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1.  B-Lymphocyte-Mediated Delayed Cognitive Impairment following Stroke 
The Journal of Neuroscience  2015;35(5):2133-2145.
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.
PMCID: PMC4315838  PMID: 25653369
B-lymphocyte; dementia; immunology; stroke
2.  Astrocytic transforming growth factor-beta signaling reduces subacute neuroinflammation after stroke in mice 
Glia  2014;62(8):1227-1240.
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 two 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.
PMCID: PMC4061255  PMID: 24733756
Astrocytes; cytokines; immunity; ischemia; inflammation
3.  Astrocytic TGFβ signaling limits inflammation and reduces neuronal damage during CNS Toxoplasma infection1 
The balance between controlling infection and limiting inflammation is particularly precarious in the brain because of its unique vulnerability to the toxic effects of inflammation. Astrocytes have been implicated as key regulators of neuroinflammation in CNS infections, including infection with Toxoplasma gondii, a protozoan parasite that naturally establishes a chronic CNS infection in mice and humans. In CNS toxoplasmosis, astrocytes are critical to controlling parasite growth. They secrete pro-inflammatory cytokines and physically encircle parasites. However, the molecular mechanisms used by astrocytes to limit neuroinflammation during toxoplasmic encephalitis have not yet been identified. Transforming growth factor beta (TGFβ) signaling in astrocytes is of particular interest because TGFβ is universally upregulated during CNS infection and serves master regulatory and primarily anti-inflammatory functions. We report here that TGFβ signaling is activated in astrocytes during toxoplasmic encephalitis, and that inhibition of astrocytic TGFβ signaling increases immune cell infiltration, uncouples pro-inflammatory and chemokine production from CNS parasite burden, and increases neuronal injury. Remarkably, we show that the effects of inhibiting astrocytic TGFβ signaling are independent of parasite burden and the ability of GFAP+ astrocytes to physically encircle parasites.
PMCID: PMC4075480  PMID: 24860191
4.  Suppression of Inflammation with Conditional Deletion of the Prostaglandin E2 EP2 Receptor in Macrophages and Brain Microglia 
The Journal of Neuroscience  2013;33(40):16016-16032.
Prostaglandin E2 (PGE2), a potent lipid signaling molecule, modulates inflammatory responses through activation of downstream G-protein coupled EP1–4 receptors. Here, we investigated the cell-specific in vivo function of PGE2 signaling through its E-prostanoid 2 (EP2) receptor in murine innate immune responses systemically and in the CNS. In vivo, systemic administration of lipopolysaccharide (LPS) resulted in a broad induction of cytokines and chemokines in plasma that was significantly attenuated in EP2-deficient mice. Ex vivo stimulation of peritoneal macrophages with LPS elicited proinflammatory responses that were dependent on EP2 signaling and that overlapped with in vivo plasma findings, suggesting that myeloid-lineage EP2 signaling is a major effector of innate immune responses. Conditional deletion of the EP2 receptor in myeloid lineage cells in Cd11bCre;EP2lox/lox mice attenuated plasma inflammatory responses and transmission of systemic inflammation to the brain was inhibited, with decreased hippocampal inflammatory gene expression and cerebral cortical levels of IL-6. Conditional deletion of EP2 significantly blunted microglial and astrocytic inflammatory responses to the neurotoxin MPTP and reduced striatal dopamine turnover. Suppression of microglial EP2 signaling also increased numbers of dopaminergic (DA) neurons in the substantia nigra independent of MPTP treatment, suggesting that microglial EP2 may influence development or survival of DA neurons. Unbiased microarray analysis of microglia isolated from adult Cd11bCre;EP2lox/lox and control mice demonstrated a broad downregulation of inflammatory pathways with ablation of microglial EP2 receptor. Together, these data identify a cell-specific proinflammatory role for macrophage/microglial EP2 signaling in innate immune responses systemically and in brain.
PMCID: PMC3787507  PMID: 24089506
5.  Blood–brain barrier dysfunction–induced inflammatory signaling in brain pathology and epileptogenesis 
Epilepsia  2012;53(0 6):37-44.
The protection of the brain from blood-borne toxins, proteins, and cells is critical to the brain’s normal function. Accordingly, a compromise in the blood–brain barrier (BBB) function accompanies many neurologic disorders, and is tightly associated with brain inflammatory processes initiated by both infiltrating leukocytes from the blood, and activation of glial cells. Those inflammatory processes contribute to determining the severity and prognosis of numerous neurologic disorders, and can both cause, and result from BBB dysfunction. In this review we examine the role of BBB and inflammatory responses, in particular activation of transforming grown factor β (TGFβ) signaling, in epilepsy, stroke, and Parkinson’s disease.
