Background: Remote ischemic preconditioning (RIPC) of a limb has been reported to protect against ischemic stroke. Our previous results demonstrated that the RIPC-mediated neuroprotection is associated with alterations in circulating immune cell populations. Here, we evaluated the effect of the spleen, the largest reservoir of immune cells, on RIPC-mediated neuroprotection against stroke. Methods: Noninvasive RIPC was achieved by four repeated cycles of 5-min blood flow constriction in the hindlimbs using a tourniquet. The blood and spleens were collected before and 1 h and 3 days after preconditioning to analyze the effect of RIPC on the spleen and the correlation between splenic and peripheral lymphocytes. Moreover, spleen weight and splenic lymphocytes were compared in stroke rats with or without RIPC. Finally, splenectomy was made 1 day or 2 weeks before RIPC and 90-min middle cerebral artery occlusion (MCAO). The infarct areas and deficits were assessed. Blood was collected 1 h after RIPC and 3 days after MCAO to explore the impact of splenectomy on RIPC- induced neuroprotection and immune changes. The contralateral and ipsilateral hemispheres were collected 3 days after MCAO to detect the infiltration of immune cells after RIPC and splenectomy. + + Results: Flow cytometry analysis demonstrated that the RIPC promptly increased the percentages of CD3 CD8 + + cytotoxic T (Tc) cells in the spleen with a relatively delayed elevation in CD3 CD161 natural killer T (NKT) and − + CD3 CD45RA B lymphocytes. The percentages of circulating lymphocytes are positively correlated with the percentages of splenic lymphocytes in normal rats. Interestingly, RIPC resulted in negative correlations between the percentages of splenic and circulating T lymphocytes, while the correlation between splenic and circulating B lymphocytes remained positive. For animals subjected to RIPC followed by MCAO, RIPC increased splenic volume with an expansion of splenic lymphocytes 3 days after MCAO. Furthermore, the removal of the spleen 1 day or 2 weeks before RIPC and MCAO reduced the protective effect of RIPC on ischemic brain injury and reversed the effects of RIPC on circulating immune cell composition. RIPC significantly reduced brain infiltration of Tc and NKT cells. Prior splenectomy showed no effect on immune cell infiltration after RIPC and stroke. Conclusion: These results reveal an immunomodulatory effect of the spleen, effecting mainly the spleen-derived lymphocytes, during RIPC-afforded neuroprotection against cerebral ischemia. Keywords: Cerebral ischemia, Spleen, Lymphocytes, Limb remote ischemic preconditioning * Correspondence: firstname.lastname@example.org; email@example.com China-America Institute of Neuroscience, Beijing Luhe Hospital, Capital Medical University, Beijing 101100, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen et al. Journal of Neuroinflammation (2018) 15:167 Page 2 of 14 Background biphasic, with an initial decrease followed by a delayed Stroke is the leading cause of lethality and permanent increase . Such changes might correlate with the disability throughout the world. Accumulating evidence post-stroke inflammatory cascade. All these studies sug- indicates that brief episodes of ischemic pre-treatment gest an important role of spleen in stroke pathology. have a protective effect on subsequent cerebral However, the effect of the spleen in RIPC-mediated neu- ischemia-reperfusion injury [1–3]. Such ischemic pre- roprotection after stroke has been unexplored. conditioning may vary greatly in terms of location, tim- In this study, we used surgical splenectomy to study ing, and duration. The remote ischemic preconditioning the impact of the spleen on RIPC-mediated neuropro- (RIPC) of the limbs elicited tolerance against brain is- tection and peripheral lymphocyte changes after stroke. chemia through brief blood flow constriction [4, 5]. Spe- We discovered that RIPC resulted in dramatic changes cifically, the noninvasive RIPC strategy of bilateral limb in the compositions of immune cells (T lymphocyte sub- occlusion by tourniquet can be applied more readily to sets and B lymphocytes) in the spleen, which correlated humans and has therefore become a focus for clinical with the lymphocyte changes in blood. Splenectomy im- translation [6–8]. RIPC has been shown to prevent the paired the protective effect of RIPC on ischemic brain ischemic brain damage in patients during intracranial injury and reversed the RIPC-induced peripheral aneurysm treatment . Individuals with peripheral vas- lymphocyte changes, while showed no effect on immune cular hypoperfusion, similar to RIPC, have significantly cell infiltration into the ischemic brain. Taken together, more favorable stroke outcomes . Moreover, we have our results reveal a novel immunomodulatory effect of reported that brief repetitive bilateral upper arm ischemic the spleen underlying RIPC-afforded protection that preconditioning improves cerebral perfusion and reduces warrants further mechanistic investigations. recurrent