1Departments of Anesthesiology 2Pharmacology and Chemical Biology 3Computational and Systems Biology 4Physics and Astronomy, and 5Structural Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA 6Department of Biomedical Sciences, Paul L. Foster School of Medicine, and 7Graduate School of Biomedical Sciences, Texas Tech University Health Sciences Center, El Paso, TX 79905, USA
Although a direct link has long been suspected between systemic immune responses and neuronal injuries after stroke, it is unclear which immune cells play an important role. A question remains as to whether the blood brain barrier (BBB) is transiently disrupted after circulatory arrest to allow peripheral immune cells to enter brain parenchyma. Here, we developed a clinically relevant cardiac arrest and resuscitation model in mice to investigate the BBB integrity using noninvasive magnetic resonance imaging. Changes in immune signals in the brain and periphery were assayed by immunohistochemistry and flow cytometry. Quantitative variance maps from T1-weighted difference images before and after blood-pool contrast clearance revealed BBB disruptions immediately after resuscitation and one day after reperfusion. Time profiles of hippocampal CA1 neuronal injuries correlated with the morphological changes of microglia activation. Cytotoxic T cells, CD11b+CD11c+ dendritic cells, and CD11b+CD45+hi monocytes and macrophages were significantly increased in the brain three days after cardiac arrest and resuscitation, suggesting direct infiltration of these cells following the BBB disruption. Importantly, these immune cell changes were coupled with a parallel increase in the same subset of immune cell populations in the bone marrow and blood. We conclude that neurovascular breakdown during the initial reperfusion phase contributes to the systemic immune cell invasion and subsequent neuropathogenesis affecting the long-term outcome after cardiac arrest and resuscitation.
Figure 1. A mouse model of cardiac arrest and resuscitation. (A) Representative trace of relative arterial blood pressure (ABP) before, during, and after a 5-min cardiac arrest and resuscitation. ABP values are normalized against the mean ABP before cardiac arrest to correct for baseline artifacts due to sharing of the same arterial line for blood monitoring and blood infusion during resuscitation. Rapid onset of circulatory arrest was initiated by an intravenous injection of the short-acting-β blocker esmolol, followed by apnea. Controlled resuscitation was achieved by infusion of oxygenated blood with a resuscitation mixture, leading to the return of spontaneous circulation (ROSC) with ~1 min. (B) Quantification of neuronal injuries based on the percentage of unhealthy neurons in the CA1 region of the hippocampus for naïve and sham-operated controls and for cardiac arrest mice 3 and 10 days after cardiac arrest and resuscitation. (C) Representative micrographs of H&E staining in paraffin sections showed that ischemia-damaged hippocampal CA1 pyramidal neurons are significantly increased on post-resuscitation day 3. A decrease in the number of unhealthy neurons is seen on post-resuscitation day 10. Data in (B) are mean ± SEM and analyzed using one-way ANOVA with pair-wise post hoc least significant difference comparison. ** P < 0.01.
Figure 2. Characterization of astrocytosis and innate immune responses in the central nervous system after cardiac arrest. Displayed here are immune-fluorescent micrographs of DAPI staining (blue) for nuclear DNA, GFAP (red, A-H) for astrocytes, and Iba1 (green, I-P) for reactive microglia, showing the overall immunoreactivity in the hippocampal region (10x, A-D and I-L) and the detailed morphology (40x E-H and M-P) 3 and 10 days after cardiac arrest and resuscitation in comparison to the naïve and time-matched sham controls. Hypertrophy of astrocytic processes (G and H) and amoeboid morphology of macrophagic changes of microglia become very pronounced 3 days after cardiac arrest and resuscitation. The GFAP (Q) and Iba1 (R) immune-reactivities are quantified by fluorescence image segmentation as mean ± SEM. **P < 0.01 and *P < 0.05. (Scale bars = 20 μm)
Figure 3. Magnetic resonance imaging (MRI) of blood-brain-barrier (BBB) integrity. (A) Displayed are representative T2- (first row) and T1-weighted (second row) images, T1-difference images (third row), and the corresponding variance maps (fourth row) before cardiac arrest (Day -1), and 0, 1, and 2 days after resuscitation. Images in each row are displayed using the same intensity scales for easy comparison. (B) Pixel variances in the variance maps are averaged (mean ± SEM) to measure the degree of the BBB leakage as a function of time. Nonparametric Kruskal-Wallis test showed that all time points are different from each other with p = 0.000 except Day 0 vs. Day 3 (p = 0.027) and Day 2 vs. Day -1 (not significantly different).
