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. Author manuscript; available in PMC: 2019 Jan 1.
Published in final edited form as: Neurobiol Aging. 2017 Sep 28;61:102–111. doi: 10.1016/j.neurobiolaging.2017.09.022

Splenectomy protects aged mice from injury after experimental stroke

Anjali Chauhan a, Abdullah Al Mamun a, Gabriel Spiegel a, Nia Harris b, Liang Zhu d, Louise D McCullough a,c,*
PMCID: PMC5947993  NIHMSID: NIHMS909269  PMID: 29059593

Abstract

Elderly stroke patients and aged animals subjected to experimental stroke have significantly worse functional recovery and higher mortality compared to younger subjects. Activation of the peripheral immune system is known to influence stroke outcome. Prior studies have shown that splenectomy reduces ischemic brain injury in young mice. As immune function changes with aging, it is unclear whether splenectomy will confer similar benefits in aged animals. We investigated the contribution of spleen to brain injury after cerebral ischemia in aged male mice. Splenic architecture and immune cell composition were altered in aged mice. Splenectomy two weeks before stroke resulted in improved neurobehavioral and infarct outcomes in aged male mice. In addition, there was a reduction in peripheral immune cell infiltration into the brain and decreased levels of peripheral inflammatory cytokines after stroke in aged splenectomized mice. Splenectomy immediately after reperfusion also improved behavioral and infarct outcomes. This study suggests that inhibition of the splenic immune response is a translationally relevant target to pursue for stroke treatment in aged individuals.

Keywords: Splenectomy, Stroke, Age, Middle cerebral artery occlusion model, Immune response

1. Introduction

Ischemic stroke is the fifth leading cause of death and the leading cause of significant disability in the United States (Kochanek et al., 2014). Age is the most important non-modifiable risk factor for stroke, with stroke rates doubling every decade after the age of 55 in both sexes (Reeves et al., 2008; Rojas et al., 2007). The aging process affects all organs, including the brain (Katsimpardi et al., 2014; Villeda et al., 2011) and leads to functional deficits in many cell types, including the innate immune cell of the central nervous system, the microglia (Hart et al., 2012; Ritzel et al., 2015). Aging also leads to an increase in systemic levels of inflammation due to an increase in circulating pro-inflammatory cytokines in peripheral tissues (Alvarez-Rodriguez et al., 2012; Chesnokova et al., 2016; Franceschi et al., 2007). We have found differences in the peripheral immune cell infiltration after stroke in the aged brain (Manwani et al., 2013). Therefore, it is imperative to include aged animals in studies that are designed to investigate potential therapies that target the immune response.

The influx of peripheral immune cells into the brain after ischemic injury is well documented, and the spleen is a major source of these peripheral immune cells (Offner et al., 2006a; Offner et al., 2006b; Seifert et al., 2012a; Seifert et al., 2012b). After an ischemic injury, sympathetic activation (Ajmo et al., 2009), production of pro-inflammatory cytokines (Offner et al., 2006a) and antigen presentation in the damaged brain (Planas et al., 2012) stimulates the release of spleen-derived immune cells into the blood, which then migrate to the injured brain (Ajmo et al., 2008; Zhang et al., 2013). Removal of the spleen in young mice two weeks prior to stroke reduced injury in both permanent and transient stroke model and reduced immune cell infiltration into the brain following middle cerebral artery occlusion (MCAo), with a decrease in infiltrating T-cells, macrophages and a reduction of pro-inflammatory cytokines (Dotson et al., 2014; Seifert et al., 2014; Zhang et al., 2013). Similarly, irradiation of the spleen after ischemic stroke reduced infarct volume, suppressed the release of splenic immune cells and decreased T cell infiltration into the injured brain (Ostrowski et al., 2012). Post-stroke splenectomy has also been reported to decrease infarct after middle cerebral artery occlusion, suggesting that this is a translationally relevant target once the exact cellular components that induce the detrimental response are identified (Belinga et al., 2016). However, all prior studies have been performed in young animals. As aging is a major risk factor for stroke, and the immune response to stroke has been shown to differ in aged animals, it is essential to examine the efficacy of splenectomy in aged mice to understand the translational value of development of therapies to inhibit the post-stroke splenic response in patients.

In this study, young and aged male mice were subjected to splenectomy or sham splenectomy surgery two weeks prior to induction of MCAo. Ninety-six hours after MCAo, mice were examined for behavioral deficits and then brains were collected for infarct assessment. Removal of the spleen resulted in improved behavior in both young and aged mice. Additional cohorts were used to measure and characterize post-stroke immune cell infiltration into the aged mice brain via flow cytometry. We observed a reduction in infiltrating immune cells in the brains of splenectomized aged mice after stroke. This decline in the leukocyte infiltration in the splenectomized aged brain was found to be driven primarily by a reduction in monocyte and neutrophil infiltration. Furthermore, post stroke splenectomy also resulted in reduced neurobehavioral deficits and infarct. These results suggest that strategies to reduce post-stroke splenic activation may indeed be beneficial in elderly stroke patients and warrant further study.

2. Methods

2.1. Ethics statement

All the animal procedures were performed in accordance with NIH guidelines for the care and use of laboratory animals and approved by the Animal Care Committee of the University of Texas Health Science Center at Houston, McGovern Medical School. The mice were randomly allocated to the study groups and all assessments were performed by an investigator blinded to surgical conditions. Animal numbers were determined by power analysis after an initial cohort was performed.

2.2. Experimental animals

C57BL/6J male mice of 8-12 weeks (total young, n= 30) and 18-22 months (total aged, n= 92) of age were pair-housed in a specific pathogen free facility (light cycle 12/12 h light/dark). Food and water were provided ad libitum.

2.3. Splenectomy

The spleen was removed under the isoflurane anesthesia. On the left dorsolateral side of the abdomen, caudal to the last rib, a longitudinal incision was made. The splenic arteries were cauterized and the spleen was removed. The abdominal wall was closed and then the skin was sutured. Sham surgery was performed by exteriorizing the spleen and then reinserting it into the abdominal cavity. After the surgery, the animals were allowed to recover for two weeks prior to induction of stroke. In young cohorts, mice were randomly divided into two groups; mice that underwent sham splenectomy (n=10) and a splenectomized group (n=10). In the aged cohorts, mice were randomly divided into sham splenectomy (n= 34) and a splenectomized aged cohort (n=24). An additional cohort of aged mice was subjected to post-stroke splenectomy (see section 2.10).