strokes in patients with intracranial arterial stenosis . These clinical evidences suggest a neuropro- Methods tective effect of RIPC in stroke. However, the mechanism Animals and grouping underlying RIPC-afforded neuroprotection is not clear. Male Sprague-Dawley (SD) rats weighing 280–320 g (Vital The observation that RIPC in bilateral limbs can lead River Laboratory Animal Technology Co. Ltd., Beijing, to brain protection draws our attention to the peripheral China) were used in this study. Animal care was carried elements that function as the linkages between brain out in accordance with guidelines approved by the Capital and hind limbs. Immune cells, particularly lymphocytes, Medical University. All efforts were made to minimize any have been verified to play an important role in the pro- suffering and to reduce the number of animals used. gression and prognosis after stroke [11–13]. Our previ- A total of 132 rats were used in this study. In order to ous study has shown that noninvasive RIPC changes the detect the effect of RIPC on the spleen under the compositions of peripheral immune cells, ameliorates physiological status, 18 rats were randomly divided into the post-middle cerebral artery occlusion (MCAO) re- three groups (n = 6 each group), from which spleens and duction of T lymphocytes in the blood, and reverses the blood were collected at different time points: pre-RIPC, reduction of B cells . These results suggest that RIPC-1 h, and RIPC-3 days. To clarify the role of RIPC changes in immune cells, especially the lymphocytes, in on the spleen under the pathologic condition of stroke, − − circulation may be a mechanism underlying the RIPC- 24 rats were divided into three groups: RIPC MCAO , − + + + mediated neuroprotection. RIPC MCAO , and RIPC MCAO (n = 8 each group). The spleen, the largest peripheral immune organ, is a At 3 days post reperfusion, the spleen weight and reservoir for a variety of immune cells. Many studies splenocytes were analyzed. In order to demonstrate the have shown that during the acute phase of stroke, the role of the spleen in the RIPC-mediated neuroprotec- spleen dramatically shrinks accompanied by the release tion, 72 rats were randomly divided into three groups: of the immune cells into the circulation [14, 15]. The re- MCAO, RIPC + MCAO, and splenectomy + RIPC + leased immune cells subsequently infiltrate into the is- MCAO (n = 24 each group). Splenectomy was performed chemic brain and contribute to brain injury [16, 17]. 1 day before RIPC, and RIPC was achieved 1 h before Observations from human studies identified that sub- MCAO. Neurological deficits and motor behaviors were stantial post-stroke reduction in splenic volume oc- assessed over the 7 days after injury. Brain infarct, curred in approximately 40% of stroke patients. Some edema, and immune cell infiltration into the brain were biological factors, including age, sex, race, and history of measured 3 days post reperfusion. Moreover, the im- stroke, influenced post-stroke splenic contraction. mune changes in circulation were detected by flow cy- Patients with splenic contraction showed higher percent- tometry from the rats of RIPC + MCAO and ages of blood lymphocytes and higher inflammatory splenectomy + RIPC + MCAO groups to explore the cytokine levels in blood [14, 18]. Moreover, the post- underlying mechanism. The other 18 rats were randomly stroke changes in splenic volume over time were divided into three groups: MCAO, RIPC + MCAO, and Chen et al. Journal of Neuroinflammation (2018) 15:167 Page 3 of 14 splenectomy + RIPC + MCAO (n = 6 each group), with Cerebral edema assessment the splenectomy performed 2 weeks before RIPC to limit Brain water content (BWC) was quantified using the wet- the role of the body’s immune system responses due to a dry method as previously described . At 96 h post major surgery like a splenectomy. reperfusion, rats from each group were decapitated and the brains were rapidly removed. BWC was estimated in 3-mm coronal sections of the ipsilateral brain (or corre- Splenectomy sponding contralateral brain), centered upon the impact Rats were anesthetized with 5% isoflurane inhalation site. Tissue was immediately weighed (wet weight), then (Lunan Pharmaceutical Group Corporation, Shandong, dehydrated at 65 °C. The sample was reweighed 48 h later China) and maintained with 2% isoflurane inhalation to obtain a dry weight. The percentage of tissue water (rodent ventilator model: ZS-M; ZS Dichuang Technol- content was calculated using the following formula: ogy Co. Ltd., Beijing, China). A ~ 1-cm incision was BWC = [(wet weight) − (dry weight)/wet weight] × 100%. made on the left side of the abdominal cavity under the rib cage. The spleen was removed by cutting the mesen- 2,3,5-Triphenyltetrazolium chloride staining tery and connective tissue, and the splenic vessels were For 2,3,5-triphenyltetrazolium chloride (TTC) staining, cauterized. For sham-control rats, incisions were made the brains were removed rapidly on ice and sliced into without