarction (MI) remodelling, yet the mechanisms of cardioprotection are unknown. We hypothesised that administration of Bifidobacterium animalis subsp. lactis 420 (B420), a probiotic with known anti-inflammatory properties, to mice will mitigate the pathological impact of MI, and that anti-inflammatory T regulatory (Treg) immune cells are necessary to impart protection against MI as a result of B420 administration. Wild-type male mice were administered B420, saline or Lactobacillus salivarius 33 (Ls-33) by gavage daily for 14 or 35 days, and underwent ischemia/reperfusion (I/R). Pretreatment with B420 for 10 or 28 days attenuated cardiac injury from I/R and reduced levels of inflammatory markers. Depletion of Treg cells by administration of anti-CD25 monoclonal antibodies eliminated B420-mediated cardio-protection. Further cytokine analysis revealed a shift from a pro-inflammatory to an anti-inflammatory environment in the probiotic treated post-MI hearts compared to controls. To summarise, B420 administration mitigates the pathological impact of MI. Next, we show that Treg immune cells are necessary to mediate B420-mediated protection against MI. Finally, we identify putative cellular, epigenetic and/or post-translational mechanisms of B420-mediated protection against MI.
• Doyle, K. P., & Buckwalter, M. S. (2017). Does B lymphocyte-mediated autoimmunity contribute to post-stroke dementia?. Brain, behavior, and immunity, 64, 1-8.
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  Post-stroke cognitive decline and dementia pose a significant public health problem, with 30% of stroke survivors suffering from dementia. The reason for this high prevalence is not well understood. Pathogenic B cell responses to the damaged CNS are one possible contributing factor. B-lymphocytes and antibodies are present in and around the stroke core of some human subjects who die with stroke and dementia, and mice that develop delayed cognitive dysfunction after stroke have clusters of B-lymphocytes in the stroke lesion, and antibody infiltration in the stroked hemisphere. The ablation of B-lymphocytes prevents post-stroke cognitive impairment in mice. Multiple drugs that target B cells are FDA approved, and so if pathogenic B cell responses are occurring in a subset of stroke patients, this is potentially treatable. However, it has also been demonstrated that regulatory B cells can be beneficial in mouse models of stroke. Consequently, it is important to understand the relative contribution of B-lymphocytes to recovery versus pathogenicity, and if this balance is heterogeneous in different individuals. Therefore, the purpose of this review is to summarize the current state of knowledge with regard to the role of B-lymphocytes in the etiology of post-stroke dementia.
• Doyle, K. P. (2016). Unraveling the pathophysiology of chronic stroke lesions could yield treatments for stroke-related dementia. Future Neurology, 11(1), 1-4.
• Nguyen, T. V., Frye, J. B., Zbesko, J. C., Stepanovic, K., Hayes, M., Urzua, A., Serrano, G., Beach, T. G., & Doyle, K. P. (2016). Multiplex immunoassay characterization and species comparison of inflammation in acute and non-acute ischemic infarcts in human and mouse brain tissue. Acta neuropathologica communications, 4(1), 100.
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  This study provides a parallel characterization of the cytokine and chemokine response to stroke in the human and mouse brain at different stages of infarct resolution. The study goal was to address the hypothesis that chronic inflammation may contribute to stroke-related dementia. We used C57BL/6 and BALB/c mice to control for strain related differences in the mouse immune response. Our data indicate that in both mouse strains, and humans, there is increased granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), interleukin-12 p70 (IL-12p70), interferon gamma-induced protein-10 (IP-10), keratinocyte chemoattractant/interleukin-8 (KC/IL-8), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatory protein-1β (MIP-1β), regulated on activation, normal T cell expressed and secreted (RANTES), and Tumor necrosis factor-α (TNF-α) in the infarct core during the acute time period. Nevertheless, correlation and two-way ANOVA analyses reveal that despite this substantial overlap between species, there are still significant differences, particularly in the regulation of granulocyte colony-stimulating factor (G-CSF), which is increased in mice but not in humans. In the weeks after stroke, during the stage of liquefactive necrosis, there is significant resolution of the inflammatory response to stroke within the infarct. However, CD68+ macrophages remain present, and levels of IL-6 and MCP-1 remain chronically elevated in infarcts from both mice and humans. Furthermore, there is a chronic T cell response within the infarct in both species. This response is differentially polarized towards a T helper 1 (Th1) response in C57BL/6 mice, and a T helper 2 (Th2) response in BALB/c mice, suggesting that the chronic inflammatory response to stroke may follow a different trajectory in different patients. To control for the fact that the average age of the patients used in this study was 80 years, they were of both sexes, and many had suffered from multiple strokes, we also present findings that reveal how the chronic inflammatory response to stroke is impacted by age, sex, and multiple strokes in mice. Our data indicate that the chronic cytokine and chemokine response to stroke is not substantially altered in 18-month old compared to 3-month old C57BL/6 mice, although T cell infiltration is attenuated. We found a significant correlation in the chronic cytokine response to stroke in males and females. However, the chronic cytokine response to stroke was mildly exacerbated by a recurrent stroke in both C57BL/6 and BALB/c mice.
• Chung, A. G., Frye, J. B., Zbesko, J. C., Constantopoulos, E., Hayes, M., Figueroa, A. G., Becktel, D. A., Antony Day, W., Konhilas, J. P., McKay, B. S., Nguyen, T. V., & Doyle, K. P. (2018). Liquefaction of the Brain following Stroke Shares a Similar Molecular and Morphological Profile with Atherosclerosis and Mediates Secondary Neurodegeneration in an Osteopontin-Dependent Mechanism. eNeuro, 5(5).
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  Here we used mouse models of heart and brain ischemia to compare the inflammatory response to ischemia in the heart, a protein rich organ, to the inflammatory response to ischemia in the brain, a lipid rich organ. We report that ischemia-induced inflammation resolves between one and four weeks in the heart compared to between eight and 24 weeks in the brain. Importantly, we discovered that a second burst of inflammation occurs in the brain between four and eight weeks following ischemia, which coincided with the appearance of cholesterol crystals within the infarct. This second wave shares a similar cellular and molecular profile with atherosclerosis and is characterized by high levels of osteopontin (OPN) and matrix metalloproteinases (MMPs). In order to test the role of OPN in areas of liquefactive necrosis, OPN mice were subjected to brain ischemia. We found that at seven weeks following stroke, the expression of pro-inflammatory proteins and MMPs was profoundly reduced in the infarct of the OPN mice, although the number of cholesterol crystals was increased. OPN mice exhibited faster recovery of motor function and a higher number of neuronal nuclei (NeuN) positive cells in the peri-infarct area at seven weeks following stroke. Based on these findings we propose that the brain liquefies after stroke because phagocytic cells in the infarct are unable to efficiently clear cholesterol rich myelin debris, and that this leads to the perpetuation of an OPN-dependent inflammatory response characterized by high levels of degradative enzymes.
• Doyle, K. P., Quach, L. N., Arceuil, H. E., & Buckwalter, M. S. (2015). Ferumoxytol administration does not alter infarct volume or the inflammatory response to stroke in mice. Neuroscience letters, 584, 236-40.
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  Ferumoxytol is an ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle that is FDA-approved as an intravenous iron replacement therapy for the treatment of iron deficiency anemia in patients with chronic kidney disease. Ferumoxytol has also been used as a contrast agent for cerebral blood volume mapping by magnetic resonance imaging (MRI), which suggests it could be used for imaging hemodynamic abnormalities after stroke. However, circulating macrophages can internalize USPIOs, and recent data indicate that the accumulation of iron in macrophages can lead them to adopt the M1 pro-inflammatory phenotype. Therefore, the uptake of intravenously administered iron particles by circulating macrophages that home to the stroke core could potentially alter the inflammatory response to stroke. To test this possibility in vivo we administered a dose of ferumoxytol previously used to obtain cerebral blood volume maps in healthy humans by steady-state susceptibility contrast (SSC) MRI to BALB/cJ mice 48h after stroke and examined cytokine levels, microglial/macrophage activation, and lesion volume in the brain 5 days later. Treatment with ferumoxytol did not lead to any differences in these parameters. These data indicate that the use of ferumoxytol as a contrast agent for brain imaging after stroke does not alter the inflammatory response to stroke in mice, and is therefore unlikely to do so in human subjects.
• Doyle, K. P., Quach, L. N., Solé, M., Axtell, R. C., Nguyen, T. V., Soler-Llavina, G. J., Jurado, S., Han, J., Steinman, L., Longo, F. M., Schneider, J. A., Malenka, R. C., & Buckwalter, M. S. (2015). B-lymphocyte-mediated delayed cognitive impairment following stroke. The Journal of neuroscience : the official journal of the Society for Neuroscience, 35(5), 2133-45.
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  Each year, 10 million people worldwide survive the neurologic injury associated with a stroke. Importantly, stroke survivors have more than twice the risk of subsequently developing dementia compared with people who have never had a stroke. The link between stroke and the later development of dementia is not understood. There are reports of oligoclonal bands in the CSF of stroke patients, suggesting that in some people a B-lymphocyte response to stroke may occur in the CNS. Therefore, we tested the hypothesis that a B-lymphocyte response to stroke could contribute to the onset of dementia. We discovered that, in mouse models, activated B-lymphocytes infiltrate infarcted tissue in the weeks after stroke. B-lymphocytes undergo isotype switching, and IgM, IgG, and IgA antibodies are found in the neuropil adjacent to the lesion. Concurrently, mice develop delayed deficits in LTP and cognition. Genetic deficiency, and the pharmacologic ablation of B-lymphocytes using an anti-CD20 antibody, prevents the appearance of delayed cognitive deficits. Furthermore, immunostaining of human postmortem tissue revealed that a B-lymphocyte response to stroke also occurs in the brain of some people with stroke and dementia. These data suggest that some stroke patients may develop a B-lymphocyte response to stroke that contributes to dementia, and is potentially treatable with FDA-approved drugs that target B cells.
• Cekanaviciute, E., Fathali, N., Doyle, K. P., Williams, A. M., Han, J., & Buckwalter, M. S. (2014). Astrocytic transforming growth factor-beta signaling reduces subacute neuroinflammation after stroke in mice. Glia, 62(8), 1227-40.
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  Astrocytes limit inflammation after CNS injury, at least partially by physically containing it within an astrocytic scar at the injury border. We report here that astrocytic transforming growth factor-beta (TGFβ) signaling is a second, distinct mechanism that astrocytes utilize to limit neuroinflammation. TGFβs are anti-inflammatory and neuroprotective cytokines that are upregulated subacutely after stroke, during a clinically accessible time window. We have previously demonstrated that TGFβs signal to astrocytes, neurons and microglia in the stroke border days after stroke. To investigate whether TGFβ affects astrocyte immunoregulatory functions, we engineered "Ast-Tbr2DN" mice where TGFβ signaling is inhibited specifically in astrocytes. Despite having a similar infarct size to wildtype controls, Ast-Tbr2DN mice exhibited significantly more neuroinflammation during the subacute period after distal middle cerebral occlusion (dMCAO) stroke. The peri-infarct cortex of Ast-Tbr2DN mice contained over 60% more activated CD11b(+) monocytic cells and twice as much immunostaining for the activated microglia and macrophage marker CD68 than controls. Astrocytic scarring was not altered in Ast-Tbr2DN mice. However, Ast-Tbr2DN mice were unable to upregulate TGF-β1 and its activator thrombospondin-1 2 days after dMCAO. As a result, the normal upregulation of peri-infarct TGFβ signaling was blunted in Ast-Tbr2DN mice. In this setting of lower TGFβ signaling and excessive neuroinflammation, we observed worse motor outcomes and late infarct expansion after photothrombotic motor cortex stroke. Taken together, these data demonstrate that TGFβ signaling is a molecular mechanism by which astrocytes limit neuroinflammation, activate TGFβ in the peri-infarct cortex and preserve brain function during the subacute period after stroke.
• Doyle, K. P., & Buckwalter, M. S. (2014). A mouse model of permanent focal ischemia: distal middle cerebral artery occlusion. Methods in molecular biology (Clifton, N.J.), 1135, 103-10.
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  Here we provide a standardized protocol for performing distal middle cerebral artery occlusion (DMCAO) in mice. DMCAO is a method of inducing permanent focal ischemia that is commonly used as a rodent stroke model. To perform DMCAO a temporal craniotomy is performed, and the middle cerebral artery (MCA) is permanently ligated at a point downstream of the lenticulostriate branches. The size of the lesion produced by this surgery is strain dependent. In C57BL/6J mice, DMCAO produces an infarct predominantly restricted to the barrel region of the somatosensory cortex, but in BALB/cJ mice, DMCAO generates a much larger lesion that incorporates more of the somatosensory cortex and part of the M1 region of the motor cortex. The larger lesion produced by DMCAO in BALB/cJ mice produces a clearer sensorimotor deficit, which is useful for investigating recovery from stroke. We also describe how to modify DMCAO in C57BL/6J mice with the application of hypoxia to generate a lesion and sensorimotor deficit that are similar in size to those produced by DMCAO alone in BALB/cJ mice. This is extremely useful for stroke experiments that require a robust sensorimotor deficit in transgenic mice created on a C57BL/6J background.
• Steinman, L., Sole, M., Shah, A. T., Nguyen, T. V., Malenka, R. C., Longo, F. M., Han, J., Gilberto, S., Doyle, K. P., Buckwalter, M. S., & Axtell, R. C. (2013). Mice develop B-lymphocyte–dependent delayed cognitive deficits weeks after stroke. Alzheimers & Dementia, 9(4). doi:10.1016/j.jalz.2013.05.231
• Csiszar, A., Podlutsky, A., Podlutskaya, N., Sonntag, W. E., Merlin, S. Z., Philipp, E. E., Doyle, K., Davila, A., Recchia, F. A., Ballabh, P., Pinto, J. T., & Ungvari, Z. (2012). Testing the oxidative stress hypothesis of aging in primate fibroblasts: is there a correlation between species longevity and cellular ROS production?. The journals of gerontology. Series A, Biological sciences and medical sciences, 67(8), 841-52.