2.4. Middle cerebral artery occlusion

Two weeks after sham surgery or splenectomy, transient focal ischemia was induced under isoflurane anesthesia in young (n=20) and aged (n=44) mice for 1 hour by occlusion of the right middle cerebral artery (Manwani et al., 2013). Body temperature was maintained at 37.0 ± 1.0°C throughout the surgery by automated temperature control feedback system. One hour after MCAo, animals were anesthetized again and reperfusion was established by withdrawal of the monofilament. Animals were then placed in a recovery cage and behavior assessments were performed 96 hours after MCAo immediately prior to sacrifice. After the induction of middle cerebral artery occlusion, the mice were administered daily injections of 0.9% sodium chloride, provided with wet mash, and body weight was recorded.

2.5. Behavioral assessment

Percentage of spleen body weight was assessed in young (sham: n=5, stroke: n=7) and aged mice (sham: n=5, stroke: n=7). Behavioral assessment was performed on stroke mice with intact (young: n=7, aged: n=7) and splenectomy (young: n=8, aged: n=8). Ninety-six hours after MCAo, neurological deficit scores were assessed by a 4-point scale where 0-no deficit, 1-forelimb weakness and torso turning to the ipsilateral side when held by tail, 2-circling to the affected side, 3-unable to bear weight on affected side and 4-no spontaneous activity or barrel rolling. The corner test was performed as described earlier (Manwani et al., 2011). Briefly, two cardboard pieces at an angle of 30 degrees were placed in front of the mouse. The mouse was allowed to enter the corner and contact of cardboard with the vibrissae led to a rear and the direction in which the mouse turned was recorded. Ambulatory and cognitive functions were assessed on the Y maze for a period of 5 minutes (Alfieri et al., 2016). Briefly, the mouse was placed in one of the arms and the arms in which it entered was recorded. A blinded investigator performed all behavioral testing.

2.6. Tissue Harvesting

Mice were euthanized, transcardially perfused with 60mL cold, sterile phosphate buffered saline (PBS); spleen, blood and the brains were harvested. The olfactory bulb, brainstem, and cerebellum were removed. The brain was then divided along the interhemispheric fissure into two hemispheres and subsequently rinsed with PBS to remove adherent cells. Spleen from young and aged uninjured mice was used to investigate the immune cell changes in the spleen with age by flow cytometry (n=5 for each group). Furthermore, changes in the spleen immune cells was assessed in the aged mice after stroke (sham: n=5, stroke: n=6). Additional cohorts of spleen intact and splenectomized sham and strokes were used to investigate the role of spleen on infiltrating immune cells in the brain after MCAo by flow cytometry.

2.7. 2,3,5-Triphenyltetrazolium chloride (TTC) staining and immunohistochemistry

Mice were euthanized at day 4 post MCAo. For TTC staining, the animals were euthanized; brains were harvested and stored at −80°C for 4 min to slightly harden the tissue. Five, 2 mm coronal sections were cut from the olfactory bulb to the cerebellum and then stained with 1.5% TTC (SIGMA, St. Louise, MO). Slices were formalin-fixed (4%) and then digitalized for assessing infarct area using Sigma Scan Pro software as previously described (Venna et al., 2012). The final infarct area are presented as percentage area (percentage of contralateral structures with correction for edema).

For immunohistochemistry- The spleen was cut into 6 μm thick tissue sections and stained with standard protocol (Aw et al., 2008). The sections were stained with primary antibody (CD3, ABCAM, 1:100) overnight at 4°C. Followed by three washes in PBS, the sections were stained with secondary antibody and directly conjugated B220 (20 μg/ml; EBIOSCIENCE). These were then mounted with Vectashield medium and viewed under Leica confocal microscope (LEICA MICROSYSTEMS LTD, UK).

2.8. Flow cytometry

Flow cytometry was performed in uninjured mice (young: n=5, aged: n=5), in aged mice (sham: n=5, stroke: n=6), and in aged mice with intact spleen (sham: n=7, stroke: n=7) and with splenectomy (sham: n=7, stroke: n=8). Blood was drawn by cardiac puncture with heparinized needles. Spleens were removed and processed by mechanical disruption on a 70um filter screen. Red blood cell lysis was achieved by three consecutive ten minute incubations with Trisammonium chloride (STEM CELL TECHNOLOGIES). The brain was placed in RPMI (LONZA) medium and mechanically and enzymatically digested in collagenase/dispase (1mg/mL) and DNAse (10mg/mL; both ROCHE DIAGNOSTICS). The cell suspension was filtered through a 70μm filter. Leukocytes were harvested from the interphase of a 70%/30% Percoll gradient. Splenic and brain leukocytes were washed with 1X PBS and stained with Live/dead discrimination stain (THERMOFISCHER SCIENTIFIC) for 30 minutes. The cells were washed twice and blocked with mouse Fc Block (1μl/50μl) (eBioscience) prior to staining with primary antibody-conjugated fluorophores (CD45, CD11b, CD3, CD4, CD8, Cd19, Ly6C and Ly6G) purchased from BIOLEGEND and were used at dilution of 1μl/50μl. Data were acquired on CytoFLEX (BECKMAN COULTER) and analyzed by FlowJo (TREESTAR INC). No less than 250,000 events were recorded for each sample. Cell specific fluorescence minus one controls were used to confirm individual antibody specificity.

2.9. ELISA

Plasma was collected at 96 hours post MCAo. Lipopolysaccharide binding protein (LBP) levels were determined by ELISA (ABNOVA) following manufacturer’s instructions. Plasma cytokines [tumor necrosis factor (TNF) α, interleukin-12 (IL-12), interleukin-6 (IL-6), interleukin-1 (IL-1) alpha, interleukin-13 (IL-13) and chemokine ligand-1 (CXCL1)] were determined using multiplex (BIORAD) and were run in duplicates. Inter-and intra-assay coefficients of variation were less than 10%.

2.10. Post stroke splenectomy

To evaluate the efficacy of post stroke splenectomy in aged mice, mice underwent sham splenectomy or splenectomy immediately after reperfusion, one hour after ischemic onset (n=16). The mice were placed back in their home cages and allowed to recover. Behavioral testing was conducted ninety-six hours after MCAo immediately prior to sacrifice.

2.11. Statistical Analysis

Descriptive statistics (mean and standard error of mean (SEM), median, or interquartile range) were provided for cell counts and neurological outcomes. Two-sample t test or Wilcoxon rank-sum test was used to compare variables between different groups (Table 1 and 2 and Fig 1A, 6A, 6B and 6D). Behavior, infarct area, cellular subtypes by flow cytometry, LBP and pro-inflammatory cytokine status data were measured within subgroups of young/aged and splenectomy/intact in MCAo mice. Two-way ANOVA or a general linear model was used depending on whether the number of mice in each category was equal, followed by post-hoc tests adjusted by Tukey method (Fig 2, 3, 4, 5B). For mortality data, Kaplan-Meier survival curves were provided and log-rank test was used to compare the mortality between splenectomy and intact groups for mice splenectomized 15 days prior to stroke (Fig 5A), and for mice splenectomized immediately after reperfusion (Fig 6C). Statistical significance was set at p<0.05. All statistical analyses were performed in SAS software (Cary, NC) or GrahPad Prism.