PMCID: PMC3703535  PMID: 23134494
Blood–brain barrier; Epileptogenesis; Inflammation; TGF-beta; Cholinergic system
6.  Delayed administration of a small molecule TrkB ligand promotes recovery after hypoxic- ischemic stroke 
Background and Purpose
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 (BDNF) stimulates neurogenesis and plasticity, processes that are implicated in stroke recovery. It binds to both the tropomyosin-related kinase B (TrkB) and p75 neurotrophin (p75NTR) receptors. However, BDNF 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 TrkB would promote neurogenesis and functional recovery after stroke.
Four-month-old mice were trained on motor tasks prior to stroke. After stroke, functional test results were used to randomize mice into two equally, and severely, impaired groups. Beginning 3 days after stroke, mice received LM22A-4 or saline vehicle daily for ten weeks.
LM22A-4 treatment significantly improved limb swing speed and accelerated the return to normal gait accuracy after stroke. LM22A-4 treatment also doubled both the number of new mature neurons and immature neurons adjacent to the stroke. Drug-induced differences were not observed in angiogenesis, dendritic arborization, axonal sprouting, glial scar formation, or neuroinflammation.
A small molecule agonist of TrkB improves functional recovery from stroke and increases neurogenesis when administered beginning three days after stroke. These findings provide proof-of-concept that targeting of TrkB alone is capable of promoting one or more mechanisms relevant to stroke recovery. LM22A-4 or its derivatives might therefore serve as “pro-recovery” therapeutic agents for stroke.
PMCID: PMC3383889  PMID: 22535263
Stroke recovery; neurotrophin; small molecule
7.  Distal Hypoxic stroke: A new mouse model of stroke with high throughput, low variability and a quantifiable functional deficit 
Journal of Neuroscience Methods  2012;207(1):31-40.
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 1 hour 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.
PMCID: PMC3348433  PMID: 22465679
New Mouse Stroke Model; Cerebral Ischemia; Hypoxia; Circle of Willis Variability
8.  Stratification substantially reduces behavioral variability in the hypoxic–ischemic stroke model 
Brain and Behavior  2012;2(5):698-706.
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, R2 = 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.
PMCID: PMC3489820  PMID: 23139913
Behavior; hypoxic–ischemic stroke; motor recovery; mouse model
9.  A Comparison of Cooling Techniques to Treat Cardiac Arrest Patients with Hypothermia 
Stroke Research and Treatment  2011;2011:690506.
Introduction. We sought to compare the performance of endovascular cooling to conventional surface cooling after cardiac arrest. Methods. Patients in coma following cardiopulmonary resuscitation were cooled with an endovascular cooling catheter or with ice bags and cold-water-circulating cooling blankets to a target temperature of 32.0–34.0°C for 24 hours. Performance of cooling techniques was compared by (1) number of hourly recordings in target temperature range, (2) time elapsed from the written order to initiate cooling and target temperature, and (3) adverse events during the first week. Results. Median time in target temperature range was 19 hours (interquartile range (IQR), 16–20) in the endovascular group versus. 10 hours (IQR, 7–15) in the surface group (P = .001). Median time to target temperature was 4 (IQR, 2.8–6.2) and 4.5 (IQR, 3–6.5) hours, respectively (P = .67). Adverse events were similar. Conclusion. Endovascular cooling maintains target temperatures better than conventional surface cooling.
PMCID: PMC3148603  PMID: 21822470
10.  TGFβ signaling in the brain increases with aging and signals to astrocytes and innate immune cells in the weeks after stroke 
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.
We performed distal middle cerebral artery occlusion strokes on 5 and 18 month old TGFβ reporter mice to get a readout of TGFβ responses after stroke in real time. To determine which cell type is the source of increased TGFβ production after stroke, brain sections were stained with an anti-TGFβ antibody, colocalized with markers for reactive astrocytes, neurons, and activated microglia. To determine which cells are responding to TGFβ after stroke, brain sections were double-labelled with anti-pSmad2, a marker of TGFβ signaling, and markers of neurons, oligodendrocytes, endothelial cells, astrocytes and microglia.