removing the spleen. seven coronal sections (2 mm thick). The sections were immersed in 2% TTC (Sigma-Aldrich, San Jose, CA) at 37 °C for 20 min and then fixed in 4% paraformaldehyde. Remote ischemic preconditioning Using the ImageJ 2× (National Institutes of Health, Noninvasive remote ischemic preconditioning (RIPC) of Bethesda, MD), the infarct size with edema correction the limbs was performed as described previously [6, 19]. was calculated as the area of the contralateral hemi- Rats were anesthetized with 5% isoflurane inhalation and sphere minus the non-infarcted area of the ipsilateral maintained with 2% isoflurane inhalation. Limb RIPC was hemisphere. Data were normalized to the non-ischemic achieved with four cycles of bilateral hindlimb ischemia brain and expressed as a percentage. (5 min/cycle, 40 min total). For each cycle, the proximal parts of the hindlimbs were tied with gauze ropes (13 cm × 13 cm) for 5 min, which was followed by 5 min of reperfu- Neurological deficit assessment sion with the gauze ropes untied. Non-RIPC animals were Neurological deficit assessment was performed by inves- exposed to the same anesthesia, and untied gauze ropes tigators blinded to the treatment groups. Rats were ex- were placed on both hindlimbs for 40 min. The precondi- amined and assessed before surgery, 0.5, 24, 48, and tioning procedure was performed 1 h before middle cere- 72 h after brain reperfusion. The Longa scoring system bral artery occlusion (MCAO) or sham surgery. was used as follows: 0 = no deficit, 1 = failure to extend left forepaw, 2 = circling to the left, 3 = dumping to the left, 4= failure of spontaneous walking and loss of con- Transient focal cerebral ischemia and reperfusion sciousness, and 5 = death. Immediately after RIPC, transient (90 min) focal cerebral ischemia was induced in male SD rats as previously de- Balance beam test scribed [20, 21]. Anesthesia was induced with 5% isoflur- The rats were placed on a narrow strip of wood ane and maintained with 2% isoflurane. Core body (30 × 1.3 cm ). The scoring standards were as follows: temperatures were maintained with a heating pad. The 1 = four limbs were all on the wood in a balanced cerebral blood flow (CBF) during the surgery was mea- situation, 2 = limbs of one side were able to grasp sured by laser Doppler perfusion monitoring with a laser the wood or shake on the wood, 3 = one or two Doppler probe (PeriFlux System 5000, Perimed AB, limbs slipped from the wood, 4 = three limbs slipped Sweden) interfaced to a laptop equipped with the from the wood, 5 = fell over after struggle on the PeriSoft data acquisition software (Perimed Systems, Inc., wood, 6 = suspended on the wood and fell over after Sweden) as previously described . The CBF data struggle, and 7 = fell over immediately without of each animal were obtained at three time points struggle at all . (baseline, ischemia, and 10 min after reperfusion) and presented as the percentages of baseline. The animals with less than 60% reduction of cerebral blood flow Foot fault test or showing no obvious sign of neurological deficits The rats were placed on a net with 2.3 × 2.3 cm mesh (neurological score less than 2) after MCAO surgery size. When the rats were walking, the number of times were excluded. Exposure of the right MCA without the front paws fell through over 2 min was recorded. occlusion was performed as sham surgery. The calculation formula = (the number of the wrong Chen et al. Journal of Neuroinflammation (2018) 15:167 Page 4 of 14 steps of the left front paw − the number of the wrong Statistical analysis steps of right front paw)/total steps. The number of rats in each experimental group was deter- mined by power analyses, informed by our past experience with similar measurements (α =0.05 and β =0.20). All Leukocyte harvest from blood, spleen, and brain data are expressed as mean ± SEM. Differences between Blood was taken from either the left or the right caudal two groups were evaluated for statistical significance using vein with 1 ml syringe and rapidly transferred to heparin aStudent’s t test. Differences between three or more saline anticoagulant tubes. For each rat, 0.5–1 ml of ven- groups were evaluated for statistical significance using ous blood was harvested. Spleen single-cell suspensions one-way ANOVA followed by Bonferroni post hoc test. were obtained by grinding followed by filtration through Normality of the distribution and homogeneity of variance a nylon mesh. For leukocyte retrieval, the samples from were assessed by the F test or Bartlett’s test before the the blood and spleen were centrifuged at 3000 rpm for Student’s t test and the ANOVA, respectively. A two- 4 min at 4 °C. The pellets were treated with the red tailed p value of < 0.05 was considered statistically signifi- blood cell lysis buffer (Beyotime Biotechnology Co. Ltd., cant. Correlation was analyzed with the Pearson Jiangsu, China) and washed twice with phosphate buffer correlation coefficient. The datasets used and/or analyzed solution (PBS, HyClone Laboratories