Table 1. Changes in the immune cell counts in the spleen of young and aged mice.

Data is presented as mean ± SEM and analyzed by Wilcoxon rank sum test.

Cell type (counts) Young (n=5) Aged (n=5) p value
Live cells 228875 ± 5254.16 219489 ± 9329.82 0.406
CD45 (Leukocytes) 226788 ± 5396.88 211120 ± 11141.33 0.425
CD45+CD11b+Ly6C+ (Monocytes) 2517.40 ± 134.01 2296.80 ± 335.15 0.178
CD45+CD11b+Ly6G+ (Neutrophils) 1163 ± 116.06 4121 ± 1402.89 0.033
CD19+ B cells 138921 ± 6418.19 150833 ± 10628.10 0.425
CD3+ T cells 73333 ± 2014.21 46780.6 ± 3443.82 0.033
CD3+CD4+ T cells 38672 ± 1309.05 24494 ± 1703.99 0.033
CD3+CD8+ T cells 26880 ± 1269.67 25441 ± 7480.09 0.178
CD4/CD8 1.44 ± 0.03 1.22 ± 0.25 0.546
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Table 2. Changes in the counts of spleen cells after MCAo in aged mice at 96 hours.

Data is presented as mean ± SEM and analyzed by Wilcoxon rank sum test.

Cell type (counts) Sham(n=5) Stroke (n=6) p value
Live cells 232063.40 ± 8102.34 218627.67 ± 8725.86 0.201
CD45 (Leukocytes) 218660.20 ± 10257.69 208531.33 ± 9636.18 0.537
CD45+CD11b+Ly6C+ (Monocytes) 1036.60 ± 150.66 294.50 ± 30.85 0.069
CD45+CD11b+Ly6G+ (Neutrophils) 5025.20 ± 1709.60 1598.67 ± 131.46 0.024
CD19+ B cells 148872.80 ± 10508.26 146782.00 ± 8296.57 0.929
CD3+ T cells 51616.40 ± 2550.28 51595.50 ± 4546.73 0.929
CD3+CD4+ T cells 24080.00 ± 1570.26 23205.83 ± 1995.52 0.658
CD3+CD8+ T cells 19905.60 ± 2455.47 22730.17 ± 2545.32 0.537
CD4/CD8 1.29 ± 0.19 1.07 ± 0.11 0.537
Percentage spleen weight 2.76 ± 0.18 2.02 ± 0.19 0.002
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Fig 1. Spleen changes with aging.

Fig 1

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(A) Increase in body weight with aging. (B) Disarrangement of white pulp with aging. Data is presented as mean ± SEM, n=8-9, and analyzed by Wilcoxon rank sum test. **p<0.01

Fig 6. Splenectomy post stroke improves functional and infarct outcomes in aged mice.

Fig 6

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(A) Reduced neurological deficit scores. (B) Decrease in corner test score was observed in the post stroke splenectomized aged mice. (C) Kaplan –Meier survival curves for MCAo intact and splenectomy mice. (D) Decrease in percentage infarct area was seen in the post stroke splenectomized aged mice at 4 days. Data is presented as mean± SEM, and analyzed by two sample t test or Wilcoxon rank sum test. Mortality data was analyzed by log rank test. n=6; *p<0.05. **p<0.01. n=6; p<0.05.

Fig 2. Splenectomy improved functional and infarct outcomes in young and aged mice.

Fig 2

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(A) Reduction in the percentage spleen body weight after MCAo in both young and aged mice. (B) Reduced neurological deficit scores. (C) Increase in number of entries and (D) percentage alterations on Y maze (E) Decrease in corner test score was observed in the splenectomized aged mice after MCAo. (F) Decrease in percentage infarct area was seen in the splenectomized aged mice at 4 days after MCAo. Data is presented as mean± SEM, n=7-8; analyzed by two-way ANOVA or general linear model followed by post hoc Tukey test, *p<0.05, **p<0.01, ***p<0.001.

Fig 3. Decline in the infiltrating immune cells in the splenectomized aged mice after MCAo.

Fig 3

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(A) Reduced counts of infiltrating CD45hi cells (B) Decreased monocyte counts (c) decrease neutrophil counts was observed in the splenectomized aged mice after MCAo as compared to spleen intact mice. Data is presented as mean± SEM, n=7-8; analyzed by two-way ANOVA or general linear model followed by post hoc Tukey test, *p<0.05, **p<0.01, ***p<0.001.

Fig 4. Decline in the peripheral pro-inflammatory cytokines in the splenectomized aged mice after MCAo.

Fig 4

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Decline in the circulating TNF α (A) and IL-12 (B) was observed in the splenectomized mice as compared to spleen intact. Reduced plasma levels of IL-1α and IL-6 was observed in the splenectomized MCAo mice compared to splenectomized sham group. Data is presented as mean± SEM analyzed by two-way ANOVA or general linear model followed by post hoc Tukey test, n=7-10; *p<0.05, **p<0.01, ***p<0.001.

Fig 5. Splenectomy does not increase mortality and LBP levels after MCAo in aged mice.

Fig 5

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(A) Shows the Kaplan –Meier survival curves. (B) Increase in plasma LBP levels was observed after MCAo in spleen intact and splenectomized aged mice. Data is presented as mean± SEM, mortality data analyzed by log-rank test and LBP data was analyzed by two-way ANOVA or general linear model followed by post hoc Tukey test, n=7-8; p<0.05.

3. Results

3.1. Splenic architecture and immune compartment changes with age

No differences were seen in spleen size with aging (n=8-9 per group), yet the body weight of the animals significantly increases with age, resulting in a decrease in the ratio of spleen/body weight (Fig 1A, sum of rank (SR)8, 9=97, 56, p=0.009). Recent studies have found that the splenic architecture becomes disorganized with aging (Aw et al., 2016; Birjandi et al., 2011). We also observed an enlargement in the T cell area in the aged spleen, consistent with previous findings from others (Fig 1B) (Aw et al., 2016).

3.2. Splenic immune compartment alters with aging

To evaluate and quantify changes in the cellular immune composition of the spleen with aging, we harvested spleens from uninjured young and aged mice (n=5, Table 1). The immune composition of the spleens were immunophenotyped using flow cytometry. Wilcoxon rank sum test showed a significant increase in the splenic neutrophil counts in the aged (SR5,5=15,40, p=0.033) as compared to young mice. Additionally, there was significant decline in splenic CD3+ T cells with age (SR5,5=40,15, p=0.033). Moreover, a significant decline in CD3CD4+ T cells was observed with age (SR5,5=40,15, p=0.033).