TGFβ signaling increased 2 fold after stroke, beginning on day 1 and peaking on day 7. This pattern of increase was preserved in old animals and absolute TGFβ signaling in the brain increased with age. Activated microglia and macrophages were the predominant source of increased TGFβ after stroke and astrocytes and activated microglia and macrophages demonstrated dramatic upregulation of TGFβ signaling after stroke. TGFβ signaling in neurons and oligodendrocytes did not undergo marked changes.
We found that TGFβ signaling increases with age and that astrocytes and activated microglia and macrophages are the main cell types that undergo increased TGFβ signaling in response to post-stroke increases in TGFβ. Therefore increased TGFβ after stroke likely regulates glial scar formation and the immune response to stroke.
PMCID: PMC2958905  PMID: 20937129
11.  Glia-dependent TGF-β signaling, acting independently of the TH17 pathway, is critical for initiation of murine autoimmune encephalomyelitis 
The Journal of Clinical Investigation  2007;117(11):3306-3315.
Autoimmune encephalomyelitis, a mouse model for multiple sclerosis, is characterized by the activation of immune cells, demyelination of axons in the CNS, and paralysis. We found that TGF-β1 synthesis in glial cells and TGF-β–induced signaling in the CNS were activated several days before the onset of paralysis in mice with autoimmune encephalomyelitis. While early production of TGF-β1 was observed in glial cells TGF-β signaling was activated in neurons and later in infiltrating T cells in inflammatory lesions. Systemic treatment with a pharmacological inhibitor of TGF-β signaling ameliorated the paralytic disease and reduced the accumulation of pathogenic T cells and expression of IL-6 in the CNS. Priming of peripheral T cells was not altered, nor was the generation of TH17 cells, indicating that this effect was directed within the brain, yet affected the immune system. These results suggest that early production of TGF-β1 in the CNS creates a permissive and dangerous environment for the initiation of autoimmune inflammation, providing a rare example of the brain modulating the immune system. Importantly, inhibition of TGF-β signaling may have benefits in the treatment of the acute phase of autoimmune CNS inflammation.
PMCID: PMC2040317  PMID: 17965773
12.  Increased T cell recruitment to the central nervous system after Aβ1–42 immunization in Alzheimer’s mice overproducing TGF-β1 
Immunotherapy targeting the Aβ peptide is a novel therapy under investigation for the treatment of Alzheimer’s disease (AD). A clinical trial using Aβ1–42 (AN1792) as the immunogen was halted due to development of meningoencephalitis in a small number of patients. The cytokine TGF-β1 is a key modulator of immune responses that is increased in the brain in AD. We show here that local overexpression of TGF-β1 in the brain increases both meningeal and parenchymal T lymphocyte number. Furthermore, TGF-β1 overexpression in a mouse model for AD (APP mice) leads to development of further T cell infiltrates when mice were immunized at a young but not old age with AN1792. Notably, only mice overproducing both Aβ (APP mice) and TGF-β1 experienced a rise in T lymphocyte number after immunization. One third of infiltrating T cells were CD4 positive. We did not observe significant differences in B lymphocyte numbers in any of the genotypes or treatment groups. These results demonstrate that TGF-β1 overproduction in the brain can promote T cell infiltration, in particular after Aβ1–42 immunization. Likewise, levels of TGF-β1 or other immune factors in brains of AD patients may influence the response to Aβ1–42 immunization.
PMCID: PMC1892201  PMID: 17079673
13.  Modelling neuroinflammatory phenotypes in vivo 
Inflammation of the central nervous system is an important but poorly understood part of neurological disease. After acute brain injury or infection there is a complex inflammatory response that involves activation of microglia and astrocytes and increased production of cytokines, chemokines, acute phase proteins, and complement factors. Antibodies and T lymphocytes may be involved in the response as well. In neurodegenerative disease, where injury is more subtle but consistent, the inflammatory response is continuous. The purpose of this prolonged response is unclear, but it is likely that some of its components are beneficial and others are harmful. Animal models of neurological disease can be used to dissect the specific role of individual mediators of the inflammatory response and assess their potential benefit. To illustrate this approach, we discuss how mutant mice expressing different levels of the cytokine transforming growth factor β-1 (TGF-β1), a major modulator of inflammation, produce important neuroinflammatory phenotypes. We then demonstrate how crosses of TGF-β1 mutant mice with mouse models of Alzheimer's disease (AD) produced important new information on the role of inflammation in AD and on the expression of different neuropathological phenotypes that characterize this disease.
PMCID: PMC500895  PMID: 15285805

Results 1-13 (13)