Inc., Logan, UT) to during the current study are available from the corre- remove red blood cells. For brain cell isolation, the sponding author upon request. contralateral and ipsilateral hemispheres were cut into small pieces, ground, and filtered through a 70-μm cell Results strainer (Miltenyi Biotec, Bergisch Gladbach, Germany) RIPC increased the number of lymphocytes in the spleen to obtain a single cell suspension. Cell pellets were re- In order to identify the effect of RIPC on the spleen, spleens suspended in 70% Percoll (Solarbio, Beijing, China), were removed at different time points (before RIPC, 1 h transferred into 15 ml tubes, and then overlaid with 30% and 3 days after RIPC) and splenic lymphocytes were ana- Percoll. The mononuclear cells were harvested after cen- lyzed by flow cytometry (Fig. 1a). RIPC increased the per- trifuging at 2000 rpm for 25 min at room temperature centages of Tc cells as soon as 1 h after RIPC (Fig. 1b). The . The leukocytes were analyzed for membrane percentage of NKT cells was significantly increased 3 days marker expression by flow cytometry. after RIPC (Fig. 1b). Remarkably, RIPC dramatically in- creased the B cell percentage in the spleen 3 days after Flow cytometry analysis RIPC. These results suggest that RIPC can increase the per- Leukocyte classification by phenotypic analysis (the sur- centages of splenic lymphocyte populations. face expression of antigen markers) was performed by flow cytometry. Leukocytes were resuspended in PBS at a The effect of RIPC on the correlation between splenic and concentration of 2 × 10 /ml and stained with the peripheral lymphocytes fluorochrome-conjugated antibodies in darkness for In our previous study, we found that RIPC alone de- 30 min at room temperature. All antibodies were creased the percentage of T lymphocytes in peripheral purchased from Biolegend (San Diego, CA), including blood after stroke, while the percentage of B cells in per- fluorescein isothiocyanate (FITC) anti-rat CD3 (201403), ipheral blood was slightly reduced in rats subjected to peridinin chlorophyll protein (PerCP) anti-rat CD8a RIPC alone . Spleen is a major reservoir of blood cells. (201712), allophycocyanin (APC) anti-rat CD161 (205606), We therefore analyzed the correlation between splenic phycoerythrin (PE) anti-rat CD45RA (202307), and PE and peripheral lymphocytes in order to detect the effect of anti-rat CD4 (203307). Appropriate isotype-matched RIPC on the communication between spleen and blood. immunoglobulins were used as negative controls. An As shown in Fig. 2, there is a positive correlation between additional file shows this in more detail (please refer to splenic and peripheral lymphocytes in normal rats. RIPC Additional file 1: Fig. S1) Cells were analyzed on a induced a negative correlation between splenic and per- FACSCalibur flow cytometer with Cell Quest software ipheral Th, Tc, and NKT cells, while the correlations be- (Becton Dickinson, San Jose, CA, USA). The lymphocytes tween splenic and circulating B lymphocytes remained were gated on the scatter plots of forward scatter (FSC-H) positive. These results suggest that RIPC may prevent the and side scatter (SSC-H), excluding debris and cell efflux of T lymphocytes into the circulation while enhan- aggregates; T cell subsets and B cells were further gated in cing B cell generation and/or release from spleen. the P1 population based on their expression of specific + + markers. We defined the CD3 CD4 population as helper RIPC increased splenic volume and lymphocyte number + + T(Th) cells,CD3 CD8 population as cytotoxic T (Tc) after MCAO − + cells, CD3 CD45RA population as B lymphocytes, and In order to further clarify the effect of RIPC on the + + CD3 CD161a population as natural killer T (NKT) cells. spleen under the pathological state of stroke, the spleens Chen et al. Journal of Neuroinflammation (2018) 15:167 Page 5 of 14 Fig. 1 Limb remote ischemic preconditioning (RIPC) increases the splenic lymphocyte populations. RIPC was conducted by four cycles (10 min/cycle, 40 min total) of bilateral hindlimb ischemia. Spleens were removed before RIPC and 1 h and 3 days after RIPC. a Representative images of flow + + + + cytometry showing different splenic lymphocytes, including helper T (Th) cells (CD3 CD4 ), cytotoxic T (Tc) cells (CD3 CD8 ), natural killer T (NKT) + + − + (CD3 CD161 ) cells, and B (CD3 D45RA ). Indicated numbers are the mean percentages of targeted cells. b Statistical analysis of splenic lymphocyte populations prior to and after RIPC. n = 6 rats per group. Data are presented as means ± SEM, *p <0.05, **p < 0.01 versus corresponding lymphocytes before RIPC from MCAO rats with or without RIPC were collected RIPC can maintain the splenic volume and the number 3 days after reperfusion (Fig. 3a). We found that the of splenic lymphocyte populations after stroke. − − spleen shrank after MCAO (RIPC MCAO vs. − + RIPC MCAO ). However, RIPC could significantly Splenectomy reversed the protective effect of RIPC on maintain the volume