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Aging and disease    2018, Vol. 9 Issue (3) : 467-479     DOI: 10.14336/AD.2018.0130
Orginal Article |
Splenectomy Fails to Provide Long-Term Protection Against Ischemic Stroke
Ran Yuanyuan1,2, Liu Zongjian1,2,*, Huang Shuo1,2, Shen Jiamei1, Li Fengwu1, Zhang Wenxiu2, Chen Chen1, Geng Xiaokun1, Ji Zhili1, Du Huishan1, Hu Xiaoming1,3,*
1China-America Institute of Neuroscience, Beijing Luhe Hospital, Capital Medical University, Beijing, China
2Central Laboratory, Beijing Luhe Hospital, Capital Medical University, Beijing, China
3Pittsburgh Institute of Brain Disorders and Recovery, and Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA
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Abstract  

Splenectomy before or immediately after stroke provides early brain protection. This study aims to explore the effect of splenectomy on long-term neurological recovery after stroke, which is currently lacking in the field. Adult male rats were randomized into splenectomy or sham groups and then subjected to 90 min of middle cerebral artery occlusion (MCAO). Spleen was removed right upon reperfusion or 3d after MCAO. Infarct volume, neurological functions, and peripheral immune cell populations were assessed up to 28d after stroke. The results show that delayed removal of spleen did not reduce brain tissue loss and showed no effect on sensorimotor function (Rotarod, beam balance, forelimb placing, grid walk, and adhesive removal tests) or cognitive function (Morris water maze). Spleen removal immediately upon reperfusion, although significantly reduced the infarct size and immune cell infiltration 3d after MCAO, also failed to promote long-term recovery. Flow cytometry analysis demonstrated that immediate splenectomy after MCAO resulted in a prolonged decrease in the percentage of CD3+CD4+ and CD3+CD8+ T cells in total lymphocytes as compared to non-splenectomy MCAO rats. In contrast, the percentage of CD3-CD45RA+ B cells was significantly elevated after splenectomy. As a result, the ratio of T/B cells was significantly reduced in stroke rats with splenectomy. In conclusion, delayed splenectomy failed to provide long-term protection to the ischemic brain or improve functional recovery. The acute neuroprotective effect achieved by early splenectomy after stroke cannot last for long term. This loss of neuroprotection might be related to the prolonged disturbance in the T cell to B cell ratio.

Keywords cerebral ischemia      splenectomy      lymphocytes      neurological function     
Corresponding Authors: Liu Zongjian,Hu Xiaoming   
About author:

These authors contributed equally to this paper.

