Medical Student Research Fellowship for Summer 2012
Mentor: Ann M. Stowe, PhD
Department: Neurology and Neurotherapeutics
Room number: NL9.118B
Mail Code: 8813
Phone number: 214-633-1884
E-mail: Ann.Stowe@utsouthwestern.edu
Project title: B cells mediate exercise-induced protection from stroke
Human subjects IRB approved project number (where applicable):
Animal subjects IRB approved project number (where applicable): 2010-0133
Project Type animal-based research
Brief Description of Project:
More than 800,000 people in the U.S. will suffer a new or recurrent stroke this year.1 Despite this prevalence, thrombolysis is the only FDA-approved medical intervention.2 Several factors can, however, influence the severity of stroke injury, including exercise. Individuals with high pre-stroke physical activity present with milder strokes and motor deficits,3 and aerobic exercise following stroke improves cognitive comprehension and motor learning4 and reduces the incidence of recurrent stroke.5 Recent studies show that exercise in healthy adults increases peripheral B cell populations6, 7 and modulates B cell receptor signaling pathways,8-10 while activated B cells are the only source of leukocyte-derived neurotrophin expression (e.g. NGF, BDNF).11 There are, however, no studies establishing a connection between altered B cell phenotypic profiles and natural protection from stroke. This is particularly surprising, as B cells also secrete IL-10, a known post-stroke neuroprotectant12 that reduces ischemic injury by “turning off” subsequent neutrophil diapedesis and pro-inflammatory chemokine production in the brain.13-15
One of the main focuses of my lab is to understand how B cells can be reprogrammed to mediate endogenous protection from stroke (i.e. ischemic tolerance). Preliminary data indicate that a naturally protective phenotype includes an enhanced presence of B cells in the ischemic hemisphere for days after stroke onset, but it is unknown whether these B cells have an upregulated secretion of pro-recovery factors such as IL10 and BDNF that could protect the brain during ischemia. This project will therefore determine if exercise training following stroke modulates resident, peripheral, and cortical B cell populations as part of an adaptive immunity to ischemic injury.
The following is the protocol we will use to test this hypothesis: Wild-type (C57Bl6/J) mice will undergo 3 wks of voluntary exercise in individual running wheels, with sedentary controls in cages with locked wheels. Animals will rest for 1 week and then a transient focal stroke will be induced in the left cortex. At 2 days post-stroke, animals will have peripheral blood collected and will be euthanized with isoflurane for transcardiac perfusion with 20ml 0.01M PBS. We will isolate lymphocytes from the ischemic hemisphere, and determine the percentage of CNS-derived B cells that produce IL10 and BDNF by intracellular staining measured using flow cytometry.16 Blood and brain leukocytes will be exposed to fluorescently conjugated primary antibodies to stain for B cells (CD19, CD45R), neutrophils (Gr), monocytes (CD11b) and T cells (CD4, TCR-β-chain). Upon quantification in a fluorescence-activated cell sorter (FACS) Aria, B cells will be isolated from both cortical and peripheral samples for further profiling. Within the same animals, protein levels of IL10 and other pro-recovery factors will be measured by ELISA in both cortical lysates and processed serum.
The timeframe for this experiment exceeds 4 weeks. Therefore, to optimize Angelica’s participation in this research, we will initiate the first group of animals prior to her entering the lab to collect samples (March/April). In addition, we plan for an experiment to conclude shortly after her start in the lab (May/June). With this experiment, Angelica will be able to participate in the flow cytometry protocol to learn the techniques for staining of peripheral blood, and for centrifugal isolation of leukocytes within the brain parenchyma. In addition, Angelica will be taught the necessary techniques (quantitative PCR, ELISA) to measure growth factor production in the isolated B cells of protected animals compared to controls. Finally, she will be able to see one group of animals run the entire voluntary exercise protocol for completion by the end of her fellowship (June/July). I fully expect for her to have enough data for an abstract and a co-authorship on a paper by the end of the summer. These studies, in conjunction with other ongoing experiments, will be the first to determine if B cells with a unique pro-recovery phenotype (e.g. enhanced IL10, BDNF) can minimize ischemic injury and promote plasticity, a protection that could also be translated to other CNS injuries and diseases. This research will also provide the foundation to investigate B cell phenotypes in sub-populations of stroke patients to determine the influence of B cells on functional recovery.