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  The present study was conducted to test predictions of the oxidative stress theory of aging assessing reactive oxygen species production and oxidative stress resistance in cultured fibroblasts from 13 primate species ranging in body size from 0.25 to 120 kg and in longevity from 20 to 90 years. We assessed both basal and stress-induced reactive oxygen species production in fibroblasts from five great apes (human, chimpanzee, bonobo, gorilla, and orangutan), four Old World monkeys (baboon, rhesus and crested black macaques, and patas monkey), three New World monkeys (common marmoset, red-bellied tamarin, and woolly monkey), and one lemur (ring-tailed lemur). Measurements of cellular MitoSox fluorescence, an indicator of mitochondrial superoxide (O2(·-)) generation, showed an inverse correlation between longevity and steady state or metabolic stress-induced mitochondrial O2(·-) production, but this correlation was lost when the effects of body mass were removed, and the data were analyzed using phylogenetically independent contrasts. Fibroblasts from longer-lived primate species also exhibited superior resistance to H(2)O(2)-induced apoptotic cell death than cells from shorter-living primates. After correction for body mass and lack of phylogenetic independence, this correlation, although still discernible, fell short of significance by regression analysis. Thus, increased longevity in this sample of primates is not causally associated with low cellular reactive oxygen species generation, but further studies are warranted to test the association between increased cellular resistance to oxidative stressor and primate longevity.
• Doyle, K. P., & Buckwalter, M. S. (2012). The double-edged sword of inflammation after stroke: what sharpens each edge?. Annals of neurology, 71(6), 729-31.
• Doyle, K. P., Fathali, N., Siddiqui, M. R., & Buckwalter, M. S. (2012). Distal hypoxic stroke: a new mouse model of stroke with high throughput, low variability and a quantifiable functional deficit. Journal of neuroscience methods, 207(1), 31-40.
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  C57BL/6J are the most commonly used strain of mouse for stroke experiments but vascular anatomy of the Circle of Willis within this strain is extremely variable and the cortex has extensive collateralization. This causes large variability in stroke models that target the middle cerebral artery proximally and confers resistance to ischemia in those that target it distally. We tested the hypothesis that by combining distal middle cerebral artery occlusion with 1h of hypoxia, we could generate a large lesion that causes a behavioral deficit with low variability. We found that this new distal hypoxic (DH) model of stroke generates a lesion with a volume of 25% of the ipsilateral hemisphere, extends to the motor cortex and causes a behavioral deficit. It also has a very clear border, exceptionally low variability, and can be performed by a single surgeon on up to 30 animals a day. Moreover, survivability is 100% in young adult animals, the model can be performed on old animals, and therapeutic intervention can reduce infarct volume. Therefore DH stroke is an excellent complement to existing stroke models and could be used for preclinical studies in C57BL/6J mice.
• Han, J., Pollak, J., Yang, T., Siddiqui, M. R., Doyle, K. P., Taravosh-Lahn, K., Cekanaviciute, E., Han, A., Goodman, J. Z., Jones, B., Jing, D., Massa, S. M., Longo, F. M., & Buckwalter, M. S. (2012). Delayed administration of a small molecule tropomyosin-related kinase B ligand promotes recovery after hypoxic-ischemic stroke. Stroke; a journal of cerebral circulation, 43(7), 1918-24.
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  Stroke is the leading cause of long-term disability in the United States, yet no drugs are available that are proven to improve recovery. Brain-derived neurotrophic factor stimulates neurogenesis and plasticity, processes that are implicated in stroke recovery. It binds to both the tropomyosin-related kinase B and p75 neurotrophin receptors. However, brain-derived neurotrophic factor is not a feasible therapeutic agent, and no small molecule exists that can reproduce its binding to both receptors. We tested the hypothesis that a small molecule (LM22A-4) that selectively targets tropomyosin-related kinase B would promote neurogenesis and functional recovery after stroke.
• Pollak, J., Doyle, K. P., Mamer, L., Shamloo, M., & Buckwalter, M. S. (2012). Stratification substantially reduces behavioral variability in the hypoxic-ischemic stroke model. Brain and behavior, 2(5), 698-706.
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  Stroke is the most common cause of long-term disability, and there are no known drug therapies to improve recovery after stroke. To understand how successful recovery occurs, dissect candidate molecular pathways, and test new therapies, there is a need for multiple distinct mouse stroke models, in which the parameters of recovery after stroke are well defined. Hypoxic-ischemic stroke is a well-established stroke model, but behavioral recovery in this model is not well described. We therefore examined a panel of behavioral tests to see whether they could be used to quantify functional recovery after hypoxic-ischemic stroke. We found that in C57BL/6J mice this stroke model produces high mortality (approximately one-third) and variable stroke sizes, but is fast and easy to perform on a large number of mice. Horizontal ladder test performance on day 1 after stroke was highly and reproducibly correlated with stroke size (P < 0.0001, R(2) = 0.7652), and allowed for functional stratification of mice into a group with >18% foot faults and 2.1-fold larger strokes. This group exhibited significant functional deficits for as long as 3 weeks on the horizontal ladder test and through the last day of testing on automated gait analysis (33 days), rotarod (30 days), and elevated body swing test (EBST) (36 days). No deficits were observed in an automated activity chamber. We conclude that stratification by horizontal ladder test performance on day 1 identifies a subset of mice in which functional recovery from hypoxic-ischemic stroke can be studied.
• Bahjat, F. R., Williams-Karnesky, R. L., Kohama, S. G., West, G. A., Doyle, K. P., Spector, M. D., Hobbs, T. R., & Stenzel-Poore, M. P. (2011). Proof of concept: pharmacological preconditioning with a Toll-like receptor agonist protects against cerebrovascular injury in a primate model of stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 31(5), 1229-42.
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  Cerebral ischemic injury is a significant portion of the burden of disease in developed countries; rates of mortality are high and the costs associated with morbidity are enormous. Recent therapeutic approaches have aimed at mitigating the extent of damage and/or promoting repair once injury has occurred. Often, patients at high risk of ischemic injury can be identified in advance and targeted for antecedent neuroprotective therapy. Agents that stimulate the innate pattern recognition receptor, Toll-like receptor 9, have been shown to induce tolerance (precondition) to ischemic brain injury in a mouse model of stroke. Here, we demonstrate for the first time that pharmacological preconditioning against cerebrovascular ischemic injury is also possible in a nonhuman primate model of stroke in the rhesus macaque. The model of stroke used is a minimally invasive transient vascular occlusion, resulting in brain damage that is primarily localized to the cortex and as such, represents a model with substantial clinical relevance. Finally, K-type (also referred to as B-type) cytosine-guanine-rich DNA oligonucleotides, the class of agents employed in this study, are currently in use in human clinical trials, underscoring the feasibility of this treatment in patients at risk of cerebral ischemia.
• Doyle, K. P., Cekanaviciute, E., Mamer, L. E., & Buckwalter, M. S. (2010). TGFβ signaling in the brain increases with aging and signals to astrocytes and innate immune cells in the weeks after stroke. Journal of neuroinflammation, 7, 62.
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  TGFβ is both neuroprotective and a key immune system modulator and is likely to be an important target for future stroke therapy. The precise function of increased TGF-β1 after stroke is unknown and its pleiotropic nature means that it may convey a neuroprotective signal, orchestrate glial scarring or function as an important immune system regulator. We therefore investigated the time course and cell-specificity of TGFβ signaling after stroke, and whether its signaling pattern is altered by gender and aging.
• West, G. A., Golshani, K. J., Doyle, K. P., Lessov, N. S., Hobbs, T. R., Kohama, S. G., Pike, M. M., Kroenke, C. D., Grafe, M. R., Spector, M. D., Tobar, E. T., Simon, R. P., & Stenzel-Poore, M. P. (2009). A new model of cortical stroke in the rhesus macaque. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 29(6), 1175-86.
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  Primate models are essential tools for translational research in stroke but are reportedly inconsistent in their ability to produce cortical infarcts of reproducible size. Here, we report a new stroke model using a transorbital, reversible, two-vessel occlusion approach in male rhesus macaques that produces consistent and reproducible cortical infarcts. The right middle cerebral artery (distal to the orbitofrontal branch) and both anterior cerebral arteries were occluded with vascular clips. Bilateral occlusion of the anterior cerebral artery was critical for reducing collateral flow to the ipsilateral cortex. Reversible ischemia was induced for 45, 60, or 90 mins (n=2/timepoint) and infarct volume and neurologic outcome were evaluated. The infarcts were located predominantly in the cortex and increased in size with extended duration of ischemia determined by T(2)-weighted magnetic resonance imaging . Infarct volume measured by 2,3,5-triphenyl tetrazolium chloride and cresyl violet staining corroborated magnetic resonance imaging results. Neurologic deficit scores worsened gradually with longer occlusion times. A subset of animals (n=5) underwent 60 mins of ischemia resulting in consistent infarct volumes primarily located to the cortex that correlated well with neurologic deficit scores. This approach offers promise for evaluating therapeutic interventions in stroke.
• Doyle, K. P., Yang, T., Lessov, N. S., Ciesielski, T. M., Stevens, S. L., Simon, R. P., King, J. S., & Stenzel-Poore, M. P. (2008). Nasal administration of osteopontin peptide mimetics confers neuroprotection in stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 28(6), 1235-48.
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  Osteopontin (OPN), a large secreted glycoprotein with an arginine, glycine, aspartate (RGD) motif, can bind and signal through cellular integrin receptors. We have shown previously that OPN enhances neuronal survival in the setting of ischemia. Here, we sought to increase the neuroprotective potency of OPN and improve the method of delivery with the goal of identifying a treatment for stroke in humans. We show that thrombin cleavage of OPN improves its ability to ligate integrin receptors and its neuroprotective capacity in models of ischemia. Thrombin-cleaved OPN is a twofold more effective neuroprotectant than the untreated molecule. We also tested whether OPN could be administered intranasally and found that it is efficiently targeted to the brain via intranasal delivery. Furthermore, intranasal administration of thrombin-treated OPN confers protection against ischemic brain injury. Osteopontin mimetics based on the peptide sequences located either N or C terminal to the thrombin cleavage site were generated and tested in models of ischemia. Treatment with successively shorter N-terminal peptides and a phosphorylated C-terminal peptide provided significant neuroprotection against ischemic injury. These findings show that OPN mimetics offer promise for development into new drugs for the treatment of stroke.
• Stenzel-poore, M. P., Simon, R. P., & Doyle, K. P. (2008). Mechanisms of ischemic brain damage.. Neuropharmacology, 55(3), 310-8. doi:10.1016/j.neuropharm.2008.01.005
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  In the United States stroke is the third leading cause of death and the leading cause of disability. Brain injury following stroke results from the complex interplay of multiple pathways including excitotoxicity, acidotoxicity, ionic imbalance, peri-infarct depolarization, oxidative and nitrative stress, inflammation and apoptosis. There are very few treatments for stroke and the development of new treatments requires a comprehensive understanding of the diverse mechanisms of ischemic brain damage that are responsible for neuronal death. Here, we discuss the underlying pathophysiology of this devastating disease and reveal the intertwined pathways that are the target of therapeutic intervention.
• Doyle, K. P., Suchland, K. L., Ciesielski, T. M., Lessov, N. S., Grandy, D. K., Scanlan, T. S., & Stenzel-Poore, M. P. (2007). Novel thyroxine derivatives, thyronamine and 3-iodothyronamine, induce transient hypothermia and marked neuroprotection against stroke injury. Stroke; a journal of cerebral circulation, 38(9), 2569-76.
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  Mild hypothermia confers profound neuroprotection in ischemia. We recently discovered 2 natural derivatives of thyroxine, 3-iodothyronamine (T(1)AM) and thyronamine (T(0)AM), that when administered to rodents lower body temperature for several hours without induction of a compensatory homeostatic response. We tested whether T(1)AM- and T(0)AM-induced hypothermia protects against brain injury from experimental stroke.
• Meller, R., Stevens, S. L., Minami, M., Cameron, J. A., King, S., Rosenzweig, H., Doyle, K., Lessov, N. S., Simon, R. P., & Stenzel-Poore, M. P. (2005). Neuroprotection by osteopontin in stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 25(2), 217-25.
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  Osteopontin (OPN) is a secreted extracellular phosphoprotein involved in diverse biologic functions, including inflammation, cell migration, and antiapoptotic processes. Here we investigate the neuroprotective potential of OPN to reduce cell death using both in vitro and in vivo models of ischemia. We show that incubation of cortical neuron cultures with OPN protects against cell death from oxygen and glucose deprivation. The effect of OPN depends on the Arg-Gly-Asp (RGD)-containing motif as the protective effect of OPN in vitro was blocked by an RGD-containing hexapeptide, which prevents integrin receptors binding to their ligands. Osteopontin treatment of cortical neuron cultures caused an increase in Akt and p42/p44 MAPK phosphorylation, which is consistent with OPN-inducing neuroprotection via the activation of these protein kinases. Indeed, the protective effect of OPN was reduced by inhibiting the activation of Akt and p42/p44 MAPK using LY294002 and U0126, respectively. The protective effect of OPN was also blocked by the protein synthesis inhibitor cycloheximide, suggesting that the neuroprotective effect of OPN required new protein synthesis. Finally, intracerebral ventricular administration of OPN caused a marked reduction in infarct size after transient middle cerebral artery occlusion in a murine stroke model. These data suggest that OPN is a potent neuroprotectant against ischemic injury.
• Doyle, K. P., Simon, R. P., Snyder, A., & Stenzel-Poore, M. P. (2003). Working with GFP in the brain. BioTechniques, 34(3), 492-4.