Figure 4. Infiltration of lymphocytes into the brain after cardiac arrest and resuscitation. (A) Flow cytometry evaluation of regulatory (CD4+) and cytotoxic (CD8+) T cell infiltration into the brain parenchyma 3 days after cardiac arrest and resuscitation, as compared to the naïve and time-matched sham controls. (B) Quantification shows nearly threefold increase in the CD8+T cell population in the brain tissue. Data are presented as mean ± SEM and analyzed using one-way ANOVA with least significant difference post hoc comparisons. *P < 0.05.
Figure 5. Infiltration of peripheral dendritic cells, monocytes, and macrophages into the brain parenchyma after cardiac arrest and resuscitation. (A-C) Gating strategies to isolate infiltrating immunocytes. (D) Representative flow cytometry data showing distinct CD11b+hiCD45+hi cell population (boxes) in the brain 3 days after cardiac arrest and resuscitation. (E) Quantification shows a twofold increase in the CD11b+CD45+ after cardiac arrest and resuscitation. (F) Among the CD11b and CD45 doubly positive cells, the subpopulation of CD11b+hiCD45+hi increased more than six-folds. This subpopulation is likely of peripheral origin. (G and H) CD11b+CD11c+ dendritic cells and CD11b+Ly6G- monocytes are also significantly increased in the brain 3 days after cardiac arrest and resuscitation. Data are presented as mean ± SEM and analyzed using one-way ANOVA with least significant difference post hoc comparison. **P < 0.01 and *P < 0.05.
Figure 6. Spatial distribution of CD45+ cells in brain parenchyma. Immunohistochemical staining reveals that major changes in CD45+ immunoreactivity (red) occur in the dentate gyrus (A, C, E) and hippocampal CA1 region (B, D, F). Cell nuclei are co-stained by DAPI (blue). In naïve (A and B) and sham operated (C and D) mice, strongly CD45 positive cells are scarce. On Day 3 after cardiac arrest and resuscitation, there is a significant increase in the number of CD45+ cells in dentate gyrus and CA1 region. The strongly positive immmunoreactivity in E-F as compared to that in A-D is likely related to the CD45+hi subpopulation in Figure 5. (Scale bar = 15 μm).
Figure 7. Flow cytometry of peripheral immune response to cardiac arrest and resuscitation. Quantitative evaluation of peripheral dendritic cells, monocytes, and macrophages in the bone marrow (A) and the blood (B). Data are presented as mean ± SEM and analyzed using one-way ANOVA with least significant difference post hoc comparison. **P < 0.01 and *P < 0.05.
Writing Group M, Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, et al. (2016). Executive Summary: Heart Disease and Stroke Statistics--2016 Update: A Report From the American Heart Association. Circulation, 133: 447-454
Biarent D, Fonteyne C, Willems A, Dupont A (2013). Post-cardiac Arrest Syndrome in Children. Curr Pediatr Rev, 9: 125-133
Hypothermia after Cardiac Arrest Study G (2002). Mild therapeutic hypothermia to improve the neurologic outcome after cardiac arrest. N Engl J Med, 346: 549-556
Nielsen N, Wetterslev J, Cronberg T, Erlinge D, Gasche Y, Hassager C, et al. (2013). Targeted temperature management at 33 degrees C versus 36 degrees C after cardiac arrest. N Engl J Med, 369: 2197-2206
Anrather J, Iadecola C (2016). Inflammation and Stroke: An Overview. Neurotherapeutics, 13: 661-670
Famakin BM (2014). The Immune Response to Acute Focal Cerebral Ischemia and Associated Post-stroke Immunodepression: A Focused Review. Aging Dis, 5: 307-326
Qian L, Yuanshao L, Wensi H, Yulei Z, Xiaoli C, Brian W, et al. (2016). Serum IL-33 Is a Novel Diagnostic and Prognostic Biomarker in Acute Ischemic Stroke. Aging Dis, 7: 614-622
Liachenko S, Tang P, Hamilton RL, Xu Y (1998). A reproducible model of circulatory arrest and remote resuscitation in rats for NMR investigation. Stroke, 29: 1229-1238; discussion 1238–1229
Liachenko S, Tang P, Hamilton RL, Xu Y (2001). Regional dependence of cerebral reperfusion after circulatory arrest in rats. J Cereb Blood Flow Metab, 21: 1320-1329
