Background
Stroke is the leading cause of lethality and permanent disability throughout the world. Accumulating evidence indicates that brief episodes of ischemic pre-treatment have a protective effect on subsequent cerebral ischemia-reperfusion injury [
1‐
3]. Such ischemic preconditioning may vary greatly in terms of location, timing, and duration. The remote ischemic preconditioning (RIPC) of the limbs elicited tolerance against brain ischemia through brief blood flow constriction [
4,
5]. Specifically, the noninvasive RIPC strategy of bilateral limb occlusion by tourniquet can be applied more readily to humans and has therefore become a focus for clinical translation [
6‐
8]. RIPC has been shown to prevent the ischemic brain damage in patients during intracranial aneurysm treatment [
9]. Individuals with peripheral vascular hypoperfusion, similar to RIPC, have significantly more favorable stroke outcomes [
10]. Moreover, we have reported that brief repetitive bilateral upper arm ischemic preconditioning improves cerebral perfusion and reduces recurrent strokes in patients with intracranial arterial stenosis [
7]. These clinical evidences suggest a neuroprotective effect of RIPC in stroke. However, the mechanism underlying RIPC-afforded neuroprotection is not clear.
The observation that RIPC in bilateral limbs can lead to brain protection draws our attention to the peripheral elements that function as the linkages between brain and hind limbs. Immune cells, particularly lymphocytes, have been verified to play an important role in the progression and prognosis after stroke [
11‐
13]. Our previous study has shown that noninvasive RIPC changes the compositions of peripheral immune cells, ameliorates the post-middle cerebral artery occlusion (MCAO) reduction of T lymphocytes in the blood, and reverses the reduction of B cells [
8]. These results suggest that changes in immune cells, especially the lymphocytes, in circulation may be a mechanism underlying the RIPC-mediated neuroprotection.
The spleen, the largest peripheral immune organ, is a reservoir for a variety of immune cells. Many studies have shown that during the acute phase of stroke, the spleen dramatically shrinks accompanied by the release of the immune cells into the circulation [
14,
15]. The released immune cells subsequently infiltrate into the ischemic brain and contribute to brain injury [
16,
17]. Observations from human studies identified that substantial post-stroke reduction in splenic volume occurred in approximately 40% of stroke patients. Some biological factors, including age, sex, race, and history of stroke, influenced post-stroke splenic contraction. Patients with splenic contraction showed higher percentages of blood lymphocytes and higher inflammatory cytokine levels in blood [
14,
18]. Moreover, the post-stroke changes in splenic volume over time were biphasic, with an initial decrease followed by a delayed increase [
18]. Such changes might correlate with the post-stroke inflammatory cascade. All these studies suggest an important role of spleen in stroke pathology. However, the effect of the spleen in RIPC-mediated neuroprotection after stroke has been unexplored.
In this study, we used surgical splenectomy to study the impact of the spleen on RIPC-mediated neuroprotection and peripheral lymphocyte changes after stroke. We discovered that RIPC resulted in dramatic changes in the compositions of immune cells (T lymphocyte subsets and B lymphocytes) in the spleen, which correlated with the lymphocyte changes in blood. Splenectomy impaired the protective effect of RIPC on ischemic brain injury and reversed the RIPC-induced peripheral lymphocyte changes, while showed no effect on immune cell infiltration into the ischemic brain. Taken together, our results reveal a novel immunomodulatory effect of the spleen underlying RIPC-afforded protection that warrants further mechanistic investigations.
Methods
Animals and grouping
Male Sprague-Dawley (SD) rats weighing 280–320 g (Vital River Laboratory Animal Technology Co. Ltd., Beijing, China) were used in this study. Animal care was carried out in accordance with guidelines approved by the Capital Medical University. All efforts were made to minimize any suffering and to reduce the number of animals used.
A total of 132 rats were used in this study. In order to detect the effect of RIPC on the spleen under the physiological status, 18 rats were randomly divided into three groups (n = 6 each group), from which spleens and blood were collected at different time points: pre-RIPC, RIPC-1 h, and RIPC-3 days. To clarify the role of RIPC on the spleen under the pathologic condition of stroke, 24 rats were divided into three groups: RIPC−MCAO−, RIPC−MCAO+, and RIPC+MCAO+ (n = 8 each group). At 3 days post reperfusion, the spleen weight and splenocytes were analyzed. In order to demonstrate the role of the spleen in the RIPC-mediated neuroprotection, 72 rats were randomly divided into three groups: MCAO, RIPC + MCAO, and splenectomy + RIPC + MCAO (n = 24 each group). Splenectomy was performed 1 day before RIPC, and RIPC was achieved 1 h before MCAO. Neurological deficits and motor behaviors were assessed over the 7 days after injury. Brain infarct, edema, and immune cell infiltration into the brain were measured 3 days post reperfusion. Moreover, the immune changes in circulation were detected by flow cytometry from the rats of RIPC + MCAO and splenectomy + RIPC + MCAO groups to explore the underlying mechanism. The other 18 rats were randomly divided into three groups: MCAO, RIPC + MCAO, and splenectomy + RIPC + MCAO (n = 6 each group), with the splenectomy performed 2 weeks before RIPC to limit the role of the body’s immune system responses due to a major surgery like a splenectomy.