1. Lloyd-Jones D, Adams RJ, Brown TM, et al. Heart disease and stroke statistics--2010 update: a report from the American Heart Association. Circulation. 2010;121(7):e46-e215.
2. Kidd PM. Integrated brain restoration after ischemic stroke--medical management, risk factors, nutrients, and other interventions for managing inflammation and enhancing brain plasticity. Altern Med Rev. 2009 Mar;14(1):14-35.
3. Krarup LH, Truelsen T, Gluud C, et al. Prestroke physical activity is associated with severity and long-term outcome from first-ever stroke. Neurology. 2008 Oct 21;71(17):1313-8.
4. Quaney BM, Boyd LA, McDowd JM, et al. Aerobic exercise improves cognition and motor function poststroke. Neurorehabil Neural Repair. 2009 Nov;23(9):879-85.
5. Spence JD. Secondary stroke prevention. Nat Rev Neurol. 2010 Sep;6(9):477-86.
6. Cordova A, Sureda A, Tur JA, Pons A. Immune response to exercise in elite sportsmen during the competitive season. J Physiol Biochem. 2010 Mar;66(1):1-6.
7. Zaldivar F, Wang-Rodriguez J, Nemet D, et al. Constitutive pro- and anti-inflammatory cytokine and growth factor response to exercise in leukocytes. J Appl Physiol. 2006 Apr;100(4):1124-33.
8. Fragala MS, Kraemer WJ, Mastro AM, et al. Glucocorticoid receptor expression on human B cells in response to acute heavy resistance exercise. Neuroimmunomodulation. 2011;18(3):156-64.
9. Radom-Aizik S, Zaldivar F, Jr., Leu SY, Cooper DM. A brief bout of exercise alters gene expression and distinct gene pathways in peripheral blood mononuclear cells of early- and late-pubertal females. J Appl Physiol. 2009 Jul;107(1):168-75.
10. Coppola A, Coppola L, dalla Mora L, et al. Vigorous exercise acutely changes platelet and B-lymphocyte CD39 expression. J Appl Physiol. 2005 Apr;98(4):1414-9.
11. Edling AE, Nanavati T, Johnson JM, Tuohy VK. Human and murine lymphocyte neurotrophin expression is confined to B cells. J Neurosci Res. 2004 Sep 1;77(5):709-17.
12. Planas AM, Gorina R, Chamorro A. Signalling pathways mediating inflammatory responses in brain ischaemia. Biochem Soc Trans. 2006 Dec;34(Pt 6):1267-70.
13. Kasama T, Strieter RM, Lukacs NW, Burdick MD, Kunkel SL. Regulation of neutrophil-derived chemokine expression by IL-10. J Immunol. 1994 Apr 1;152(7):3559-69.
14. Mann MK, Maresz K, Shriver LP, Tan Y, Dittel BN. B cell regulation of CD4+CD25+ T regulatory cells and IL-10 via B7 is essential for recovery from experimental autoimmune encephalomyelitis. J Immunol. 2007 Mar 15;178(6):3447-56.
15. Pils MC, Pisano F, Fasnacht N, et al. Monocytes/macrophages and/or neutrophils are the target of IL-10 in the LPS endotoxemia model. Eur J Immunol. 2010 Feb;40(2):443-8.
16. Monson NL, Cravens P, Hussain R, et al. Rituximab therapy reduces organ-specific T cell responses and ameliorates experimental autoimmune encephalomyelitis. PLoS ONE. 2011;6(2):e17103.
17. Stowe AM, Altay T, Freie AB, Gidday JM. Repetitive hypoxia extends endogenous neurovascular protection for stroke. Ann Neurol. 2011 Jun;69(6):975-85.
Previous Research Activities or Publications with Medical Students:
Peer-reviewed articles
Abstracts