Presentations

• Doyle, K. (2023). Targeting foam cells to improve stroke recovery. Seminar Presentation at the Miami Project to Cure Paralysis/Miller School of Medicine, University of Miami. Miami, Florida: Miami Project to Cure Paralysis/Miller School of Medicine, University of Miami.
• Doyle, K. (2023, April).

  Unlocking the Future of Stroke Recovery: Target Identification and Translational Research Best Practices

  
  . Seminar Presentation to NeuroTrauma Biosciences, Alpharetta, GA. Zoom.
• Doyle, K. (2023, March).

  Targeting foam cells to improve stroke recovery

  . Center for Cerebrovascular Research Seminar, UCSFCenter for Cerebrovascular Research, UCSF.
• Doyle, K. (2023, May).

  Translational Stroke Workshop

  . Workshop/Seminar, Stanford University. Stanford, CA: Stanford University.
• Doyle, K. P. (2023, February).

  Chronic inflammation post-stroke and its relation to dementia.

  . International Stroke Conference. Dallas, Texas: American Heart Association.
• Doyle, K. (2022, May). Understanding Neurodegeneration after stroke, plasma biomarkers, and the role of B-lymphocytes. University of Edinburgh. Edinburgh, Scotland: Leducq Foundation, Stroke IMPaCT Network.
• Doyle, K. (2022, October). Managing the fire within: Treating the chronic inflammatory response to stroke to improve recovery. University of Kentucky. Lexington, Kentucky: University of Kentucky.
• Doyle, K. (2021, January). The Chronic Consequences of Stroke. Neuroscience Grand Rounds. University of Arizona: Neurology Department.
• Doyle, K. (2020, January). Is there a link between the inflammatory response to stroke and post-stroke dementia?. Arizona Alzheimer's Consortium 18th Annual Retreat. Lake Havasu, Arizona: Arizona Alzheimer's Consortium.
• Doyle, K. (2020, June). Neuroimmunology and Neuroinflammation Lecture. Dine College Summer Lecture Series. Dine College via Zoom: NINIDS Neuroscience Development for Advancing the Careers of a Diverse Research Workforce.
• Doyle, K. (2020, June). The Chronic Consequences of Stroke. Invited Seminar (Tulane University School of Medicine)Tulane University School of Medicine.
• Doyle, K. (2019, July/Summer). The pathophysiology of chronic stroke infarcts: What happens after brain tissue dies?. Invited Seminar (University of Manchester). Manchester, UK: University of Manchester.
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  Relatively little is known about the long-term consequences of stroke on brain aging and the development of Alzheimer’s disease (AD). In this talk, data will be presented that indicates that the inflammatory response to ischemia follows a different trajectory compared to the inflammatory response to ischemia in other organs and tissues, and that this may be due to the high cholesterol content of myelin. Data will then be presented that shines a light on how the chronic inflammatory response to stroke, chronic blood brain barrier impairment, impaired paravascular clearance, and axonal degeneration impact the development of AD pathology in stroke survivors.
• Doyle, K. (2019, May/Spring). Inflammation and inflammasome activation following stroke. GLIA Scientific Meeting. Childrens’ Hospital of Philadelphia, Philadelphia, PA: Childrens’ Hospital of Philadelphia.
• Doyle, K. (2019, October/Fall). The Pathophysiology of Chronic Stroke: What Happens After Brain Tissue Dies?. xMAP Connect. San Diego, CA: Luminex Corportaion.
• Doyle, K. (2018, April). Does crystalline cholesterol derived from myelin debris cause liquefaction of the brain following stroke?. Neuroscience DataBlitz. Museum of Contemporary Art, Tucson, AZ: University of Arizona College of Medicine.
• Doyle, K. (2018, April). The Chronic Sequelae of Stroke. Neurology Department Faculty Meeting. University of Arizona College of Medicine.
• Doyle, K. (2018, November). Liquefaction of the brain following stroke shares a similar molecular and morphological profile with atherosclerosis. Society for Neuroscience, Satellite symposium on neuroinflammation, degeneration, and disease. San Diego, California: Society for Neuroscience.
• Doyle, K. (2018, September). The Pathophysiology of Chronic Stroke Infarcts: What Happens After Brain Tissue Dies?. Invited Seminar, Department of Neurology, Stanford, California. Stanford University, California.
• Falk, T., Falk, T., Sherman, S. J., Sherman, S. J., Heien, M. L., Heien, M. L., Doyle, K., Doyle, K., Farrell, D. C., Farrell, D. C., Smidt, S. I., Smidt, S. I., Cristiani, S., Cristiani, S., Bartlett, M. J., & Bartlett, M. J. (2018, Fall). VEGF-B overexpression in PINK1 gene knock out rats improves motor function: Is this effect due to neuroprotection or to functional improvement of dopaminergic neurons?. Society for Neuroscience.
• Falk, T., Sherman, S. J., Heien, M. L., Doyle, K., Farrell, D. C., Smidt, S. I., Cristiani, S., & Bartlett, M. J. (2018, Fall). VEGF-B overexpression in PINK1 gene knock out rats improves motor function: Is this effect due to neuroprotection or to functional improvement of dopaminergic neurons?. Society for Neuroscience Meeting.
• Doyle, K. (2017, September). How long does it take for the brain to heal following stroke?. Department of Animal Care Seminar Series. Department of Animal Care, University of Arizona, Tucson, Arizona: Department of Animal Care.
• Doyle, K. (2017, September). How long does it take for the brain to heal following stroke?. Department of Pharmacology Seminar Series. University of Arizona, Tucson, Arizona: Department of Pharmacology.
• Doyle, K. (2015, March). B-Lymphocyte Mediated Delayed Cognitive Impairment following Stroke. Frontiers in Immunobiology & Immunopathogenesis Symposium. University of Arizona, Tucson, AZ: Department of Immunobiology.
• Doyle, K., Sole, M., Nguyen, T., Quach, L., Soler-Llavina, G., Jurado, S., Han, J., Steinman, L., Longo, F., Malenka, R., & Buckwalter, M. (2015, June). Mice develop B-lymphocyte dependent delayed cognitive deficits weeks after stroke. 2015 Alzheimer's Disease Congress. London, United Kingdom: Life Science Events.
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  Each year, ten million people worldwide survive the neurologic injury associated with a stroke. Importantly, stroke survivors have almost twice the risk of subsequently developing dementia. The link between stroke and the later development of dementia is not understood. For example, it is unknown how long inflammation persists after stroke. In this study, we tested the hypothesis that chronic inflammation in the brain contributes to the onset of dementia. We utilized a mouse model of stroke (DH, or Distal Hypoxic stroke) that generates a large cortical lesion and no immediate cognitive deficits. We characterized inflammatory responses (n=7-10 mice per group), long-term potentiation (n=6-9), and cognition (n=10) before and after stroke in wildtype C57BL/6J mice. We found that the inflammatory response to stroke persisted for months and was characterized by the presence of macrophages and B and T lymphocytes in the stroke lesion. Moreover, immunoglobulin was observed in the lesion and surrounding tissue, and there was prolonged inflammation in the area of Wallerian degeneration. We found that immunoglobulin accumulated in the hippocampus and mice developed a delayed deficit in hippocampal long-term potentiation and delayed short-term memory deficits one to seven weeks after stroke. Notably, delayed cognitive impairment was mitigated in C57BL/6J-muMT mice that lacked mature B lymphocytes and in wild-type mice treated with an anti-CD20 B cell-depleting antibody after stroke. Thus, the inflammatory response to stroke persisted for months in mice, and such mice developed a B lymphocyte-mediated autoimmune response that contributed to delayed cognitive dysfunction.
• Nguyen, T., Doyle, K., Nguyen, T., & Doyle, K. (2015, October). Advancing neuroimmunology: Untangling biomarkers in the brain. Science.
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  Unraveling how the immune and nervous systems interact has helped advance our understanding of many mechanisms within the brain, from development to diseases. Inflammation and immune responses in the nervous system have been linked to a variety of brain disorders, including neurodegeneration, traumatic brain injury, stroke, and cognitive decline. Our knowledge of immune-related pathologies in the brain and the ability to identify biomarkers for disease and inflammation are key for forming meaningful mechanistic conclusions. In this webinar, our expert panel will share their experience researching neuroinflammation and neurodegeneration-related biomarkers, and will discuss methodologies for tracking pathological changes and quantifying specific inflammatory mediators, such as T cells, cytokines, and antibodies. During this webinar, the speakers will discuss:Best practices and the challenges of studying biomarkers in the brainTechniques for measuring immune-related moleculesProtocols for analyzing human samples as well as rodent models

Poster Presentations

• Becktel, D., Zbesko, J., Frye, J., Chung, A., Calderon, K., Nguyen, T., & Doyle, K. (2022, June). Targeting foam cells to attenuate the chronic inflammatory response to ischemic stroke. Brain & Brain PET. Glasgow, United Kingdom: International Society for Cerebral Blood Flow and Metabolism (ISCBFM).
• Loppi, S., Tavera-Garcia, M., Becktel, D., Maiyo, B., Johnson, K., Schnellmann, R. G., & Doyle, K. (2022, June). Brain metabolism is altered for at least 12 weeks following stroke. Brain & Brain PET. Glasgow, United Kingdom: International Society for Cerebral Blood Flow and Metabolism (ISCBFM).
• Loppi, S., Tavera-Garcia, M., Becktel, D., Maiyo, B., Johnson, K., Schnellmann, R. G., & Doyle, K. (2022, October). Increased fatty acid metabolism and decreased glycolysis are hallmarks of metabolic reprogramming in myeloid cells in the brain during recovery from experimental stroke. International Conference on Brain Energy Metabolism. Santa Fe, New Mexico: International Conference on Brain Energy Metabolism.
• Hay, M., Doyle, K., Ossanna, N., Falk, T., Bartlett, M., Polt, R. L., Mansour, H. M., Konhilas, J. P., Hoyer-Kimura, C., Hay, M., Doyle, K., Ossanna, N., Falk, T., Bartlett, M., Polt, R. L., Mansour, H. M., Konhilas, J. P., & Hoyer-Kimura, C. (2021, March/Spring). Novel Therapeutic and Inflammatory Biomarkers for Vascular Contributions to Cognitive Impairment and Dementia.. AD/PD 2021 15th International Conference on Alzheimer’s and Parkinson’s Diseases Virtual ConferenceAD/PD.
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  Oral e-Poster presentation
• Becktel, D. A., Zbesko, J. C., Frye, J. B., Chung, A., Calderon, K., Nguyen, T., & Doyle, K. (2019, June/Summer). Cyclodextrin treatment substantially attenuates the chronic inflammatory response to ischemic stroke in mice. Neural Environment in Disease: Glial Responses and Neuroinflammation. Keystone, Colorado: Keystone Symposia.
  More info

  Ischemic stroke is a leading cause of physical disability and dementia for Americans, and a stroke occurs every 40 seconds in the United States. In response to ischemia, the brain degenerates by the process of liquefactive necrosis. Liquefactive necrosis is a chronic inflammatory response that occurs for unknown reasons, exacerbates post-stroke injury, and persists for months following stroke. Our published data indicate that liquefactive necrosis shares a similar molecular and morphological profile with atherosclerosis. We hypothesize that following stroke, cholesterol derived from myelin debris overwhelms the processing capability of phagocytic cells in the brain, leading to the formation of lipid-laden foam cells, generation of intracellular cholesterol crystals, secretion of pro-inflammatory cytokines, and production of degradative enzymes. Furthermore, we propose that this sustained inflammatory response, coupled with concurrent cell death, causes post-stroke secondary neurodegeneration and impairs functional recovery. Therefore, the goal of this research was to determine whether cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (CD), an FDA-approved drug that can solubilize and entrap lipophilic substances, prevents the accumulation of cholesterol within foamy macrophages and initiates anti-inflammatory mechanisms after ischemic stroke. To accomplish this goal, we utilized a mouse model of chronic stroke in conjunction with immunostaining, multiplex immunoassays, RNA sequencing, lipidomics, and behavioral analyses. We discovered that treatment with CD for six weeks following stroke significantly reduced the area of liquefactive necrosis, decreased T-lymphocyte, B-lymphocyte, and plasma cell accumulation in the infarct, and attenuated the expression of multiple degradative enzymes. We also found that cyclodextrin-treated mice had improved cognitive function at 7 weeks following stroke compared to vehicle-treated mice. These findings suggest that CD treatment may help the immune system process lipid debris following stroke, and may be a potential method of enhancing stroke recovery and preventing the development of post-stroke dementia.
• Becktel, D. A., Zbesko, J. C., Frye, J. B., Chung, A., Calderon, K., Nguyen, T., & Doyle, K. (2019, June/Summer). Cyclodextrin treatment substantially attenuates the chronic inflammatory response to ischemic stroke in mice. Neurodegenerative Diseases: New Insights and Therapeutic Opportunities. Keystone, Colorado: Keystone Symposia.
  More info