and weight of the spleens ischemic brain injury − + + + (RIPC MCAO vs. RIPC MCAO ), as shown in Fig. 3b, c. Rats subjected to RIPC demonstrated significant reduc- Moreover, in rats without RIPC, the number of splenic tion in infarct volumes (Fig. 4a, b) and cerebral edema lymphocytes decreased after MCAO, accompanying the (Fig. 4c) compared to animals without RIPC 3 days reduced spleen volume. In contrast, the numbers of T after MCAO. Pre-stroke splenectomy attenuated the cell subsets and B lymphocytes in the spleens RIPC-induced reduction in brain infarct sizes and significantly increased in MCAO rats which underwent edema (Fig. 4b, c). Meanwhile, cerebral blood flow, de- RIPC (Fig. 3d–g). Together, these results suggest that tected during the MCAO surgery, was reduced to less Chen et al. Journal of Neuroinflammation (2018) 15:167 Page 6 of 14 Fig. 2 Correlation analyses between splenic and peripheral lymphocytes in rats with or without RIPC. Spleens and blood from caudal vein were collected from rats with or without RIPC. Different lymphocyte populations were detected by flow cytometry. Correlations between peripheral and splenic lymphocytes were analyzed. The data from rats without RIPC are presented in blue, and the data from rats with RIPC are presented in red Fig. 3 The effect of RIPC on splenic volume and lymphocyte populations followed by MCAO. a Experimental protocols. RIPC was conducted 1 h before MCAO. Non-RIPC animals were exposed to the same anesthesia for 40 min. Brain ischemia was induced by 90 min MCAO. Sham-operated animals underwent anesthesia and surgical exposure of the right middle cerebral artery without occlusion. Three groups of animals were prepared: − − − + + + RIPC MCAO ,RIPC MCAO , and RIPC MCAO . b Representative spleen images from each group. c Spleen weight of rats in each group. d–g − − Quantification of the number of different splenic lymphocytes. Data are expressed as fold change of control group (RIPC MCAO ). n = 8 rats per − − # ## − + group. Data are presented as means ± SEM, *p <0.05, **p < 0.01 versus RIPC MCAO rats, p <0.05, p < 0.01 versus RIPC MCAO rats Chen et al. Journal of Neuroinflammation (2018) 15:167 Page 7 of 14 Fig. 4 (See legend on next page.) Chen et al. Journal of Neuroinflammation (2018) 15:167 Page 8 of 14 (See figure on previous page.) Fig. 4 Splenectomy removes the protective effect of RIPC on ischemic brain injury. Splenectomy was conducted 1 day before 40 min RIPC and 90 min MCAO. Non-splenectomy animals underwent anesthesia and surgical exposure of the spleen without removal. a Experimental protocols. Three groups of animals were prepared: MCAO, RIPC + MCAO, and splenectomy + RIPC + MCAO (n = 18 per group). b Representative TTC images of animals from each group, n = 8 per group. The infarct areas are outlined with blue lines. c Brain water content of contralateral and ipsilateral hemispheres from each rat was detected through the wet-dry method. The calculating formula is BWC = [(wet weight) − (dry weight)/wet weight] × 100%, n = 4 per group. d Cerebral blood flow during the MCAO was measured at three time points: baseline, ischemia, and reperfusion. Data are normalized to baseline and expressed as percentages. e Body weight of all rats from each group was measured over the 7 days after MCAO. f–h Neurological function and sensorimotor behaviors were assessed by neurological deficit score, beam balance score, and foot fault index, n = 6 rats per group. Data # ## are presented as means ± SEM, *p < 0.05, **p < 0.01 versus MCAO group. p <0.05, p < 0.01 versus RIPC + MCAO rats than 40% of baseline during ischemia and reestablished vs. ④ in Fig. 6e–h). Splenectomy showed no significant to 70% of baseline after reperfusion without significant effect on RIPC-induced T lymphocyte changes in stroke differences between groups (Fig. 4d). Moreover, consist- rats (Fig. 6i). ent with the increased brain injury, rats subjected to splenectomy + RIPC exhibited decreased body weight The effect of RIPC and splenectomy on immune cell (Fig. 4e) and impaired neurological function and infiltration into the ischemic brain sensorimotor behavior, as manifested by the neuro- MCAO significantly increased the infiltration of Tc and logical deficit score, beam balance score, and foot fault NKT cells in the ipsilateral hemispheres, which was index, lasting at least through 7 days post reperfusion inhibited by RIPC (Fig. 7c–f). Splenectomy prior to (Fig. 4f–h). RIPC showed no significant effect on T lymphocyte infil- The body’s immune system is still responding to splen- tration into the ischemic brains as compared to RIPC ectomy when MCAO was performed 1 day later, pos- alone group. RIPC or splenectomy had minimal effect sibly causing immune cell fluctuations and confounding on Th cell infiltration after stroke (Fig. 7a, b). There was the data on the effects of splenectomy on RIPC and no obvious infiltration of B cells into the brain 3 days stroke. Therefore, splenectomy was performed 2 weeks after stroke (Fig. 7g, h). Although RIPC and