3.3. Removal of the spleen prior to stroke improves functional outcomes and reduces infarct volume in young and aged mice

In order to understand the contribution of the spleen to stroke pathophysiology, we removed the spleen 15 days before stroke in young (n=7-8) and aged male mice (n=7-8). Ninety-six hours after MCAo, significant stroke effect (F (1, 22) = 10.8, p=0.003) was observed on the percentage spleen body weight. Post hoc analysis showed that percentage spleen body weight declined in both young and aged MCAo mice (Fig 2A), suggesting mobilization of spleen derived immune cells. Ninety-six hours after MCAo, the mortality in the splenectomized and intact young group was 17.65% and 13.33% respectively (χ2 1=1.28, p=0.741).

There was significant splenectomy (F (1, 26) = 20.03, p=0.0001) and age (F (1, 26) = 4.23, p=0.049) effect on the neurological deficit score. Post hoc test showed splenectomy in young (q7,7=3.75, p=0.061) and aged animals (q8,8=5.26, p=0.005) resulted in improved neurological deficit scores ninety-six hours after MCAo (Fig 2C).

A significant splenectomy (F (1, 26) = 14.07, p = 0.001) and age (F (1, 26) = 29.51, p<0.0001) effect was observed on ambulatory behavior on Y maze. There was a decrease in the overall ambulatory activity in aged MCAo animals (Fig 2B). No difference in the cognitive function (percentage alterations) on Y maze was observed between splenectomized compared to spleen intact MCAo mice (Fig 2D).

There was a significant splenectomy effect on the corner test score (F (1, 26) = 28.3, p<0.0001). Splenectomized young and aged mice performed better in the corner test (Fig 2E) as compared to spleen-intact young (q7,7=5.69, p=0.002) and aged mice (q8,8=4.94, p=0.009).

Importantly, the results demonstrated that the infarct size in the cortex, striatum and hemisphere was significantly reduced in the spleen intact aged group as compared to spleen intact young mice (q7,6=6.44, p=0.0007, q7,6=10.57, p<0.0001,q7,6=6.69, p=0.0006, respectively, Fig. 2F), reflecting to smaller infarcts with age however worse functional outcomes. However, splenectomy in both young and aged mice resulted in smaller infarcts (Young: q7,6=3.43, p=0.093, q7,6= 11.5, p<0.0001, q7,6=4.32, p=0.0282; Aged: q6,6=8.88, p<0.0001, q6,6=8.24, p<0.0001, q6,6=6.24, p=0.0013; Fig. 2F), suggesting that spleen plays a significant negative role in stroke pathophysiology in young and aged animals.

3.4. Stroke leads to mobilization of splenic monocytes and neutrophils in aged mice

As our study and previous studies have shown that the spleen contracts after MCAo secondary to the mobilization of immune cells to the site of injury (Dotson et al., 2015; Seifert et al., 2014; Zhang et al., 2013), we next investigated changes in spleen composition in aged mice after MCAo. Table 2 shows the changes in splenic cellular composition after MCAo. Ninety-six hours after MCAo, the percentage spleen body weight was significantly decreased in the stroke group when compared to sham (n=5-6 per group, SR5,6=43,23, p=0.002) as assessed by Wilcoxon rank sum test. Flow cytometry data demonstrated a reduction in the splenic monocyte counts (SR5,6=45, 21, p=0.069) and significant decline in neutrophils in the MCAo group (SR5,6=45,21, p=0.024), suggesting extravasation of these cellular elements from the spleen. There was no difference in the splenic composition of CD3+ T and CD19+ B cells in the aged mice at 96 hours after MCAo, suggesting that the decrease in monocytes and neutrophils is cell-specific, rather than secondary to an overall contraction of the spleen.

3.5. Removal of the spleen before stroke in aged mice reduces infiltration of peripheral immune cells into the brain

In additional cohorts, we used flow cytometry to examine the effect of splenectomy on immune cell infiltration into the brain after MCAo in aged mice. There was significant effect of stroke (F (1, 23) = 29.1, p<0.0001) and an interaction between splenectomy and stroke (F (1, 23) = 7.15, p=0.0136) on the infiltration of CD45hi cells in the brain after MCAo (n=7-8). Interestingly, post hoc analysis showed a significant decline in the overall infiltration of peripheral immune cells (CD45hi) in the splenectomized aged mice as compared to spleen intact group 96 hours after MCAo (q8,7=4.71, p=0.014; Fig 3A). Moreover, a significant interaction between stroke and splenectomy on infiltration of inflammatory monocytes (F (1, 23) = 47.3, p<0.0001) and neutrophils (F (1, 23) = 12, p=0.002) was observed at 96 hours post MCAo. There was significant decrease in the inflammatory monocyte (q8,7=14.7, p<0.0001, Fig. 3B) and neutrophil counts (q8,7=7.36, p= 0.0002, Fig. 3C) in the splenectomized animals as compared to spleen intact mice, suggesting that the reduced peripheral immune cells were predominantly monocytes and neutrophils.

3.6. Splenectomy reduces the levels of circulating pro-inflammatory cytokines in aged mice after MCAo

Aged animals have a chronic low-level baseline elevation in many pro-inflammatory factors compared to young (defined by many as “inflammaging”). Induction of stroke in aged mice has shown to increase circulating cytokines at 24 hours to 72 hours (McCullough et al., 2016);(Verma et al., 2016). At 96 hours MCAo, we observed altered levels of pro-inflammatory cytokines (Suppl. Table 1). The levels of TNF- α, IL-12, IL-13, CXCL-1 was increased though not significant suggesting that the peak of inflammation has passed. We examined the effect of splenectomy on peripheral inflammation in aged mice ninety-six hours after MCAo (n=7-10). There was a significant effect of splenectomy on the peripheral TNF- α (F (1, 32) = 5.83, p=0.022) and IL-12 (F (1, 34) = 8.09, p=0.008) levels. Additionally, there was significant interaction between stroke and splenectomy in IL-1 alpha (F (1, 34) = 5.66, p=0.023) and IL-6 (F (1, 26) = 4.7, p=0.040) levels. Moreover, a trend towards interaction between stroke and splenectomy (F (1, 34) = 3.51, p=0.070) was seen in peripheral IL-12 levels. There was a significant decrease in many crucial pro-inflammatory cytokines (TNF-α, IL-12, IL-1a, IL-13, IL-6 and CXCL1) in the splenectomized aged mice as compared to spleen intact groups at 4 days after MCAo, suggesting that the removal of spleen lead to reduced peripheral pro-inflammatory status in the splenectomized aged mice after MCAo (Fig 4).