  Ischemic stroke is a leading cause of physical disability and dementia for Americans, and a stroke occurs every 40 seconds in the United States. In response to ischemia, the brain degenerates by the process of liquefactive necrosis. Liquefactive necrosis is a chronic inflammatory response that occurs for unknown reasons, exacerbates post-stroke injury, and persists for months following stroke. Our published data indicate that liquefactive necrosis shares a similar molecular and morphological profile with atherosclerosis. We hypothesize that following stroke, cholesterol derived from myelin debris overwhelms the processing capability of phagocytic cells in the brain, leading to the formation of lipid-laden foam cells, generation of intracellular cholesterol crystals, secretion of pro-inflammatory cytokines, and production of degradative enzymes. Furthermore, we propose that this sustained inflammatory response, coupled with concurrent cell death, causes post-stroke secondary neurodegeneration and impairs functional recovery. Therefore, the goal of this research was to determine whether cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (CD), an FDA-approved drug that can solubilize and entrap lipophilic substances, prevents the accumulation of cholesterol within foamy macrophages and initiates anti-inflammatory mechanisms after ischemic stroke. To accomplish this goal, we utilized a mouse model of chronic stroke in conjunction with immunostaining, multiplex immunoassays, RNA sequencing, lipidomics, and behavioral analyses. We discovered that treatment with CD for six weeks following stroke significantly reduced the area of liquefactive necrosis, decreased T-lymphocyte, B-lymphocyte, and plasma cell accumulation in the infarct, and attenuated the expression of multiple degradative enzymes. We also found that cyclodextrin-treated mice had improved cognitive function at 7 weeks following stroke compared to vehicle-treated mice. These findings suggest that CD treatment may help the immune system process lipid debris following stroke, and may be a potential method of enhancing stroke recovery and preventing the development of post-stroke dementia.
• Becktel, D. A., Zbesko, J. C., Frye, J. B., Chung, A., Nguyen, T., & Doyle, K. (2019, March/Spring). Cyclodextrin treatment substantially reduces the chronic inflammatory response following ischemic stroke in adult and aged mice. Frontiers in Immunobiology & Immunopathogenesis Symposium 2019. University of Arizona, Tucson: Department of Immunobiology, University of Arizona.
  More info

  Ischemic stroke is a leading cause of death for Americans, as evidenced by a stroke occurrence every 40 seconds in the United States. In response to ischemia, the brain degenerates by the process of liquefactive necrosis. Liquefactive necrosis is a chronic inflammatory response that occurs for unknown reasons, exacerbates post-stroke injury, and persists for months following stroke. Our published data indicate that liquefactive necrosis shares a similar molecular and morphological profile with atherosclerosis. It is known that an elevated level of cholesterol causes atherosclerosis and leads to distinctive atherosclerotic features such as dysregulated lipid metabolism within macrophages, activation of Toll-like receptor (TLR) signaling, and production of high concentrations of degradative enzymes. We hypothesize that following stroke, cholesterol derived from myelin debris overwhelms the processing capability of phagocytic cells in the brain, leading to the formation of lipid-laden foam cells, generation of intracellular cholesterol crystals, secretion of pro-inflammatory cytokines, and production of degradative enzymes. Furthermore, we propose that this sustained inflammatory response coupled with concurrent cell death causes post-stroke secondary neurodegeneration and impairs functional recovery.Therefore, the goal of this research was to investigate if cyclodextrin, an FDA-approved drug shown to increase cholesterol solubility, prevents the accumulation of cholesterol within foamy macrophages and initiates anti-inflammatory mechanisms after ischemic stroke. To accomplish this goal we utilized a mouse model of chronic stroke in conjunction with immunostaining, multiplex immunoassay, RNA sequencing, lipidomics, and behavioral analyses. We also addressed age as a biological variable by performing these studies in both adult and aged mice. We discovered that treatment with cyclodextrin following stroke significantly reduced the area of liquefactive necrosis, decreased T-lymphocyte, B-lymphocyte, and plasma cell accumulation in the infarct, and attenuated the expression of several matrix metalloproteinase degradative enzymes. We also found that cyclodextrin-treated mice exhibited improved cognitive function at 7 weeks following stroke compared to vehicle treated mice. In conclusion, cyclodextrin, a compound developed to solubilize and entrap lipophilic substances improves recovery following stroke in adult and aged mice. For these reasons, cyclodextrin is a promising therapy for the treatment of post-stroke chronic inflammation and secondary neurodegeneration.
• Calderon, K., Chung, A., Frye, J. B., Becktel, D. A., Zbesko, J. C., Hayes, M. I., Nguyen, T., & Doyle, K. (2019, January/Winter). Cyclodextrin treatment substantially alters the inflammatory response to stroke. 30th Annual Undergraduate Biology Research Program Conference. University of Arizona: University of Arizona Undergraduate Biology Research Program.
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  The goal of this study is to investigate cyclodextrin as a pharmacological approach for improving recovery from ischemic stroke. Approximately 800,000 people suffer a stroke each year in the United States, many of whom later develop long-term disabilities, such as post-stroke dementia and Alzheimer’s disease. Ischemic stroke accounts for around 87% of all stroke cases and occurs when an artery that supplies blood to the brain is blocked or restricted by a thrombosis, embolism, or systemic hypoperfusion. Reduced blood supply results in insufficient levels of oxygen and nutrients to maintain cellular homeostasis, thereby causing excitotoxicity, oxidative stress, apoptosis, and inflammation in the surrounding tissue. In response to this damage, brain tissue undergoes liquefactive necrosis, a process through which proteinases and other enzymes degrade tissue and transform it into a viscous mass. In prior studies, we demonstrated that stroke infarcts in the stage of liquefactive necrosis are sites of chronic inflammation, which we suspect prolongs the healing process and contributes to secondary neurodegeneration, the probable cause of long-term disabilities in stroke victims. In a mouse model of stroke, we previously found a second wave of inflammation between 4 and 8 weeks post-stroke, as demonstrated by elevated cytokine and chemokine expression and which coincided with an accumulation of cholesterol crystals within the infarct. Cholesterol crystals form within the lysosomes of a foamy macrophage when cholesterol ingested by the foamy macrophage from myelin debris exceeds the efflux capacity of the cell. The formation of cholesterol crystals destabilizes the lysosome, which produces a signal that activates the NLRP3 inflammasome. Activation of the NLRP3 inflammasome leads to the secretion of IL-1, one cytokine that prolongs inflammation. Because of this association between cholesterol crystal accumulation and chronic inflammation, we hypothesize that stroke recovery in mice can be improved by preventing cholesterol overloading within phagocytic cells through treatment with cyclodextrin, an FDA-approved drug shown to increase cholesterol solubility, promote removal of cholesterol from foamy macrophages, and initiate anti-inflammatory mechanisms.
• Zbesko, J. C., Frye, J. B., Becktel, D. A., Nguyen, T., & Doyle, K. (2019, June/Summer). The role of B-lymphocytes in post-stroke cognitive decline. Neural Environment in Disease: Glial Responses and Neuroinflammation. Keystone, Colorado: Keystone Symposia.
  More info

  Stroke is one of the leading causes of adult disability in the United States, costing an estimated 33 billion dollars annually. Up to 30% of stroke patients experience cognitive decline in the first year after their stroke. There are no FDA approved drugs that can prevent post-stroke cognitive decline, a sub-category of vascular dementia, in part due to the mechanism(s) being poorly understood. Recently our lab published the first paper to show that an adaptive B-lymphocyte response to stroke can cause delayed cognitive dysfunction in mice, and that a similar adaptive immune response occurs in the brains of some human stroke patients that suffer from vascular dementia. This is the first evidence that in some people, post-stroke dementia may be caused by a B-lymphocyte response to the stroke infarct. However, the precise mechanism by which B-lymphocytes become activated following stroke, and their target antigen(s) is still unknown. Our proposed mechanism is that B-lymphocyte mediated cognitive impairment following stroke is T-cell independent and results in the production of pathogenic central nervous system (CNS) antibodies that bind to lipid antigens. To test this hypothesis, we used the distal middle cerebral artery occlusion plus hypoxia model of stroke (DH stoke) in CD4 knockout and MHCII knockout mice, and probed protein and lipid arrays using antibodies from stroked and unstroked mice. Our results indicate that stroke elicits an IgA antibody response that is directed against oxidized low-density lipoprotein, and that it is T cell independent. Ongoing experiments are being performed in mice that lack mature B-lymphocytes (muMT mice) to determine if these antibodies both aide in the clearance of lipid rich myelin debris within the infarct, while antagonistically causing secondary damage to surviving brain tissue. The completion of these experiments will lead to a greater understanding of the mechanisms by which B-lymphocytes contribute to the development of cognitive decline in the weeks and months after stroke, which is necessary for the development of treatments that target B-lymphocyte responses to stroke.
• Zbesko, J. C., Frye, J. B., Becktel, D. A., Nguyen, T., & Doyle, K. (2019, June/Summer). The role of B-lymphocytes in post-stroke cognitive decline. Neurodegenerative Diseases: New Insights and Therapeutic Opportunities. Keystone, Colorado: Keystone Symposia.
  More info