Splenectomy
Rats were anesthetized with 5% isoflurane inhalation (Lunan Pharmaceutical Group Corporation, Shandong, China) and maintained with 2% isoflurane inhalation (rodent ventilator model: ZS-M; ZS Dichuang Technology Co. Ltd., Beijing, China). A ~ 1-cm incision was made on the left side of the abdominal cavity under the rib cage. The spleen was removed by cutting the mesentery and connective tissue, and the splenic vessels were cauterized. For sham-control rats, incisions were made without removing the spleen.
Remote ischemic preconditioning
Noninvasive remote ischemic preconditioning (RIPC) of the limbs was performed as described previously [
6,
19]. Rats were anesthetized with 5% isoflurane inhalation and maintained with 2% isoflurane inhalation. Limb RIPC was achieved with four cycles of bilateral hindlimb ischemia (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 reperfusion with the gauze ropes untied. Non-RIPC animals were exposed to the same anesthesia, and untied gauze ropes were placed on both hindlimbs for 40 min. The preconditioning procedure was performed 1 h before middle cerebral artery occlusion (MCAO) or sham surgery.
Transient focal cerebral ischemia and reperfusion
Immediately after RIPC, transient (90 min) focal cerebral ischemia was induced in male SD rats as previously described [
20,
21]. Anesthesia was induced with 5% isoflurane and maintained with 2% isoflurane. Core body temperatures were maintained with a heating pad. The cerebral blood flow (CBF) during the surgery was measured by laser Doppler perfusion monitoring with a laser Doppler probe (PeriFlux System 5000, Perimed AB, Sweden) interfaced to a laptop equipped with the PeriSoft data acquisition software (Perimed Systems, Inc., Sweden) as previously described [
22]. The CBF data of each animal were obtained at three time points (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 or showing no obvious sign of neurological deficits (neurological score less than 2) after MCAO surgery were excluded. Exposure of the right MCA without occlusion was performed as sham surgery.
Cerebral edema assessment
Brain water content (BWC) was quantified using the wet-dry method as previously described [
23]. At 96 h post 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 corresponding contralateral brain), centered upon the impact site. Tissue was immediately weighed (wet weight), then dehydrated at 65 °C. The sample was reweighed 48 h later to obtain a dry weight. The percentage of tissue water content was calculated using the following formula: BWC = [(wet weight) − (dry weight)/wet weight] × 100%.
2,3,5-Triphenyltetrazolium chloride staining
For 2,3,5-triphenyltetrazolium chloride (TTC) staining, the brains were removed rapidly on ice and sliced into 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. Using the ImageJ 2× (National Institutes of Health, Bethesda, MD), the infarct size with edema correction was calculated as the area of the contralateral hemisphere minus the non-infarcted area of the ipsilateral hemisphere. Data were normalized to the non-ischemic brain and expressed as a percentage.
Neurological deficit assessment
Neurological deficit assessment was performed by investigators blinded to the treatment groups. Rats were examined and assessed before surgery, 0.5, 24, 48, and 72 h after brain reperfusion. The Longa scoring system 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 consciousness, and 5 = death.
Balance beam test
The rats were placed on a narrow strip of wood (30 × 1.3 cm
2). The scoring standards were as follows: 1 = four limbs were all on the wood in a balanced situation, 2 = limbs of one side were able to grasp the wood or shake on the wood, 3 = one or two limbs slipped from the wood, 4 = three limbs slipped from the wood, 5 = fell over after struggle on the wood, 6 = suspended on the wood and fell over after struggle, and 7 = fell over immediately without struggle at all [
24].
The rats were placed on a net with 2.3 × 2.3 cm mesh size. When the rats were walking, the number of times the front paws fell through over 2 min was recorded. The calculation formula = (the number of the wrong steps of the left front paw − the number of the wrong steps of right front paw)/total steps.