3.7. Splenectomy does not increase mortality and lipopolysaccharide binding protein (LBP) levels after MCAo in aged mice

One of the major concerns with splenectomy is alteration in the host defense against infections (Dragomir et al., 2016; Rubin et al., 2014). Infection after MCAo is major cause of mortality in hospitalized patients especially in the elderly (Westendorp et al., 2011), hence we assessed mortality and lipopolysaccharide binding protein (LBP) levels, a potential marker of sepsis (Tschaikowsky et al., 2011) after MCAo in our aged mice. There was no mortality after splenectomy in the aged mice at 15 days, prior to MCAo. Moreover, there was no difference in the mortality in the aged MCAo group as compared to spleen intact at 4 days after MCAo (n=17 and 27, p=0.101, Fig 5A). There was a significant stroke effect on the LBP levels (F (1, 22) = 89.2, p<0.0001). There was significant increase in the plasma LBP levels in both the spleen intact and splenectomized aged MCAo mice as compared to sham (n=7-8 per group, q 6,7=9, p<0.0001; q 6,7=9.89, p=0.0001; Fig 5B) however, no difference in LBP levels was observed between spleen intact and splenectomized group.

3.8. Post stroke splenectomy improves functional and infarct outcomes in age mice

Aged mice that underwent splenectomy immediately after reperfusion had reduced behavioral deficits as compared to MCAo intact cohort at ninety-six hours after stroke (n=6 per group, SR50, 28, p=0.052, Fig 6A). Additionally, turning bias in the corner test score was less in the aged post stroke splenectomy mice at day 4 as compared to sham splenectomy (n=6 per group, t=2.48 df=6.98, p=0.042, Fig 6B) as evident by unpaired Welch-corrected t test analysis. Moreover, a significant infarct reduction was observed in the cortex (t=3.4 df=8.97, p=0.007), striatum (t=4.33 df=8.22, p=0.002) and hemisphere (n=6 per group, t=3.64 df=9.91, p=0.005) of the aged MCAo splenectomized mice as compared to the MCAo intact group (Fig 6D). There was no difference in the mortality between the groups (n=8 per group, χ2 1=2.44, p=0.882, Fig 6C)

4. Discussion

Aging leads to low-grade chronic inflammation (Franceschi et al., 2000), and experimental models have shown that baseline leukocyte extravasation into tissues, including the brain, increases with advancing age (Ritzel et al., 2016; Stichel et al., 2007). After cerebral ischemia, leukocytes isolated from both the blood and brain of aged animals are notably pro-inflammatory, and even at baseline demonstrate increased capability to produce pro-inflammatory cytokines when stimulated ex vivo (Ritzel et al., 2016). When subjected to similar ischemic duration and severity, aged mice have significantly smaller infarcts but worse functional outcomes in the reversible MCAo model compared to young mice (Crapser et al., 2016; Manwani et al., 2011), which may be due to enhanced glial scar formation (Badan et al., 2003; Popa-Wagner et al., 2007), or immune cell dysregulation (Kapetanovic et al., 2015; Lee et al., 2016; Matt et al., 2016). As the vast majority of studies performed in aged animals have shown poorer recovery and higher mortality, which is also seen in patient populations, it is clear that age significantly worsens post-stroke pathophysiology.

In this study, we explored the effect of age on splenic cellular composition and contribution to ischemic injury. We found that the splenic architecture becomes disorganized, characterized by an enlargement of the T-cell area, a finding that has been corroborated by others (Aw et al., 2016; Birjandi et al., 2011). On further evaluation, we found that the neutrophil count was higher in the spleen of aged mice, indicating that the immune composition of the spleen changes significantly with aging. Studies have shown that the aging increases the predominance of myeloid progenitor cells in several immune cell niches (Dykstra et al., 2011; Nacionales et al., 2014). Both the bone marrow and blood are more neutrophilic in aged mice when compared to young mice (Eash et al., 2009; Shao et al., 2011), part of a process known as inflammaging (Franceschi et al., 2014). In addition, the frequency of splenic CD3+ T cells was decreased in aged animals, suggesting that the adaptive lymphoid immune response may also significantly change with age. Recent studies have highlighted that aged T cells have a dysfunctional phenotype, rendering a detrimental or diminished response towards injury when compared to T cells from young animals (Fulop et al., 2014; Jiang et al., 2011; Mirza et al., 2011). In addition, previous studies have shown that aged spleen tend to produce higher amounts of pro-inflammatory cytokines reflecting cellular senescence (Campisi et al., 2011; Park et al., 2014). While studies have shown that removal of the spleen in young animals before or after ischemic stroke (Belinga et al., 2016) is protective, there are no prior studies examining the role of the spleen in aged mice after experimental stroke. Demonstrating that altering the splenic response to stroke also benefits aged animals is imperative before further refinement and therapeutic development are considered.

Activation of astrocytes and microglia after stroke promotes local inflammation and release pro-inflammatory cytokines, which in turn leads to recruitment of peripheral immune cells, many of which are of splenic origin (Offner et al., 2006a; Pennypacker et al., 2015). Hence, in the current study, we explored the contribution of the spleen to ischemic injury in aged animals after MCAo. We observed a decrease in the percentage spleen/body weight in young and aged mice at 96 hours post-stroke, likely due to immune activation and release of peripheral immune cells from the spleen into the systemic circulation, which is consistent to what has been previously described in young mice. This suggests that the aged spleen is responsive to the same signals from the ischemic brain as are young animals (Offner et al., 2006b; Yan et al., 2014). Our data demonstrated that 96 hours post stroke, splenic monocytes and neutrophils decreased in aged mice, suggesting that these cells may be released from the spleen and recruited to the site of injury. Importantly, CD3+ T and CD19+ B cell frequencies did not differ between the sham and stroke group, suggesting that at 96 hours post stroke, the primary spleen-derived brain-infiltrating immune cells were monocytes and neutrophils.

To determine the role of the spleen in stroke pathology in aged animals, we removed the spleen 15 days prior to stroke in young and aged mice. We observed a significant improvement in behavior and significantly reduced infarct volume in the splenectomized young and aged mice. Specifically, the neurological deficit score was lower, overall motor activity was greater and turning bias in the corner test score was significantly less in splenectomized MCAo mice as compared to the spleen intact group. We performed splenectomy on young animals to ensure that we could replicate prior findings that splenectomy is beneficial, as reproducibility of key findings is an important first step to successful translation. Our data also demonstrated that splenectomy significantly reduced the infiltration of peripheral immune cells (CD45hi) into the brain in aged mice, and removal of the spleen appears to reduce a source of infiltrating immune cells (Gronberg et al., 2013; Seifert et al., 2012a; Seifert et al., 2012b) There was a significant increase in monocyte counts after MCAo in spleen-intact and splenectomized groups as compared to their respective sham controls, but the monocyte counts in the splenectomized stroke mice were significantly lower than the spleen-intact group. Similar to monocyte counts, neutrophil counts were significantly lower in the splenectomized stroke animals as compared to spleen intact cohorts.