  Stroke is one of the leading causes of adult disability in the United States, costing an estimated 33 billion dollars annually. Up to 30% of stroke patients experience cognitive decline in the first year after their stroke. There are no FDA approved drugs that can prevent post-stroke cognitive decline, a sub-category of vascular dementia, in part due to the mechanism(s) being poorly understood. Recently our lab published the first paper to show that an adaptive B-lymphocyte response to stroke can cause delayed cognitive dysfunction in mice, and that a similar adaptive immune response occurs in the brains of some human stroke patients that suffer from vascular dementia. This is the first evidence that in some people, post-stroke dementia may be caused by a B-lymphocyte response to the stroke infarct. However, the precise mechanism by which B-lymphocytes become activated following stroke, and their target antigen(s) is still unknown. Our proposed mechanism is that B-lymphocyte mediated cognitive impairment following stroke is T-cell independent and results in the production of pathogenic central nervous system (CNS) antibodies that bind to lipid antigens. To test this hypothesis, we used the distal middle cerebral artery occlusion plus hypoxia model of stroke (DH stoke) in CD4 knockout and MHCII knockout mice, and probed protein and lipid arrays using antibodies from stroked and unstroked mice. Our results indicate that stroke elicits an IgA antibody response that is directed against oxidized low-density lipoprotein, and that it is T cell independent. Ongoing experiments are being performed in mice that lack mature B-lymphocytes (muMT mice) to determine if these antibodies both aide in the clearance of lipid rich myelin debris within the infarct, while antagonistically causing secondary damage to surviving brain tissue. The completion of these experiments will lead to a greater understanding of the mechanisms by which B-lymphocytes contribute to the development of cognitive decline in the weeks and months after stroke, which is necessary for the development of treatments that target B-lymphocyte responses to stroke.
• Chung, A., Frye, J. B., Zbesko, J. C., Constantopoulos, E., Hayes, M. I., Figueroa, A. G., Day, W. A., Konhilas, J. P., Mckay, B. S., Nguyen, T., & Doyle, K. (2018, June). Liquefaction of the brain following stroke shares a similar molecular and morphological profile with atherosclerosis and mediates secondary neurodegeneration in an osteopontin dependent mechanism. Keystone Symposia Conference: New Frontiers in Neuroinflammation: What Happens when CNS and Periphery Meet?. Keystone, Colorado: Keystone Symposia Conference.
• Chung, A., Frye, J. B., Zbesko, J. C., Constantopoulos, E., Hayes, M. I., Figueroa, A. G., Day, W. A., Konhilas, J. P., Mckay, B. S., Nguyen, T., & Doyle, K. (2018, November). Liquefaction of the brain following stroke shares characteristics with atherosclerosis. Society for Neuroscience Conference. San Diego, California: Society for Neuroscience.
• Falk, T., Sherman, S. J., Heien, M. L., Doyle, K., Parent, K. L., Farrell, D. C., Muller, D. C., Silashki, B. D., Cristiani, S., & Bartlett, M. J. (2018, Summer). AAV2/1-hVEGF-B overexpression improves motor outcomes in PINK1 gene knockout rat: An insight into potential mechanisms.. 2nd Pan American Parkinson's Disease and Movement Disorders Congress.
• Falk, T., Sherman, S. J., Steece-Collier, K., Doyle, K., Dollish, H. K., Flores, A. J., & Bartlett, M. J. (2018, Summer). Sub-anesthetic ketamine prevents levodopa-induced dyskinesia and improves motor function in a 6-OHDA rat model of Parkinson’s disease. 2nd Pan American Parkinson's Disease and Movement Disorders Congress.
• Nguyen, T., Hayes, M. I., Frye, J. B., Zbesko, J. C., Belichenko, N. P., Longo, F. M., & Doyle, K. (2018, November). Alzheimer’s disease pathology is a chronic sequela of ischemic stroke in two mouse models of mixed dementia. Society for Neuroscience Conference. San Diego, California: Society for Neuroscience.
• Sherman, S. J., Steece-Collier, K., Heien, M. L., Doyle, K., Stancati, J. A., Dollish, H. K., Flores, A. J., Bartlett, M. J., & Falk, T. (2018, Fall). Neuroplastic effects in the striatum contribute to the suppression of L-DOPA-induced dyskinesia by sub-anesthetic ketamine. Society for Neuroscience Meeting.
• Sherman, S. J., Steece-Collier, K., Heien, M. L., Doyle, K., Stancati, J. A., Dollish, H. K., Flores, A. J., Bartlett, M. J., & Falk, T. (2018, Fall). Neuroplastic effects in the striatum contribute to the suppression of L-DOPA-induced dyskinesia by sub-anesthetic ketamine. Society for Neuroscience.
• Zbesko, J. C., Nguyen, T., Yang, T., Frye, J., Hussein, O., Hayes, M. I., Chung, A., Day, W. A., Stepanovic, K., Krumberger, M., Mona, J., Longo, F. M., & Doyle, K. (2018, March). Glial scars are permeable to the neurotoxic environment of chronic stroke infarcts. American Society for Neurochemistry. Riverside, California: American Society for Neurochemistry.
• Bartlett, M. J., Falk, T., Flores, A. J., Sherman, S. J., Ye, T., Cowen, S. L., Dollish, H. K., Doyle, K., Dollish, H. K., Doyle, K., Ye, T., Cowen, S. L., Flores, A. J., Sherman, S. J., Bartlett, M. J., & Falk, T. (2017, Summer). Mechanisms of sub-anesthetic ketamine infusions to reduce levodopa-induced dyskinesia: effects on striatal mTOR signaling and beta band oscillations in striatum and motor cortex.. International Parkinson and Movement Disorders Society Abstracts.
• Bartlett, M. J., Flores, A. J., Dollish, H. K., Doyle, K., Pottinger, A., Morrison, H. W., Sherman, S. J., & Falk, T. (2017, December). Neuroplastic effects contribute to the suppression of L-DOPA-induced dyskineasis by sub-anesthetic ketamine. Neurobiology, Aging, Dementias and Movement Disorders Division Meeting. Scottsdale, Arizona: Arizona Wellbeing Commons Division.
• Falk, T., Sherman, S. J., Cowen, S. L., Doyle, K., Dollish, H. K., Ye, T., Flores, A. J., & Bartlett, M. J. (2017, Summer). Mechanisms of sub-anesthetic ketamine infusions to reduce levodopa-induced dyskinesia: effects on striatal mTOR signaling and beta band oscillations in striatum and motor cortex.. International Parkinson and Movement Disorders Society Abstracts.
• Falk, T., Sherman, S. J., Doyle, K., Dollish, H. K., Bartlett, M. J., & Flores, A. J. (2017, November). Role of BDNF and mTOR pathways in the suppression of L-DOPA-induced dyskineasis by sub-anesthetic ketamine.. Society for Neuroscience Abstracts.
• Falk, T., Sherman, S. J., Heien, M. L., Doyle, K., Farrell, D. C., Parent, K. L., Silashki, B. D., Muller, D. C., & Bartlett, M. J. (2017, November). AAV2/1 VEGF-B overexpression improves motor function and prevents dopamine loss in PINK1 gene knock out rats.. Society for Neuroscience Abstracts.
• Zbesko, J. C., Nguyen, T., Hussain, O., Day, W. A., Frye, J., Hayes, M. I., Krumberger, M., Mona, J. K., Stepanovic, K., & Doyle, K. (2017, June). Glial Scars are permeable to neurodegenerative factors present in areas of liquefactive necrosis following stroke. Neuroinflammation: Concepts, Characteristics, Consequences. Keystone Symposia Conference. Keystone, Colorado: Keystone Symposia.
• Doyle, K. P., Becker, K. J., Schneider, J. A., & Buckwalter, M. S. (2016, October). Adaptive immune responses to stroke cause post-stroke cognitive decline in mice and are associated with it in stroke survivors. VasCog Congress. Amsterdam, The Netherlands: VAS COG Society, The international society of vascular behavioural and cognitive disorders.
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  Objective: Stroke increases the risk of subsequent incident dementia, and is associated with immunoglobulins in cerebrospinal fluid. We tested the hypothesis that antibody producing B-lymphocytes contribute to delayed cognitive impairment after stroke. Methods: We used wildtype mice to develop a mouse model of delayed cognitive impairment after stroke. B-lymphocytes were ablated genetically and 5 days after stroke with an anti-CD20 antibody. Immunohistochemisty for adaptive immune responses was also performed on ischemic lesions in brain sections from Rush University’s Memory and Aging Project and the Religious Orders Study. Finally, we assessed autoantibodies to the white matter proteins myelin basic protein and proteolipid protein in 58 stroke survivors with MMSEs administered 30, 90, 180, and 365 days after stroke. Results: We describe a model of post-stroke cognitive impairment in mice. There is normal cognition and hippocampal electrophysiology (long-term potentiation) one week after a cortical stroke adjacent to the hippocampus. Within seven weeks, cognitive impairment and progressive loss of long-term potentiation begin, in parallel with the accumulation of antibody-producing B-lymphocytes in the stroke core, and antibodies in the core and surrounding brain parenchyma. We prevented delayed cognitive impairment in mice by ablating B-lymphocytes using both genetic and treatment models. In human brain tissue there are antibody-producing B-lymphocyte plasmablasts in and around the stroke core. Lastly, serum autoantibody titers against myelin basic protein but not proteolipid protein were the only predictor of MMSE decline in the year after stroke in multivariate analysis. Conclusions: B-lymphocyte mediated adaptive immune responses cause post-stroke cognitive decline in mice and are associated with cognitive decline after stroke in humans. Studies are needed to test a causal relationship between cognitive decline and autoimmunity in humans, and to develop biomarkers for treatment trials. References: Doyle et al., 2015 J. Neuroscience 35(5):2133–2145. Becker et al., 2016 J. Neuroimmunology 295–296:9–11.
• Krumberger, M., Zbesko, J., Hussein, O., Nguyen, T., & Doyle, K. (2016, March). Following stroke neurodegenerative factors leak from newly formed blood vessels, but are prevented from entering healthy brain tissue by endocytic glial scars. American Society for Neurochemistry Conference. Denver, Colorado: American Society for Neurochemistry.
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  Stroke is the leading cause of long-term disability in the United States, affecting 795,000 people annually. Following stroke, the blood brain barrier (BBB) is breached in the area of the lesion. One method of repair involves the formation of new blood vessels in the damaged region to deliver immune cells to participate in the clearance of dead tissue. Another method of repair involves reactive astrocytes joining together to seal off the damaged region to protect the surviving neuropil. Both of these processes are important components of the healing response to stroke. However, there have been no studies on the possibility that the formation of the glial scar and its segregation of the lesion from the rest of the brain, prevents the blood vessels that form in the lesion from developing into mature brain blood vessels that have tight junctions and astrocytic end-feet, which are key components of the BBB. Therefore, to extend our knowledge about restoration of the BBB following stroke, C57BL/6J mice underwent a distal hypoxic (DH) model of stroke and the structure and function of blood vessels within the lesion, and the structure and function of the glial scar, were evaluated at multiple time points for seven weeks following stroke.
• Nguyen, T. V., Frye, J. B., Zbesko, J. C., Urzua, A., Stepanovic, K., Hayes, M., & Doyle, K. P. (2016, November). Characterization of Inflammation in Acute and Non-Acute Ischemic Infarcts in Human and Mouse Brain Tissue. Society for Neuroscience. San Diego, California: Society for Neuroscience.