splenectomy prior to RIPC and MCAO to confirm the role of the induced a robust elevation of peripheral B cell popula- spleen in RIPC-afforded neuroprotection. Splenectomy tion (Fig. 6b, c), they had no significant effect in B 2 weeks before RIPC and MCAO fully removed the pro- lymphocyte infiltration 3 days after MCAO (Fig. 7g, h). tective effect of RIPC on ischemic brain injury (Fig. 5). Taken together, these results demonstrate that splenec- Discussion tomy removed the protective effect of RIPC on ischemic Ischemic stroke results in a rapid systemic inflammatory brain injury, suggesting an important role for the spleen response, which exacerbates the initial infarct [26, 27]. in RIPC-mediated neuroprotection against stroke. Modulation of immune and inflammatory responses rep- resents an important target for improving clinical out- Splenectomy removed RIPC-induced changes in comes after stroke . Our previous study indicated peripheral lymphocyte population after MCAO that RIPC dramatically altered the levels of multiple im- Our previous studies revealed dramatic immune changes mune cell populations and cytokines in blood . How- in peripheral blood composition after RIPC neuroprotec- ever, how RIPC induced these alterations in the tion against cerebral ischemia . Here, we explored the peripheral immune system has remained elusive. effect of splenectomy on RIPC-induced changes in circu- Over the past decade, the contribution of the spleen to lating immune populations after stroke (Fig. 6a). As ischemic brain damage has gained considerable attention shown in Fig. 6b, c, RIPC robustly elevated the percent- in stroke research. There is increasing evidence that the age of peripheral B cells in animals without splenectomy spleen-mediated peripheral immune changes contribute after MCAO (① vs. ②). Splenectomy alone significantly to progressive injury in a number of acute neurological elevated the blood B cell population after RIPC (① vs. disorders, including spinal cord injury, hemorrhagic ③), whereas levels of B cells in animals with splenec- stroke, and traumatic brain injury [29–32]. A recent tomy remained unchanged after MCAO (③ vs. ④). The study demonstrated that multipotent adult progenitor increase of blood B cell composition was maximized cells enhance recovery after stroke by modulating the after splenectomy + RIPC so there is no further increase splenic immune response . However, whether RIPC after MCAO (②-① vs. ④-③ in Fig. 6d). modulates the splenic response is not clear. In this Splenectomy increased the blood composition of Th study, we discovered that in normal subjects, RIPC cells after RIPC (① vs. ③ in Fig. 6e), and RIPC signifi- increased the splenic lymphocyte populations; mean- cantly decreased the peripheral T lymphocytes both in while, RIPC decreased the T lymphocytes and robustly animals without or with splenectomy (① vs. ② and ③ elevated the percentage of B cells in peripheral blood Chen et al. Journal of Neuroinflammation (2018) 15:167 Page 9 of 14 Fig. 5 Splenectomy 2 weeks before RIPC and MCAO also removed the protective effect of RIPC on ischemic brain injury. Splenectomy was conducted 2 weeks before 40 min RIPC and 90 min MCAO. Non-splenectomy animals underwent anesthesia and surgical exposure of the spleen without removal. a Experimental protocols. Three groups of animals were prepared: MCAO, RIPC + MCAO, and splenectomy + RIPC + MCAO (n = 6 each group). b Representative TTC images of animals from each group. The infarct areas are outlined with blue lines. c Neurological deficit assessment was performed with the Longa scoring system during the 3 days after MCAO. d Body weight of all rats from each group were measured during the 3 days # ## after MCAO. Data are presented as means ± SEM, *p <0.05, **p < 0.01 versus MCAO group. p <0.05, p < 0.01 versus RIPC + MCAO rats (Figs. 1 and 2). Under the pathologic condition of We have shown previously that RIPC could reduce stroke, RIPC also maintained the splenic volume and brain infarct early after stroke . Splenectomy 2 weeks lymphocyte numbers (Fig. 3). Moreover, removal of before stroke has been shown to be neuroprotective thespleen1 dayor2weeksbeforeRIPCdeprived [34–36], while splenectomy shortly before stroke showed RIPC-afforded neuroprotection 3 days post reperfu- minimal protection . Interestingly, in our study, sion, which lasted through day 7 after stroke (Figs. 4 splenectomy performed either 1 day or 2 weeks before and 5). Splenectomy also reversed RIPC-induced RIPC and MCAO reduced and eliminated RIPC- peripheral lymphocyte changes (Fig. 6). These results afforded neuroprotection, respectively. Splenectomy suggest that immunomodulation of the splenic shortly before MCAO may initiate an acute activation of response by RIPC may create a special immune envir- the immune response, leading to tissue damage that can- onment that influences the progression of ischemic cels its protective effects in the brain . This is sup- stroke. ported by the fact that peripheral lymphocytes increased Chen et al. Journal