We then investigated the peripheral inflammatory status after MCAo in spleen-intact and splenectomized mice 96 hours after MCAo. Pro-inflammatory cytokines are known to play a detrimental role in neuroinflammation after MCAo (Kim et al., 2014; Pradillo et al., 2012; Stanimirovic et al., 2000; Whiteley et al., 2009). These inflammatory mediators are detected by vagus nerve and inform the brain about peripheral inflammation (Tracey, 2002; Watkins et al., 1995). IL-6, TNF-α, IL-12, IL-1 alpha and CXCl1 have all previously been shown to play a detrimental role in stroke (Boutin et al., 2001; Losy et al., 2005; Whiteley et al., 2009) and are secreted by monocytes, macrophages and neutrophils (Cai et al., 2016; Chimen et al., 2017; Farahi et al., 2017; Knob et al., 2016; Nowicki et al., 2017; Sica et al., 2012). Interestingly, splenectomy in both sham and stroke animals resulted in a decline in pro-inflammatory cytokines, suggesting a role of spleen in cytokine modulation. Previous data has shown that suppression of TNF-α and IL-12 levels with specific inhibitors reduces brain injury after MCAo (Konoeda et al., 2010; Tuttolomondo et al., 2014). We observed a significant decrease in the IL-12 and TNF-α levels in splenectomized stroke mice compared to the spleen-intact mice after MCAo, suggesting a reduced systemic inflammatory state after MCAo in mice without a spleen.

Removal of the spleen alters the host immune response to infections, and risk of infections post stroke is the major cause of mortality in stroke patients (Dragomir et al., 2016; Rubin et al., 2014; Westendorp et al., 2011). However, we did not observe an increase in mortality after splenectomy in our aged cohorts, mortality in the splenectomized aged MCAo was not different that spleen intact aged MCAo group suggesting that at 4 days post stroke, removal of spleen did not increase mortality in aged mice. LBP, is a biomarker of endotoxemia (Gonzalez-Quintela et al., 2013; Nien et al., 2017; Schumann, 2011), that binds to LPS and activates toll like receptor-4 and leads to production of pro inflammatory cytokines IL-6 and TNF-α (Cohen, 2002; van Deventer et al., 1990). LBP levels were not different between spleen intact and splenectomized aged mice in sham groups, suggesting that splenectomy did not increase the risk for endotoxemia in our mice at this time point. However, induction of stroke lead to increase in plasma LBP levels in both splenectomized and spleen intact mice. The spleen is a major source of TNF-α during endotoxemia, and we found reduced TNF-α level in the plasma levels of splenectomized aged MCAo mice.

The spleen has a rich supply of sympathetic enervation and induction of stroke enhances sympathetic signals to spleen, leading to splenic atrophy (Yan et al., 2014) and mobilization of immune cells to site of injury. Removal of spleen lead to decline in infiltrating immune cells monocytes and neutrophils in the splenectomized aged MCAo animals at 96 hours post stroke. Hence, one of the possible mechanism for reduced mobilization of these immune cells to brain might be lack of brain derived sympathetic relay of information in stroke to the spleen in aged splenectomized mice. However, further studies investigating the role of sympathetic system on spleen in stroke in aged animals needs to be validated.

To acknowledge the role of spleen in post stroke pathology we removed the spleen in an additional cohort of aged mice immediately after reperfusion. We observed an improvement in neurobehavioral outcomes and infarct area at 4 days after MCAo, similar to what has been see by others in young mice (Belinga et al., 2016). This suggests that manipulation of the splenic response to stroke is a viable target for further therapeutic development.

Although this study provides an insight into the role of spleen in aged stroke animals, our study has some limitations. Firstly, we investigated the effect of splenectomy at a relatively early time-point (96 hours post stroke). Although removal of spleen resulted in reduced stroke injury, splenectomy itself has been shown to result in several complications including increased bacterial infections and thrombosis, which may occur at later time points (Lin et al., 2015; Pommerening et al., 2015). Secondly, in this study, we did not include female animals, as this study was conducted with the primary objective of evaluating the role of spleen in an aged stroke model to ensure that this was a viable target in an appropriate animal model.

Further studies are needed to determine whether sex differences exist in the contribution of the spleen to post-ischemic injury in aged animals and the mechanism by which this occurs.

It does appear that the mechanism driving the beneficial effects of splenectomy in young animals remains operative in aged animals. The intention is not to translate the splenectomy as a potential approach to reduce stroke injury in patients but to explore if further refinement is merited to develop viable therapeutic targets for use in stroke patients.

5. Conclusion

In conclusion, we observed that removal of spleen 15 days prior to stroke, or immediately after stroke, resulted in improved functional and infarct outcomes in aged animals. Additionally, splenectomy resulted in decreased infiltrating immune cells into the brain, predominantly due to the reduced infiltration of neutrophils and inflammatory monocytes. In addition, peripheral inflammatory cytokine levels were lower in aged splenectomized mice 4 days after stroke, suggesting that the spleen may be a potential source of damaging pro-inflammatory cytokines in ischemic stroke. Although removal of spleen is not a viable option in stroke patients, this study highlights the detrimental effects of spleen derived immune cells in the stroke injury, hence, developing immunotherapies modulating monocytes and neutrophils at earlier time point in the stroke patients might offer benefits.

Supplementary Material

1

Fig S1: Gating strategy for immune cells in the brain.

Supplementary Table 1: Circulating pro-inflammatory cytokines levels in sham and MCAo aged mice at 96 hours stroke. Data is presented as mean± SEM and analyzed by Wilcoxon rank sum test.

2
3

  • Spleen contributes to stroke induced cerebral injury

  • Splenectomy prior to stroke improved functional outcomes and reduced infarct area

  • Splenectomy reduced infiltration of monocytes and neutrophils after stroke.

  • Splenectomy prior to ischemia reduced levels of circulating cytokines after stroke.

  • Splenectomy post stroke improved functional and infarct outcomes in aged mice.

Acknowledgments

The authors thank Meaghan Roy-O’Reilly for her assistance with the multiplex experiments. This work was supported by 16POST27490032 AHA post-doctoral fellowship (to AC) and RO1 NSO94543 and R21 NSO88969 (to LDM)

Footnotes

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Conflict of interest:

Authors declare no conflict of interest.

