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Aging and disease    2018, Vol. 9 Issue (3) : 412-425     DOI: 10.14336/AD.2017.0926
Orginal Article |
Invasion of Peripheral Immune Cells into Brain Parenchyma after Cardiac Arrest and Resuscitation
Zhang Can1, Brandon Nicole R.1, Koper Kerryann1, Tang Pei1,2,3, Xu Yan1,2,4,5,*, Dou Huanyu6,7,*
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
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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.

Keywords immune responses      cerebral global ischemia      CD11b+CD45+      CD8+      blood-brain barrier      MRI inflammation     
Corresponding Authors: Xu Yan,Dou Huanyu   
About author:

These two authors contributed equally to this work.

Issue Date: 05 June 2018
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Zhang Can
Brandon Nicole R.
Koper Kerryann
Tang Pei
Xu Yan
Dou Huanyu
Cite this article:   
Zhang Can,Brandon Nicole R.,Koper Kerryann, et al. Invasion of Peripheral Immune Cells into Brain Parenchyma after Cardiac Arrest and Resuscitation[J]. Aging and disease, 2018, 9(3): 412-425.
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Lymphocytic Markers
CD3eSyrian Hamster/MouseV450BD Biosciences
CD4Rat/MousePerCP-Cy5.5BD Biosciences
CD8aRat/MouseBUV395BD Biosciences
CD25Rat/MousePE-Cy7BD Biosciences
CD45Rat/MouseAPC-Cy7BD Biosciences
FoxP3Rat/MouseAlexa Fluor647BD Biosciences
CD28Syrian Hamster/MousePEBD Biosciences
Phagocytic Markers
CD11bRat/MouseBUV395BD Biosciences
CD11cArmenian Hamster/MouseAPCeBiosciences
CD45Rat/MouseAPC-Cy7BD Biosciences
CD80Armenian Hamster/MouseFITCBD Biosciences
CD86Rat/MouseV450BD Biosciences
F4/80Rat/MousePEBD Biosciences
Ly-6CRat/MousePerCP-Cy5.5BD Biosciences
Ly-6GRat/MouseBV711BD Biosciences
Table 1  Antibodies Used in Flow Cytometry.
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.
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