Leukocyte harvest from blood, spleen, and brain
Blood was taken from either the left or the right caudal vein with 1 ml syringe and rapidly transferred to heparin saline anticoagulant tubes. For each rat, 0.5–1 ml of venous blood was harvested. Spleen single-cell suspensions were obtained by grinding followed by filtration through a nylon mesh. For leukocyte retrieval, the samples from the blood and spleen were centrifuged at 3000 rpm for 4 min at 4 °C. The pellets were treated with the red blood cell lysis buffer (Beyotime Biotechnology Co. Ltd., Jiangsu, China) and washed twice with phosphate buffer solution (PBS, HyClone Laboratories Inc., Logan, UT) to remove red blood cells. For brain cell isolation, the contralateral and ipsilateral hemispheres were cut into small pieces, ground, and filtered through a 70-μm cell strainer (Miltenyi Biotec, Bergisch Gladbach, Germany) to obtain a single cell suspension. Cell pellets were resuspended in 70% Percoll (Solarbio, Beijing, China), transferred into 15 ml tubes, and then overlaid with 30% Percoll. The mononuclear cells were harvested after centrifuging at 2000 rpm for 25 min at room temperature [
25]. The leukocytes were analyzed for membrane marker expression by flow cytometry.
Flow cytometry analysis
Leukocyte classification by phenotypic analysis (the surface expression of antigen markers) was performed by flow cytometry. Leukocytes were resuspended in PBS at a concentration of 2 × 10
5/ml and stained with the fluorochrome-conjugated antibodies in darkness for 30 min at room temperature. All antibodies were purchased from Biolegend (San Diego, CA), including fluorescein isothiocyanate (FITC) anti-rat CD3 (201403), peridinin chlorophyll protein (PerCP) anti-rat CD8a (201712), allophycocyanin (APC) anti-rat CD161 (205606), phycoerythrin (PE) anti-rat CD45RA (202307), and PE anti-rat CD4 (203307). Appropriate isotype-matched immunoglobulins were used as negative controls. An additional file shows this in more detail (please refer to Additional file
1: Fig. S1) Cells were analyzed on a FACSCalibur flow cytometer with Cell Quest software (Becton Dickinson, San Jose, CA, USA). The lymphocytes were gated on the scatter plots of forward scatter (FSC-H) and side scatter (SSC-H), excluding debris and cell aggregates; T cell subsets and B cells were further gated in the P1 population based on their expression of specific markers. We defined the CD3
+CD4
+ population as helper T (Th) cells, CD3
+CD8
+ population as cytotoxic T (Tc) cells, CD3
−CD45RA
+ population as B lymphocytes, and CD3
+CD161a
+ population as natural killer T (NKT) cells.
Statistical analysis
The number of rats in each experimental group was determined by power analyses, informed by our past experience with similar measurements (α = 0.05 and β = 0.20). All data are expressed as mean ± SEM. Differences between two groups were evaluated for statistical significance using a Student’s t test. Differences between three or more groups were evaluated for statistical significance using one-way ANOVA followed by Bonferroni post hoc test. Normality of the distribution and homogeneity of variance were assessed by the F test or Bartlett’s test before the Student’s t test and the ANOVA, respectively. A two-tailed p value of < 0.05 was considered statistically significant. Correlation was analyzed with the Pearson correlation coefficient. The datasets used and/or analyzed during the current study are available from the corresponding author upon request.
Discussion
Ischemic stroke results in a rapid systemic inflammatory response, which exacerbates the initial infarct [
26,
27]. Modulation of immune and inflammatory responses represents an important target for improving clinical outcomes after stroke [
28]. Our previous study indicated that RIPC dramatically altered the levels of multiple immune cell populations and cytokines in blood [
8]. However, how RIPC induced these alterations in the peripheral immune system has remained elusive.
Over the past decade, the contribution of the spleen to ischemic brain damage has gained considerable attention in stroke research. There is increasing evidence that the spleen-mediated peripheral immune changes contribute to progressive injury in a number of acute neurological disorders, including spinal cord injury, hemorrhagic stroke, and traumatic brain injury [
29‐
32]. A recent study demonstrated that multipotent adult progenitor cells enhance recovery after stroke by modulating the splenic immune response [
33]. However, whether RIPC modulates the splenic response is not clear. In this study, we discovered that in normal subjects, RIPC increased the splenic lymphocyte populations; meanwhile, RIPC decreased the T lymphocytes and robustly elevated the percentage of B cells in peripheral blood (Figs.
1 and
2). Under the pathologic condition of stroke, RIPC also maintained the splenic volume and lymphocyte numbers (Fig.
3). Moreover, removal of the spleen 1 day or 2 weeks before RIPC deprived RIPC-afforded neuroprotection 3 days post reperfusion, which lasted through day 7 after stroke (Figs.
4 and
5). Splenectomy also reversed RIPC-induced peripheral lymphocyte changes (Fig.
6). These results suggest that immunomodulation of the splenic response by RIPC may create a special immune environment that influences the progression of ischemic stroke.