of Neuroinflammation (2018) 15:167 Page 10 of 14 Fig. 6 The effect of splenectomy on peripheral lymphocytes before RIPC and MCAO. a Experimental protocols. Two groups of animals were adopted: non-splenectomy + RIPC + MCAO and splenectomy + RIPC + MCAO. Blood was collected from the caudal tail vein at 1 h after preconditioning (① and ③) and 3 days after brain reperfusion (② and ④). b–d Flow cytometry analysis of B lymphocytes. e–i Flow cytometry analysis of T lymphocyte subsets. ②-① reflects the change of lymphocyte populations prior to and after RIPC + MCAO without splenectomy; ④-③ reflects the change of lymphocyte populations prior to and after MCAO with splenectomy and RIPC. Data are expressed as means ± SEM for 12 independent experiments. # ## *p <0.05, **p < 0.01 versus ① or ②-①; p <0.05, p < 0.01 versus ③ in the blood after splenectomy. Such immune cell eleva- a peripheral immune cell source should not be ignored. tion might be due to the mobilization of immune cells Interestingly, our data further showed that splenectomy from other organs or tissues. For example, bone marrow 2 weeks before RIPC and MCAO fully removed the pro- is the primary site of hematopoiesis. All types of myeloid tective effect of RIPC on ischemic brain injury (Fig. 5), and lymphoid lineages are created in the bone marrow. suggesting that the spleen may contribute to RIPC- Under normal conditions, lymphoid cells must migrate induced protection against CNS injury through compli- to secondary lymph organs, such as the spleen and cated mechanisms of immune modulation over time. lymph nodes, to complete maturation. However, when Lymphocytes, especially T lymphocytes, play a de- the spleen was removed, the role of the bone marrow as structive role after stroke [39, 40]. In our study, we Chen et al. Journal of Neuroinflammation (2018) 15:167 Page 11 of 14 Fig. 7 The effect of RIPC and splenectomy on immune cell infiltration into the ischemic brains. Three groups of animals were prepared: MCAO, RIPC + MCAO, and Sp-RIPC + MCAO (n = 6 per group). Splenectomy was conducted 1 day before 40 min RIPC and 90 min MCAO. Non-splenectomy animals underwent anesthesia and surgical exposure of the spleen without removal. The brains were collected at 3 days after MCAO. a, c, e, g Representative plots of FACS showing Th, Tc, NKT, and B cells in contralateral and ipsilateral hemispheres. Indicated numbers are the mean percentages of targeted cells. b, d, f, h Statistical analysis of different lymphocyte populations in contralateral and ipsilateral hemispheres. Data are normalized to the mean of respective contralateral brains and presented as means ± SEM, *p < 0.05, **p < 0.01 versus corresponding lymphocytes in contralateral hemispheres found that exposure to RIPC could dramatically reduce decrease of T lymphocytes in the periphery might be the number of T cells especially CD8 T cells and NKT due to the retention of circulating cells in the spleen and cells in the blood prior to stroke. Interestingly, we found impaired outflow of splenic lymphocytes into the blood. an increase of splenic T cells after RIPC in this study. It is unclear from the current study how RIPC increases Correlation analysis indicates that the splenic splenic T or B lymphocytes so robustly after RIPC. We lymphocytes were proportional to the blood know that CD4 and CD8 T cells are mobilized from the lymphocytes under normal physiological conditions, spleen and redistributed to the non-lymphoid tissues fol- which is consistent with the public knowledge that lowing recognition of cognate antigens in the splenic circulating immune cells continuously migrate into and white pulp . Existing literature shows that bacteria/ out of the resting spleen . However, RIPC reversed virus infection can induce recruitment of monocytes, the positive correlation between splenic and peripheral NK cells, and neutrophils to the spleen, which is import- T lymphocytes, suggesting that the RIPC-induced ant for the infection prognosis through downstream Chen et al. Journal of Neuroinflammation (2018) 15:167 Page 12 of 14 adaptive immunity regulation [42–45]. However, Conclusion whether RIPC can also induce recruitment of immune Our study suggests that the spleen plays a pivotal role cells to the spleen or inhibit the egress of spleen im- in RIPC-mediated alterations in the peripheral im- mune cells into the circulation warrants further explor- mune system and following neuroprotection. Immu- ation. Moreover, in our previous research, we found a nomodulation of the splenic response by noninvasive dramatic reduction in the percentage of circulating T cell remote ischemic preconditioning may create a favor- + + subsets (including CD4 T cell, CD8 T cell, and NKT able systemic immune milieu to protect against ische- cells) after stroke, which was ameliorated by RIPC after mic brain injury and could serve as a platform to stroke . We found here that RIPC reduced the develop therapeutic approaches for stroke as well as infiltration of Tc and NKT cells into the ischemic brain, other