References

  1. Ajmo CT, Jr, Collier LA, Leonardo CC, Hall AA, Green SM, Womble TA, Cuevas J, Willing AE, Pennypacker KR. Blockade of adrenoreceptors inhibits the splenic response to stroke. Exp Neurol. 2009;218:47–55. doi: 10.1016/j.expneurol.2009.03.044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ajmo CT, Jr, Vernon DO, Collier L, Hall AA, Garbuzova-Davis S, Willing A, Pennypacker KR. The spleen contributes to stroke-induced neurodegeneration. J Neurosci Res. 2008;86:2227–2234. doi: 10.1002/jnr.21661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alfieri JA, Silva PR, Igaz LM. Early Cognitive/Social Deficits and Late Motor Phenotype in Conditional Wild-Type TDP-43 Transgenic Mice. Front Aging Neurosci. 2016;8:310. doi: 10.3389/fnagi.2016.00310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Alvarez-Rodriguez L, Lopez-Hoyos M, Munoz-Cacho P, Martinez-Taboada VM. Aging is associated with circulating cytokine dysregulation. Cell Immunol. 2012;273:124–132. doi: 10.1016/j.cellimm.2012.01.001. [DOI] [PubMed] [Google Scholar]
  5. Aw D, Hilliard L, Nishikawa Y, Cadman ET, Lawrence RA, Palmer DB. Disorganization of the splenic microanatomy in ageing mice. Immunology. 2016;148:92–101. doi: 10.1111/imm.12590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Aw D, Silva AB, Maddick M, von Zglinicki T, Palmer DB. Architectural changes in the thymus of aging mice. Aging Cell. 2008;7:158–167. doi: 10.1111/j.1474-9726.2007.00365.x. [DOI] [PubMed] [Google Scholar]
  7. Badan I, Buchhold B, Hamm A, Gratz M, Walker LC, Platt D, Kessler C, Popa-Wagner A. Accelerated glial reactivity to stroke in aged rats correlates with reduced functional recovery. J Cereb Blood Flow Metab. 2003;23:845–854. doi: 10.1097/01.WCB.0000071883.63724.A7. [DOI] [PubMed] [Google Scholar]
  8. Belinga VF, Wu GJ, Yan FL, Limbenga EA. Splenectomy following MCAO inhibits the TLR4-NF-kappaB signaling pathway and protects the brain from neurodegeneration in rats. J Neuroimmunol. 2016;293:105–113. doi: 10.1016/j.jneuroim.2016.03.003. [DOI] [PubMed] [Google Scholar]
  9. Birjandi SZ, Ippolito JA, Ramadorai AK, Witte PL. Alterations in marginal zone macrophages and marginal zone B cells in old mice. J Immunol. 2011;186:3441–3451. doi: 10.4049/jimmunol.1001271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Boutin H, LeFeuvre RA, Horai R, Asano M, Iwakura Y, Rothwell NJ. Role of IL-1alpha and IL-1beta in ischemic brain damage. J Neurosci. 2001;21:5528–5534. doi: 10.1523/JNEUROSCI.21-15-05528.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cai S, Kandasamy M, Rahmat JN, Tham SM, Bay BH, Lee YK, Mahendran R. Lactobacillus rhamnosus GG Activation of Dendritic Cells and Neutrophils Depends on the Dose and Time of Exposure. J Immunol Res. 2016;2016:7402760. doi: 10.1155/2016/7402760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Campisi J, Andersen JK, Kapahi P, Melov S. Cellular senescence: a link between cancer and age-related degenerative disease? Semin Cancer Biol. 2011;21:354–359. doi: 10.1016/j.semcancer.2011.09.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chesnokova V, Pechnick RN, Wawrowsky K. Chronic peripheral inflammation, hippocampal neurogenesis, and behavior. Brain Behav Immun. 2016;58:1–8. doi: 10.1016/j.bbi.2016.01.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chimen M, Yates CM, McGettrick HM, Ward LS, Harrison MJ, Apta B, Dib LH, Imhof BA, Harrison P, Nash GB, Rainger GE. Monocyte Subsets Coregulate Inflammatory Responses by Integrated Signaling through TNF and IL-6 at the Endothelial Cell Interface. J Immunol. 2017;198:2834–2843. doi: 10.4049/jimmunol.1601281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cohen J. The immunopathogenesis of sepsis. Nature. 2002;420:885–891. doi: 10.1038/nature01326. [DOI] [PubMed] [Google Scholar]
  16. Crapser J, Ritzel R, Verma R, Venna VR, Liu F, Chauhan A, Koellhoffer E, Patel A, Ricker A, Maas K, Graf J, McCullough LD. Ischemic stroke induces gut permeability and enhances bacterial translocation leading to sepsis in aged mice. Aging (Albany NY) 2016;8:1049–1063. doi: 10.18632/aging.100952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dotson AL, Wang J, Saugstad J, Murphy SJ, Offner H. Splenectomy reduces infarct volume and neuroinflammation in male but not female mice in experimental stroke. J Neuroimmunol. 2015;278:289–298. doi: 10.1016/j.jneuroim.2014.11.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dotson AL, Zhu W, Libal N, Alkayed NJ, Offner H. Different immunological mechanisms govern protection from experimental stroke in young and older mice with recombinant TCR ligand therapy. Front Cell Neurosci. 2014;8:284. doi: 10.3389/fncel.2014.00284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Dragomir M, Petrescu DGE, Manga GE, Calin GA, Vasilescu C. Patients After Splenectomy: Old Risks and New Perspectives. Chirurgia (Bucur) 2016;111:393–399. doi: 10.21614/chirurgia.111.5.393. [DOI] [PubMed] [Google Scholar]
  20. Dykstra B, Olthof S, Schreuder J, Ritsema M, de Haan G. Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells. J Exp Med. 2011;208:2691–2703. doi: 10.1084/jem.20111490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Eash KJ, Means JM, White DW, Link DC. CXCR4 is a key regulator of neutrophil release from the bone marrow under basal and stress granulopoiesis conditions. Blood. 2009;113:4711–4719. doi: 10.1182/blood-2008-09-177287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Farahi N, Paige E, Balla J, Prudence E, Ferreira RC, Southwood M, Appleby SL, Bakke P, Gulsvik A, Litonjua AA, Sparrow D, Silverman EK, Cho MH, Danesh J, Paul DS, Freitag DF, Chilvers ER. Neutrophil-mediated IL-6 receptor trans-signaling and the risk of chronic obstructive pulmonary disease and asthma. Hum Mol Genet. 2017 doi: 10.1093/hmg/ddx053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Franceschi C, Bonafe M, Valensin S, Olivieri F, De Luca M, Ottaviani E, De Benedictis G. Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci. 2000;908:244–254. doi: 10.1111/j.1749-6632.2000.tb06651.x. [DOI] [PubMed] [Google Scholar]