Xu Y, Liachenko SM, Tang P, Chan PH (2009). Faster recovery of cerebral perfusion in SOD1-overexpressed rats after cardiac arrest and resuscitation. Stroke, 40: 2512-2518
Vazzana M, Parrinello D, Cammarata M (2003). Chemiluminescence response of beta-glucan stimulated leukocytes isolated from different tissues and peritoneal cavity of Dicentrarchus labrax. Fish Shellfish Immunol, 14: 423-434
Vazzana M, Celi M, Tramati C, Ferrantelli V, Arizza V, Parrinello N (2014). In vitro effect of cadmium and copper on separated blood leukocytes of Dicentrarchus labrax. Ecotoxicology and environmental safety, 102: 113-120
Deng G, Yonchek JC, Quillinan N, Strnad FA, Exo J, Herson PS, et al. (2014). A novel mouse model of pediatric cardiac arrest and cardiopulmonary resuscitation reveals age-dependent neuronal sensitivities to ischemic injury. J Neurosci Methods, 222: 34-41
Liachenko S, Tang P, Xu Y (2003). Deferoxamine improves early postresuscitation reperfusion after prolonged cardiac arrest in rats. J Cereb Blood Flow Metab, 23: 574-581
Neumar RW, Bircher NG, Sim KM, Xiao F, Zadach KS, Radovsky A, et al. (1995). Epinephrine and sodium bicarbonate during CPR following asphyxial cardiac arrest in rats. Resuscitation, 29: 249-263
Hirko AC, Dallasen R, Jomura S, Xu Y (2008). Modulation of inflammatory responses after global ischemia by transplanted umbilical cord matrix stem cells. Stem Cells, 26: 2893-2901
Pérez-Álvarez MJ, Maza Mdel C, Anton M, Ordoñez L, Wandosell F (2012). Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia. J Neuroinflammation, 9: 157
Torres-Platas SG, Comeau S, Rachalski A, Bo GD, Cruceanu C, Turecki G, et al. (2014). Morphometric characterization of microglial phenotypes in human cerebral cortex. J Neuroinflammation, 11: 12
Fumagalli S, Perego C, Pischiutta F, Zanier ER, De Simoni MG (2015). The ischemic environment drives microglia and macrophage function. Front Neurol, 6: 81
Deng G, Carter J, Traystman RJ, Wagner DH, Herson PS (2014). Pro-inflammatory T-lymphocytes rapidly infiltrate into the brain and contribute to neuronal injury following cardiac arrest and cardiopulmonary resuscitation. J Neuroimmunol, 274: 132-140
Goverman J (2009). Autoimmune T cell responses in the central nervous system. Nat Rev Immunol, 9: 393-407
Chu HX, Kim HA, Lee S, Moore JP, Chan CT, Vinh A, et al. (2014). Immune cell infiltration in malignant middle cerebral artery infarction: comparison with transient cerebral ischemia. J Cereb Blood Flow Metab, 34: 450-459
Suidan GL, McDole JR, Chen Y, Pirko I, Johnson AJ (2008). Induction of blood brain barrier tight junction protein alterations by CD8 T cells. PLoS One, 3: e3037
Suidan GL, Dickerson JW, Chen Y, McDole JR, Tripathi P, Pirko I, et al. (2010). CD8 T cell-initiated vascular endothelial growth factor expression promotes central nervous system vascular permeability under neuroinflammatory conditions. J Immunol, 184: 1031-1040
Gupta A, Nair S, Schweitzer AD, Kishore S, Johnson CE, Comunale JP, et al. (2012). Neuroimaging of cerebrovascular disease in the aging brain. Aging Dis, 3: 414-425
Ni J, Wu Z, Peterts C, Yamamoto K, Qing H, Nakanishi H (2015). The Critical Role of Proteolytic Relay through Cathepsins B and E in the Phenotypic Change of Microglia/Macrophage. J Neurosci, 35: 12488-12501
Katsumoto A, Lu H, Miranda AS, Ransohoff RM (2014). Ontogeny and functions of central nervous system macrophages. J Immunol, 193: 2615-2621
Dou H, Grotepas CB, McMillan JM, Destache CJ, Chaubal M, Werling J, et al. (2009). Macrophage delivery of nanoformulated antiretroviral drug to the brain in a murine model of neuroAIDS. J Immunol, 183: 661-669
Dou H, Ellison B, Bradley J, Kasiyanov A, Poluektova LY, Xiong H, et al. (2005). Neuroprotective mechanisms of lithium in murine human immunodeficiency virus-1 encephalitis. J Neurosci, 25: 8375-8385
Dou H, Birusingh K, Faraci J, Gorantla S, Poluektova LY, Maggirwar SB, et al. (2003). Neuroprotective activities of sodium valproate in a murine model of human immunodeficiency virus-1 encephalitis. J Neurosci, 23: 9162-9170