acute CNS injuries in the future. which is consistent with our hypothesis that RIPC reduced the influx of immune cells after stroke. Additional file Splenectomy showed minimal effects on RIPC-induced reduction of T cells in blood or infiltration into the is- Additional file 1: The flow cytometry scatter plots for isotype controls that were used as negative control. (PDF 120 kb) chemic brain after stroke, suggesting that splenectomy removed the neuroprotective effect of RIPC through Abbreviations mechanisms probably independent of immune cell infil- APC: Allophycocyanin; BWC: Brain water content; CBF: Cerebral blood flow; tration after stroke. CNS: Central nervous system; FITC: Fluorescein isothiocyanate; FSC: Forward In our study, B cells show the most dramatic changes scatter; MCAO: Middle cerebral artery occlusion; PE: Phycoerythrin; PerCP: Peridinin chlorophyll protein; RIPC: Remote ischemic preconditioning; after RIPC. Our previous data revealed an elevation of cir- SD: Sprague-Dawley; Sp: Splenectomy; SSC: Side scatter; Tc: Cytotoxic T; culating B cells in RIPC animals, which may be essential for Th: Helper T; TTC: 2,3,5-Triphenyltetrazolium chloride RIPC-afforded protection against ischemia . In this study, there is also an intriguing increase of splenic B lymphocytes Acknowledgements The authors thank Mr. Tao Xu for the technical support of the cytometry at 3 days after RIPC. Correlation analysis shows that the B flow analysis and Xuan Liu and Haiteng Ji for the animal care and lymphocytes are elevated in the spleen and periphery at the maintenance. We thank Michael Hezel for his editorial assistance. same time even after RIPC, indicating that an upregulation of B cells in the blood might be due to increased B cell gen- Funding This work was supported by the China National Natural Science Funds (No. eration and release from the spleen. It is noted that splenec- 81571152 and 81670380) and the Luhe Hospital Research Foundation (No. tomy also mediated a high percentage of B cells in the lhq201502). blood. However, these non-splenic B cells might come from different sources, such as the bone marrow or lymph nodes, Availability of data and materials The datasets used and/or analyzed during the current study are available and possess functions distinct from B cells in the spleen. from the corresponding author on reasonable request. RIPC and splenectomy showed minimal effect on B cell in- filtration. Different from the detrimental role of T lympho- Authors’ contributions CC designed and performed the experiments, collected and analyzed data, cytes in ischemic brain injury, recent findings demonstrate and drafted the manuscript. WJ performed all the animal experiments, that B lymphocytes play a heterogeneous role in the adap- including splenectomy, MCAO surgery, and RIPC procedure. ZJL contributed tive immune response to stroke . On the one hand, to the experimental design and manuscript. FWL conducted the behavior tests. YJ performed some experiments, including the detection of brain many studies show a special population of regulatory B infarct and edema. YLZ performed some of MCAO surgery and detected the lymphocytes secrete IL-10 and are protective in cerebral cerebral blood flow. YYR participated in the collection of blood samples. MY ischemia/reperfusion injury [46, 47]. A recent study performed flow cytometry analysis and edited the manuscript. XKG contributed to the experimental design. XMJ critically revised the reported that repetitive hypoxic preconditioning induces an manuscript. HSD supervised the research group and revised the manuscript. immunosuppressive B cell phenotype prior to stroke onset, XMH designed and supervised the study and critically revised the thereby preventing the infiltration of other immune cells manuscript. All authors read and approved the final manuscript. into the brain and protecting the brain against ischemic Ethics approval attack . On the other hand, B cell and antibody All animal experiments were approved by the Capital Medical University accumulation in the infarct region correlated with Institutional Animal Care and Use Committee and were performed in impairments in hippocampal long-term potentiation, accordance with guidelines approved by the Capital Medical University for the Care and Use of Laboratory Animals. resulting in short-term memory deficit weeks after stroke , which suggests the post-stroke detrimen- Competing interests tal effects of B cells. It is therefore warranted to ex- The authors declare that they have no competing interests. plore whether the robustly increased peripheral B lymphocytes after RIPC or splenectomy possessed Publisher’sNote distinct functions and played critical roles in stroke Springer Nature remains neutral with regard to jurisdictional claims in outcomes. published maps and institutional affiliations. Chen et al. 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Journal of Neuroinflammation – Springer Journals
Published: May 28, 2018
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