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  Approximately 30% of stroke survivors develop dementia following a stroke. We hypothesize that one of the mechanisms that contributes to this delayed cognitive decline is a chronic inflammatory response that persists at the site of the stroke lesion and causes bystander damage to surrounding tissue. To address this hypothesis, we present a comprehensive characterization of the cytokine and chemokine response to stroke in the human brain at different stages of wound healing. Our data indicate that inflammation following stroke may not be as efficiently self-limiting as it is in other tissues. In addition, due to most basic and preclinical research being conducted in rodent models of stroke, we also present a comparison of the cytokine and chemokine response to stroke in human and mouse brains at two different stages of wound healing, using C57BL/6 and BALB/c mice to control for strain related differences in the immune response. Our data indicate that the acute inflammatory response to stroke is largely conserved in mice as well as in humans. In each mouse strain, and in humans, there is a significant increase of GM-CSF, IL-6, IL-12p70, IP-10, KC/IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α in the infarct core during the acute time period. However, in the weeks after stroke, during the stage of liquefactive necrosis, the chronic inflammatory response to stroke diverges in both mouse strains and humans; only the sustained elevation of IL-6 and MCP-1 is conserved in mice and humans, with each strain of mouse, and each species, displaying a unique profile of chronically elevated pro-inflammatory cytokines in the area of infarction. Furthermore, because nearly 75% of all strokes occur in patients over the age of 65, and approximately 25% of strokes are recurrent strokes, we also present findings that reveal how the inflammatory response to stroke is impacted by age and multiple strokes in mice. Our data indicate that the chronic inflammatory response to stroke is not exacerbated by age in 18-month old mice compared to 3-month old mice but is significantly exacerbated by a recurrent stroke.
• Nguyen, T., Hayes, M., Frye, J., & Doyle, K. (2016, March). Exacerbation of inflammation, neurodegeneration, and delayed cognitive impairment in an aged mouse model of stroke. American Society for Neurochemistry Conference. Denver, Colorado: American Society for Neurochemistry.
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  Up to 30% of stroke patients develop cognitive decline in the first year after their stroke, and the etiology of this sub-category of vascular dementia is unknown. To address this problem we recently developed a model of delayed cognitive dysfunction following stroke (DH stroke), and discovered that chronic inflammation at the site of stroke lesions could be an underlying cause. However, our published research was only performed in young adult animals, yet stroke is predominantly a disease of the elderly. Little is known about how the inflammatory response to stroke changes with age, despite aging being associated with alterations in the regulation and function of the immune system. Therefore the goal of the present study was to test aged animals, to determine if they also experience delayed cognitive decline following our stroke model, if it is more rapid and severe, and if they have a more pronounced chronic inflammatory response in the brain and systemic circulation. To accomplish this goal we compared the chronic inflammatory response, and the development of delayed cognitive deficits in our stroke model, in 3-month old and 18-month old mice. Using immunostaining, multiplex immunoassay, and behavioral tests, we discovered that 18-month old C57BL/6 male mice have significantly less scarring, but more inflammation in the brain at chronic time points following stroke. This correlates with poorer recovery on tests of motor function, and an accelerated onset of delayed memory deficits in the weeks after stroke. Specifically, we discovered that there is significantly decreased GFAP+ immunostaining in the area of the glial scar in 18-month old mice, significantly increased CD3+ T cell infiltration into the brain, and significantly increased proinflammatory cytokine expression within the stroke lesion at 7 weeks post-stroke. This correlates with substantially more brain atrophy, a lack of motor recovery, and an accelerated onset of short-term memory deficits in 18-month old mice compared to 3-month old mice. In light of these findings we posit that poorer compartmentalization of stroke lesions away from uninfarcted brain tissue, and more pronounced chronic inflammation following stroke, are sequlae of age that are promising targets for developing stroke treatments specifically tailored to elderly patients.
• Zbesko, J., Nguyen, T., Frye, J., & Doyle, K. (2016, March). Characterization of the inflammatory and pathological profile of ischemic infarcts in humans with post-stroke dementia. American Society for Neurochemistry Conference. Denver, Colorado: American Society for Neurochemistry.
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  Each year, ten million people worldwide survive the neurological injury associated with stroke. Importantly, of the 75% of people who survive a stroke, up to 30% develop a progressive decline in memory and cognitive function within the first year. Afflicted patients are grouped with other patients suffering from cognitive dysfunction relating to a vascular mechanism, and are labeled as suffering from vascular dementia. Vascular dementia is the second most common form of dementia in the U.S., and the pathogenesis of delayed dementia after stroke, which affects over 1 million Americans, is unknown. However, there is a clear association between blood-brain-barrier dysfunction and increased inflammation in the blood of patients who develop dementia after stroke, and it has long been suspected that a cause may be the development of a chronic and injurious inflammatory response to necrotic brain tissue. Therefore, the goals of this study are to: (i) advance our understanding of inflammation and pathological markers related to dementia in the human brain after stroke, (ii) investigate the extent of variability between individuals, (iii) determine if some individuals develop an autoimmune response to stroke, and (iv) ascertain whether the astroglial scar that forms in the brain after stroke interferes with the influx and efflux of proteins associated with neurodegeneration. Using immunohistochemistry, scanning electron microscopy, and Milliplex® immunoassay techniques, we discovered that for months, and possibly years after stroke, human stroke lesions contain abundant T- and B-lymphocytes, pro-inflammatory cytokines, autoantibodies, and toxic amyloid-beta and tau peptides. We posit that the prolonged presence of these factors associated with neurodegeneration in the stroke lesion, and their leakage into neighboring tissue, is a cause of dementia in some humans diagnosed with stroke related dementia.
• Doyle, K., Quach, L., Sole, M., Axtell, R., Nguyen, T., Soler-Llavina, G., Jurado, S., Han, J., Steinman, L., Longo, F., Schneider, J., Malenka, R., & Buckwalter, M. (2015, January). Immune mediated delayed cognitive impairment following stroke. Neuroinflammation in Diseases of the Central Nervous System. Taos, New Mexico: Keystone.
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  Each year, ten million people worldwide survive the neurologic injury associated with a stroke. Importantly, stroke survivors have almost twice the risk of subsequently developing dementia. The link between stroke and the later development of dementia is not understood. For example, it is unknown how long inflammation persists after stroke. In this study, we tested the hypothesis that chronic inflammation in the brain contributes to the onset of dementia. We utilized a mouse model of stroke (DH, or Distal Hypoxic stroke) that generates a large cortical lesion and no immediate cognitive deficits. We characterized inflammatory responses (n=7-10 mice per group), long-term potentiation (n=6-9), and cognition (n=10) before and after stroke in wildtype C57BL/6J mice. We found that the inflammatory response to stroke persisted for months and was characterized by the presence of macrophages and B and T lymphocytes in the stroke lesion. Moreover, immunoglobulin was observed in the lesion and surrounding tissue, and there was prolonged inflammation in the area of axonal degeneration. We found that immunoglobulin accumulated in the hippocampus and mice developed a delayed deficit in hippocampal long-term potentiation and delayed short-term memory deficits one to seven weeks after stroke. Notably, delayed cognitive impairment was mitigated in C57BL/6J-muMT mice that lacked mature B lymphocytes and in wild-type mice treated with an anti-CD20 B cell-depleting antibody after stroke. Thus, the inflammatory response to stroke persisted for months in mice, and such mice developed a B lymphocyte-mediated autoimmune response that contributed to delayed cognitive dysfunction.
• Hussein, O., Frye, J., Contreras, N., Ahmed, N., Nguyen, T., & Doyle, K. (2015, January). Scanning and transmission electron microscopy of the glial scar after stroke. Neuroinflammation in Diseases of the Central Nervous System. Taos, New Mexico: Keystone.
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  To extend our knowledge about the function of the astroglial scar that forms in the brain after stroke we used electron microscopy to reveal its ultrastructure. BALB/cJ mice underwent distal middle cerebral artery occlusion and the structure of the glial scar was evaluated one, four, and seven weeks later. We discovered that hypertrophied astrocytic processes form a dense mesh that is effective at keeping large phagocytic immune cells away from adjacent healthy tissue, but is porous to smaller immune cells that by electron microscopy strongly resemble lymphocytes. To uncover the identity of these cells we used immunohistochemistry, and discovered that for weeks after stroke CD3+ T lymphocytes, but not B lymphocytes, leak out of the lesion and home to areas of degenerating white matter. Furthermore, immunohistochemistry revealed that immunoglobulin also leaks out of the glial scar into adjacent non-infarcted tissue, where its presence correlates with progressive dysfunction in the hippocampus. Together, these findings indicate that the astroglial scar is an effective border at containing larger inflammatory cells, but is porous to smaller immune cells such as T lymphocytes, and to immunoglobulin for weeks after stroke.
• Nguyen, T., Frye, J., Contreras, N., & Doyle, K. (2015, January). Characterization of inflammation in acute and non-acute ischemic infarcts in human brain tissue. Neuroinflammation in Diseases of the Central Nervous System. Taos, New Mexico: Keystone.