Issue Date: 05 June 2018
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Ran Yuanyuan
Liu Zongjian
Huang Shuo
Shen Jiamei
Li Fengwu
Zhang Wenxiu
Chen Chen
Geng Xiaokun
Ji Zhili
Du Huishan
Hu Xiaoming
Cite this article:   
Ran Yuanyuan,Liu Zongjian,Huang Shuo, et al. Splenectomy Fails to Provide Long-Term Protection Against Ischemic Stroke[J]. Aging and disease, 2018, 9(3): 467-479.
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http://www.aginganddisease.org/EN/10.14336/AD.2018.0130     OR     http://www.aginganddisease.org/EN/Y2018/V9/I3/467
Figure 1.  Delayed splenectomy after ischemic stroke shows no effect on acute and long-term brain tissue loss. Rats were subjected to 90 min MCAO followed by delayed splenectomy at 3 days after ischemic stroke. (A) Illustration of the experimental timelines. (B) Body weight was examined up to 28 days after MCAO. n=6 rats for sham groups; n=9 rats for MCAO groups. ***p<0.001: Sham vs MCAO; +++p<0.001: Sham+Splenectomy vs MCAO+Splenectomy; ##p<0.01: MCAO vs MCAO+Splenectomy group by one-way ANOVA repeated measurement. (C) Representative images of spleen from sham+splenectomy and MCAO+splenectomy groups at 3 days after MCAO. (D) Quantification of spleen weight at 3 days after stroke. n=9 for each group. #p<0.05 by Student’s t-test. (E) Representative images of TTC-stained coronal brain sections from MCAO and MCAO + splenectomy groups at 5 days after MCAO. (F) Quantification of infract volume 5 days after MCAO. n=5-6 for each group. #p<0.05 by Student’s t-test. (G) Representative images of MAP-2 staining in MCAO and MCAO + splenectomy groups at 28 days after MCAO. (H) Quantification of brain tissue loss at 28 days after MCAO. n=5-6 for each group. Values are mean ± SEM.
Figure 2.  Delayed splenectomy after ischemic stroke failed to improve long-term neurological functions. Rats were exposed to 90 min of MCAO, followed by splenectomy or mock surgery right upon reperfusion as described in Materials and Methods. Sham-operated rats with or without splenectomy were used as control. All rats were allowed to survive for 28 days. (A) Rotarod test. The latency to fall off Rotarod was recoded. (B-C) The adhesive removal test. The time to touch (B) and the time to remove the tapes (C) were recorded. (D) Beam balance test. The performance on beam was scored 1-6. (E) The forelimb placing test on left (lesion side) forelimb. The number of successful placing out of 10 trials was recorded. (F) The grid walk test was assessed by counting the number of forelimb falling. (A-F) n=6 rats for sham groups; n=9 rats for MCAO groups. (G-I) Learning ability was examined by the Morris water maze test at 24-29 days after MCAO. n=5-6 rats per group. (G) Representative images of the swim paths at 28 days after MCAO (H) Latency to locate the submerged platform at 23-28 days after MCAO. (I) Swim speed at 28 days after MCAO. Values are mean ± SEM. Sham vs MCAO group: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by two-way ANOVA repeated measurements. Sham+Splenectomy vs MCAO+Splenectomy group: +p ≤ 0.05, +++p ≤ 0.001 by two-way ANOVA repeated measurements.
Figure 3.  Splenectomy immediately after ischemic stroke reduces brain infarct early after MCAO but shows no effect on long-term brain damage. Rats were subjected to 90 min of MCAO followed by immediate splenectomy. (A) Illustration of the experimental timelines. (B) Representative images of TTC-stained coronal brain sections at 3 days after 90 min MCAO. (C) Quantification of infarct areas in each slice. n=5-6 rats per group. #p<0.05 by two-way ANOVA. (D) Quantification of infract volume. n=5-6 rats per group. #p<0.05 by Student’s t-test. (E) Representative images of MAP-2-stained coronal brain sections at 28 days after MCAO. (F) Quantification of infarct volume at 28 days after MCAO. n=5-6 rats per group. Values are mean ± SEM.
Figure 4.  Splenectomy immediately after ischemic stroke cannot improve long-term functional recovery after MCAO. Rats were subjected to 90 min of MCAO followed by immediate splenectomy. (A) Body weight was recorded up to 28 days after MCAO. (B) The longa score was examined. (C) Beam balance test. The performance on beam was scored 1-6. (D) The forelimb placing test on left (lesion side) forelimb. The number of successful placing out of 10 trials was recorded. (E) The grid walk test was assessed by counting the number of forelimb falling. n=7 for MCAO groups; n=9 for MCAO+Splenectomy groups. (F) Cognitive ability was revealed by the Morris water maze test at 24-29 days after MCAO. n=6 rats per group. Values are mean ± SEM. #p ≤ 0.05, ##p ≤ 0.01 by two-way ANOVA repeated measurements.
Figure 5.  Immediate splenectomy, but not delayed splenectomy, reduces peripheral immune cell infiltration into the ischemic brain. Rats were subjected to 90 min MCAO followed by immediate splenectomy or delayed splenectomy (3d after MCAO) as described in Materials and Methods. (A) Representative gating strategy for CD45+ leukocytes, CD45+CD3+CD45RA- T cells, CD45+CD3-CD45RA+ B cells, and CD45+CD43+ monocytes. (B-E) Flow cytometry analysis on the number of immune cell infiltration at 3 days after MCAO and immediate splenectomy. (B) Quantification of the number of CD45+ leukocytes. (C) Quantification of the number of CD45+CD3+CD45RA- T cells. (D) Quantification of the number of CD45+CD3-CD45RA+ B cells. (E) Quantification of the number of CD45+CD43+ monocytes. (F-I) Flow cytometry analysis on the number of immune cell infiltration in rats with or without delayed splenectomy at 5 days after MCAO. (F) Quantification of the number of CD45+ leukocytes. (G) Quantification of the number of CD45+CD3+CD45RA- T cells. (H) Quantification of the number of CD45+CD3-CD45RA+ B cells. (I) Quantification of the number of CD45+CD43+ monocytes. n=6 rats per group. Values are mean ± SEM. #p<0.05, ##p<0.01 by 2-tailed Student’s t test.
Figure 6.  Splenectomy results in elevated ratio of B cells in lymphocytes for at least 28 days after MCAO. Rats were subjected to 90 min of MCAO followed by immediate splenectomy. (A-C) Representative flow cytometry plots of CD3+CD45RA- T cells (A), CD3-CD45RA+ B cells (A), CD3+CD4+ Th cells (B), CD3+CD8+ Tc cells (C) in total lymphocyte in MCAO and MCAO+splenectomy groups at pre-MCAO, 3, 14 and 28 days after MCAO. (D) The percentage of T and B cells among leukocytes at 3 days after MCAO. n=9-12 rats per group. *p<0.05: Sham vs MCAO; +p<0.05, ++p<0.01: Sham+splenectomy vs MCAO + splenectomy by one-way ANOVA. (E) The percentage of T+B cells in total leukocytes in MCAO and MCAO + splenectomy groups over time. (F-J) The percentage of CD3+CD45RA- T cells (F), CD3+CD4+ Th cells (G), CD3+CD8+ Tc (H), CD3-CD45RA+ B cells (I) in lymphocytes in MCAO and MCAO+splenectomy groups over time. (J) The ratio of T/B cells in MCAO and MCAO+splenectomy groups over time. Values are mean ± SEM. #p<0.05, ##p<0.01, ###p<0.001 by two-way ANOVA. n=9-12 rats per group.
Figure 7.  Splenectomy shows no effect on percentage of monocytes or NK cells for at least 28 days after MCAO. Rats were subjected to 90 min of MCAO followed by immediate splenectomy. (A-B) Representative flow cytometry plots of CD43+ monocytes (A) and CD3-CD161a+ NK cells (B) in total leukocytes in MCAO and MCAO+splenectomy groups at pre-MCAO, 3, 14, and 28 days after MCAO. (C) The percentage of CD43+ monocytes in total leukocytes in MCAO and MCAO+splenectomy groups over time. (D) The percentage of CD3-CD161a+ monocytes in total leukocytes in MCAO and MCAO+splenectomy groups over time. n=9-12 rats per group.
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