We have shown previously that RIPC could reduce brain infarct early after stroke [
8]. Splenectomy 2 weeks before stroke has been shown to be neuroprotective [
34‐
36], while splenectomy shortly before stroke showed minimal protection [
37]. Interestingly, in our study, splenectomy performed either 1 day or 2 weeks before RIPC and MCAO reduced and eliminated RIPC-afforded neuroprotection, respectively. Splenectomy shortly before MCAO may initiate an acute activation of the immune response, leading to tissue damage that cancels its protective effects in the brain [
38]. This is supported by the fact that peripheral lymphocytes increased in the blood after splenectomy. Such immune cell elevation might be due to the mobilization of immune cells from other organs or tissues. For example, bone marrow is the primary site of hematopoiesis. All types of myeloid and lymphoid lineages are created in the bone marrow. Under normal conditions, lymphoid cells must migrate to secondary lymph organs, such as the spleen and lymph nodes, to complete maturation. However, when the spleen was removed, the role of the bone marrow as a peripheral immune cell source should not be ignored. Interestingly, our data further showed that splenectomy 2 weeks before RIPC and MCAO fully removed the protective effect of RIPC on ischemic brain injury (Fig.
5), suggesting that the spleen may contribute to RIPC-induced protection against CNS injury through complicated mechanisms of immune modulation over time.
Lymphocytes, especially T lymphocytes, play a destructive role after stroke [
39,
40]. In our study, we found that exposure to RIPC could dramatically reduce the number of T cells especially CD8
+ T cells and NKT cells in the blood prior to stroke. Interestingly, we found an increase of splenic T cells after RIPC in this study. Correlation analysis indicates that the splenic lymphocytes were proportional to the blood lymphocytes under normal physiological conditions, which is consistent with the public knowledge that circulating immune cells continuously migrate into and out of the resting spleen [
41]. However, RIPC reversed the positive correlation between splenic and peripheral T lymphocytes, suggesting that the RIPC-induced decrease of T lymphocytes in the periphery might be due to the retention of circulating cells in the spleen and impaired outflow of splenic lymphocytes into the blood. It is unclear from the current study how RIPC increases splenic T or B lymphocytes so robustly after RIPC. We know that CD4 and CD8 T cells are mobilized from the spleen and redistributed to the non-lymphoid tissues following recognition of cognate antigens in the splenic white pulp [
41]. Existing literature shows that bacteria/virus infection can induce recruitment of monocytes, NK cells, and neutrophils to the spleen, which is important for the infection prognosis through downstream adaptive immunity regulation [
42‐
45]. However, whether RIPC can also induce recruitment of immune cells to the spleen or inhibit the egress of spleen immune cells into the circulation warrants further exploration. Moreover, in our previous research, we found a dramatic reduction in the percentage of circulating T cell subsets (including CD4
+ T cell, CD8
+ T cell, and NKT cells) after stroke, which was ameliorated by RIPC after stroke [
8]. We found here that RIPC reduced the infiltration of Tc and NKT cells into the ischemic brain, which is consistent with our hypothesis that RIPC reduced the influx of immune cells after stroke. Splenectomy showed minimal effects on RIPC-induced reduction of T cells in blood or infiltration into the ischemic brain after stroke, suggesting that splenectomy removed the neuroprotective effect of RIPC through mechanisms probably independent of immune cell infiltration after stroke.
In our study, B cells show the most dramatic changes after RIPC. Our previous data revealed an elevation of circulating B cells in RIPC animals, which may be essential for RIPC-afforded protection against ischemia [
8]. In this study, there is also an intriguing increase of splenic B lymphocytes at 3 days after RIPC. Correlation analysis shows that the B lymphocytes are elevated in the spleen and periphery at the same time even after RIPC, indicating that an upregulation of B cells in the blood might be due to increased B cell generation and release from the spleen. It is noted that splenectomy also mediated a high percentage of B cells in the blood. However, these non-splenic B cells might come from different sources, such as the bone marrow or lymph nodes, and possess functions distinct from B cells in the spleen. RIPC and splenectomy showed minimal effect on B cell infiltration. Different from the detrimental role of T lymphocytes in ischemic brain injury, recent findings demonstrate that B lymphocytes play a heterogeneous role in the adaptive immune response to stroke [
12]. On the one hand, many studies show a special population of regulatory B lymphocytes secrete IL-10 and are protective in cerebral ischemia/reperfusion injury [
46,
47]. A recent study reported that repetitive hypoxic preconditioning induces an immunosuppressive B cell phenotype prior to stroke onset, thereby preventing the infiltration of other immune cells into the brain and protecting the brain against ischemic attack [
48]. On the other hand, B cell and antibody accumulation in the infarct region correlated with impairments in hippocampal long-term potentiation, resulting in short-term memory deficit weeks after stroke [
49], which suggests the post-stroke detrimental effects of B cells. It is therefore warranted to explore whether the robustly increased peripheral B lymphocytes after RIPC or splenectomy possessed distinct functions and played critical roles in stroke outcomes.