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  Inflammation in the human brain after stroke is relatively uncharacterized. Therefore, it is unknown whether there is a canonical inflammatory response to stroke in the human brain, or if inflammation in the brain after stroke follows a different trajectory in different individuals. In support of the latter, there is evidence to indicate that some humans develop an adaptive immune response to brain antigens after stroke. For instance, brain antigens have been detected in lymphoid tissue after acute stroke, myelin reactive T lymphocytes have been found in some patients after stroke, and the presence of myelin reactive mononuclear cells in the circulation 90 days after stroke is associated with worse outcome. There are also several reports of oligoclonal bands in the cerebrospinal fluid of stroke patients, which suggests that in some people a B lymphocyte response to stroke may occur. Therefore, the goals of this study were to advance our understanding of inflammation in the human brain after stroke, investigate the extent of variability between individuals, and determine if some individuals develop an autoimmune response to stroke. Using both immunohistochemistry and multiplex immunoassay techniques, we characterized immune cell infiltration and measured cytokine levels in acute and non-acute ischemic infarcts in post-mortem human brain tissue.
• Doyle, K., Sole, M., Axtell, R., Nguyen, T., Quach, L., Soler-Llavina, G., Jurado, S., Han, J., Shah, A., Steinman, L., Longo, F., Malenka, R., & Buckwalter, M. (2014, March). Immune mediated delayed cognitive impairment following stroke. American Society for Neurochemistry Conference. Los Angeles, CA: American Society for Neurochemistry.
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  Each year, ten million people worldwide survive the neurologic injury associated with a stroke. Importantly, stroke survivors have almost twice the risk of subsequently developing dementia. The link between stroke and the later development of dementia is not understood. For example, it is unknown how long inflammation persists after stroke. In this study, we tested the hypothesis that chronic inflammation in the brain contributes to the onset of dementia. We utilized a mouse model of stroke (DH, or Distal Hypoxic stroke) that generates a large cortical lesion and no immediate cognitive deficits. We characterized inflammatory responses (n=7-10 mice per group), long-term potentiation (n=6-9), and cognition (n=10) before and after stroke in wildtype C57BL/6J mice. We found that the inflammatory response to stroke persisted for months and was characterized by the presence of macrophages and B and T lymphocytes in the stroke lesion. Moreover, immunoglobulin was observed in the lesion and surrounding tissue, and there was prolonged inflammation in the area of Wallerian degeneration. We found that immunoglobulin accumulated in the hippocampus and mice developed a delayed deficit in hippocampal long-term potentiation and delayed short-term memory deficits one to seven weeks after stroke. Notably, delayed cognitive impairment was mitigated in C57BL/6J-muMT mice that lacked mature B lymphocytes and in wild-type mice treated with an anti-CD20 B cell-depleting antibody after stroke. Thus, the inflammatory response to stroke persisted for months in mice, and such mice developed a B lymphocyte-mediated autoimmune response that contributed to delayed cognitive dysfunction.
• Doyle, K., Sole, M., Axtell, R., Nguyen, T., Quach, L., Soler-Llavina, G., Jurado, S., Han, J., Shah, A., Steinman, L., Longo, F., Malenka, R., & Buckwalter, M. (2014, March). Mice develop B-lymphocyte dependent delayed cognitive deficits weeks after stroke. Frontiers in Immunobiology & Immunopathogenesis Symposium. University of Arizona, Tucson: Department of Immunobiology.
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  Each year, ten million people worldwide survive the neurologic injury associated with a stroke. Importantly, stroke survivors have almost twice the risk of subsequently developing dementia. The link between stroke and the later development of dementia is not understood. For example, it is unknown how long inflammation persists after stroke. In this study, we tested the hypothesis that chronic inflammation in the brain contributes to the onset of dementia. We utilized a mouse model of stroke (DH, or Distal Hypoxic stroke) that generates a large cortical lesion and no immediate cognitive deficits. We characterized inflammatory responses (n=7-10 mice per group), long-term potentiation (n=6-9), and cognition (n=10) before and after stroke in wildtype C57BL/6J mice. We found that the inflammatory response to stroke persisted for months and was characterized by the presence of macrophages and B and T lymphocytes in the stroke lesion. Moreover, immunoglobulin was observed in the lesion and surrounding tissue, and there was prolonged inflammation in the area of Wallerian degeneration. We found that immunoglobulin accumulated in the hippocampus and mice developed a delayed deficit in hippocampal long-term potentiation and delayed short-term memory deficits one to seven weeks after stroke. Notably, delayed cognitive impairment was mitigated in C57BL/6J-muMT mice that lacked mature B lymphocytes and in wild-type mice treated with an anti-CD20 B cell-depleting antibody after stroke. Thus, the inflammatory response to stroke persisted for months in mice, and such mice developed a B lymphocyte-mediated autoimmune response that contributed to delayed cognitive dysfunction.
• Nguyen, T., Frye, J., Hussein, O., & Doyle, K. (2014, November). Scanning and transmission electron microscopy of the glial scar after stroke. Society for Neuroscience Conference. Washington DC: Society for Neuroscience.
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  “It is the pervading law of all things organic and inorganic, of all things physical and metaphysical, of all things human and all things superhuman, of all true manifestations of the head, of the heart, of the soul, that the life is recognizable in its expression, that form ever follows function. This is the law.” The American Architect Louis Sullivan coined the phrase “form ever follows function” to encapsulate the principle that in architecture the shape of an object should reflect it’s intended function or purpose. In nature form also “ever follows function”, and so to extend our knowledge about the function of the astroglial scar that forms in the brain after stroke we used electron microscopy to reveal its ultrastructure. BALB/cJ mice underwent distal middle cerebral artery occlusion and the structure of the glial scar was evaluated one and four weeks later. We discovered that hypertrophied astocytic processes form a dense mesh that is effective at keeping large phagocytic immune cells away from adjacent healthy tissue, but is porous to smaller immune cells that by electron microscopy strongly resemble lymphocytes. To uncover the identity of these cells we used immunohistochemistry, and discovered that for weeks after stroke CD3+ T lymphocytes leak out of the lesion and home to areas of degenerating white matter. Furthermore, immunohistochemistry revealed that immunoglobulin also leaks out of the glial scar into adjacent non-infarcted tissue, where its presence correlates with progressive dysfunction in the hippocampus and delayed loss of neurons between weeks one and seven after stroke. Together, these findings indicate that the astroglial scar is an effective border at containing larger inflammatory cells, but is porous to smaller immune cells such as T lymphocytes, and to immunoglobulin.
• Nguyen, T., Frye, J., Stepanovic, K., & Doyle, K. (2014, November). Characterization of inflammation in acute and non-acute ischemic infarcts in human brain tissue. Society for Neuroscience Conference. Washngton, DC: Society for Neuroscience.
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  Inflammation in the human brain after stroke is relatively uncharacterized. Therefore, it is unknown whether there is a canonical inflammatory response to stroke in the human brain, or if inflammation in the brain after stroke follows a different trajectory in different individuals. In support of the latter, there is evidence to indicate that some humans develop an adaptive immune response to brain antigens after stroke. For instance, brain antigens have been detected in lymphoid tissue after acute stroke, myelin reactive T lymphocytes have been found in some patients after stroke, and the presence of myelin reactive mononuclear cells in the circulation 90 days after stroke is associated with worse outcome. There are also several reports of oligoclonal bands in the cerebrospinal fluid of stroke patients, which suggests that in some people a B lymphocyte response to stroke may occur. Therefore, the goals of this study were to advance our understanding of inflammation in the human brain after stroke, investigate the extent of variability between individuals, and determine if some individuals develop an autoimmune response to stroke. Using both immunohistochemistry and multiplex immunoassay techniques, we characterized immune cell infiltration and measured cytokine levels in acute and non-acute ischemic infarcts in post-mortem human brain tissue.
• Doyle, K., Sole, M., Axtell, R., Soler-Llavina, G., Jurado, S., Nguyen, T., Shah, A., Han, J., Steinman, L., Longo, F., Malenka, R., & Buckwalter, M. (2013, November). Modeling post-stroke dementia in mice: B-lymphocyte dependent cognitive deficits appear weeks after stroke. Society for Neuroscience. San Diego: Society for Neuroscience.
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  Worldwide, 10 million people survive stroke each year and many subsequently develop dementia. Even after accounting for other dementia risk factors, stroke itself doubles the risk of developing dementia in the years afterwards. This is termed post-stroke dementia. It isn’t known how stroke increases the risk of later developing dementia in people. We utilized a mouse model of stroke (DH, or Distal Hypoxic stroke) that generates a large cortical lesion and no immediate cognitive deficits to model this condition. We characterized inflammatory responses (n=7-10 mice per group), long-term potentiation (n=6-9), and cognition (n=10) before and after stroke in wildtype C57BL/6J mice. Sham controls underwent identical procedures but did not have occlusion of the middle cerebral artery. All experiments and analyses were performed in a blinded fashion. We found that in mice the inflammatory response to stroke continues for at least 3 months, and is characterized by the presence of macrophages and B- and T-lymphocytes in the stroke lesion, and the accumulation of immunoglobulin G (IgG) in the surrounding tissue. In conjunction, between weeks 1 and 7 after stroke mice develop a deficit in long-term potentiation in the hippocampus, where IgG also accumulates, and exhibit behavioral impairment on Y maze and object location testing. Long-term potentiation deficits continue to worsen between 7 weeks and 3 months after stroke. This delayed development of post-stroke cognitive impairment also occurs in a distinct wildtype mouse strain, Balb/c, after standard permanent distal middle cerebral artery stroke. Finally, delayed cognitive impairment, as measured by both Y maze and object location testing, does not occur in C57BL/6J-muMT mice that lack mature B-lymphocytes. Our data indicate that after stroke, dead brain tissue stimulates a long-lasting autoimmune response, with B-lymphocytes playing a key role. B-lymphocytes may cause neuronal dysfunction via brain-reactive autoantibodies, neurotoxic cytokines and/or by activating T-lymphocytes. These data are exciting because there are FDA-approved treatments to prevent autoimmune B-lymphocyte responses.

Reviews

• Doyle, K., & Buckwalter, M. S. (2016. Does B lymphocyte-mediated autoimmunity contribute to post-stroke dementia?.
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  Post-stroke cognitive decline and dementia pose a significant public health problem, with 30% of stroke survivors suffering from dementia. The reason for this high prevalence is not well understood. Pathogenic B cell responses to the damaged CNS are one possible contributing factor. B-lymphocytes and antibodies are present in and around the stroke core of some human subjects who die with stroke and dementia, and mice that develop delayed cognitive dysfunction after stroke have clusters of B-lymphocytes in the stroke lesion, and antibody infiltration in the stroked hemisphere. The ablation of B-lymphocytes prevents post-stroke cognitive impairment in mice. Multiple drugs that target B cells are FDA approved, and so if pathogenic B cell responses are occurring in a subset of stroke patients, this is potentially treatable. However, it has also been demonstrated that regulatory B cells can be beneficial in mouse models of stroke. Consequently, it is important to understand the relative contribution of B-lymphocytes to recovery versus pathogenicity, and if this balance is heterogeneous in different individuals. Therefore, the purpose of this review is to summarize the current state of knowledge with regard to the role of B-lymphocytes in the etiology of post-stroke dementia.
• Doyle, K. P., Simon, R. P., & Stenzel-Poore, M. P. (2008. Mechanisms of ischemic brain damage(pp 310-8).
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  In the United States stroke is the third leading cause of death and the leading cause of disability. Brain injury following stroke results from the complex interplay of multiple pathways including excitotoxicity, acidotoxicity, ionic imbalance, peri-infarct depolarization, oxidative and nitrative stress, inflammation and apoptosis. There are very few treatments for stroke and the development of new treatments requires a comprehensive understanding of the diverse mechanisms of ischemic brain damage that are responsible for neuronal death. Here, we discuss the underlying pathophysiology of this devastating disease and reveal the intertwined pathways that are the target of therapeutic intervention.