  24. Franceschi C, Campisi J. Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases. J Gerontol A Biol Sci Med Sci. 2014;69(Suppl 1):S4–9. doi: 10.1093/gerona/glu057. [DOI] [PubMed] [Google Scholar]
  25. Franceschi C, Capri M, Monti D, Giunta S, Olivieri F, Sevini F, Panourgia MP, Invidia L, Celani L, Scurti M, Cevenini E, Castellani GC, Salvioli S. Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans. Mech Ageing Dev. 2007;128:92–105. doi: 10.1016/j.mad.2006.11.016. [DOI] [PubMed] [Google Scholar]
  26. Fulop T, Le Page A, Fortin C, Witkowski JM, Dupuis G, Larbi A. Cellular signaling in the aging immune system. Curr Opin Immunol. 2014;29:105–111. doi: 10.1016/j.coi.2014.05.007. [DOI] [PubMed] [Google Scholar]
  27. Gonzalez-Quintela A, Alonso M, Campos J, Vizcaino L, Loidi L, Gude F. Determinants of serum concentrations of lipopolysaccharide-binding protein (LBP) in the adult population: the role of obesity. PLoS One. 2013;8:e54600. doi: 10.1371/journal.pone.0054600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Gronberg NV, Johansen FF, Kristiansen U, Hasseldam H. Leukocyte infiltration in experimental stroke. J Neuroinflammation. 2013;10:115. doi: 10.1186/1742-2094-10-115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hart AD, Wyttenbach A, Perry VH, Teeling JL. Age related changes in microglial phenotype vary between CNS regions: grey versus white matter differences. Brain Behav Immun. 2012;26:754–765. doi: 10.1016/j.bbi.2011.11.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jiang J, Fisher EM, Murasko DM. CD8 T cell responses to influenza virus infection in aged mice. Ageing Res Rev. 2011;10:422–427. doi: 10.1016/j.arr.2011.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kapetanovic R, Bokil NJ, Sweet MJ. Innate immune perturbations, accumulating DAMPs and inflammasome dysregulation: A ticking time bomb in ageing. Ageing Res Rev. 2015;24:40–53. doi: 10.1016/j.arr.2015.02.005. [DOI] [PubMed] [Google Scholar]
  32. Katsimpardi L, Litterman NK, Schein PA, Miller CM, Loffredo FS, Wojtkiewicz GR, Chen JW, Lee RT, Wagers AJ, Rubin LL. Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science. 2014;344:630–634. doi: 10.1126/science.1251141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kim JY, Kawabori M, Yenari MA. Innate inflammatory responses in stroke: mechanisms and potential therapeutic targets. Curr Med Chem. 2014;21:2076–2097. doi: 10.2174/0929867321666131228205146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Knob CD, Silva M, Gasparoto TH, Oliveira CE, Amor NG, Arakawa NS, Costa FB, Campanelli AP. Effects of budlein A on human neutrophils and lymphocytes. J Appl Oral Sci. 2016;24:271–277. doi: 10.1590/1678-775720150540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kochanek KD, Murphy SL, Xu J, Arias E. Mortality in the United States, 2013. NCHS Data Brief. 2014:1–8. [PubMed] [Google Scholar]
  36. Konoeda F, Shichita T, Yoshida H, Sugiyama Y, Muto G, Hasegawa E, Morita R, Suzuki N, Yoshimura A. Therapeutic effect of IL-12/23 and their signaling pathway blockade on brain ischemia model. Biochem Biophys Res Commun. 2010;402:500–506. doi: 10.1016/j.bbrc.2010.10.058. [DOI] [PubMed] [Google Scholar]
  37. Lee KA, Shin KS, Kim GY, Song YC, Bae EA, Kim IK, Koh CH, Kang CY. Characterization of age-associated exhausted CD8(+) T cells defined by increased expression of Tim-3 and PD-1. Aging Cell. 2016;15:291–300. doi: 10.1111/acel.12435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lin JN, Lin CL, Lin MC, Lai CH, Lin HH, Yang CH, Kao CH. Increased Risk of Hemorrhagic and Ischemic Strokes in Patients With Splenic Injury and Splenectomy: A Nationwide Cohort Study. Medicine (Baltimore) 2015;94:e1458. doi: 10.1097/MD.0000000000001458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Losy J, Zaremba J, Skrobanski P. CXCL1 (GRO-alpha) chemokine in acute ischaemic stroke patients. Folia Neuropathol. 2005;43:97–102. [PubMed] [Google Scholar]
  40. Manwani B, Liu F, Scranton V, Hammond MD, Sansing LH, McCullough LD. Differential effects of aging and sex on stroke induced inflammation across the lifespan. Exp Neurol. 2013;249:120–131. doi: 10.1016/j.expneurol.2013.08.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Manwani B, Liu F, Xu Y, Persky R, Li J, McCullough LD. Functional recovery in aging mice after experimental stroke. Brain Behav Immun. 2011;25:1689–1700. doi: 10.1016/j.bbi.2011.06.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Matt SM, Johnson RW. Neuro-immune dysfunction during brain aging: new insights in microglial cell regulation. Curr Opin Pharmacol. 2016;26:96–101. doi: 10.1016/j.coph.2015.10.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. McCullough LD, Mirza MA, Xu Y, Bentivegna K, Steffens EB, Ritzel R, Liu F. Stroke sensitivity in the aged: sex chromosome complement vs. gonadal hormones. Aging (Albany NY) 2016;8:1432–1441. doi: 10.18632/aging.100997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Mirza N, Pollock K, Hoelzinger DB, Dominguez AL, Lustgarten J. Comparative kinetic analyses of gene profiles of naive CD4+ and CD8+ T cells from young and old animals reveal novel age-related alterations. Aging Cell. 2011;10:853–867. doi: 10.1111/j.1474-9726.2011.00730.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Nacionales DC, Gentile LF, Vanzant E, Lopez MC, Cuenca A, Cuenca AG, Ungaro R, Li Y, Baslanti TO, Bihorac A, Moore FA, Baker HV, Leeuwenburgh C, Moldawer LL, Efron PA. Aged mice are unable to mount an effective myeloid response to sepsis. J Immunol. 2014;192:612–622. doi: 10.4049/jimmunol.1302109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nien HC, Hsu SJ, Su TH, Yang PJ, Sheu JC, Wang JT, Chow LP, Chen CL, Kao JH, Yang WS. High Serum Lipopolysaccharide-Binding Protein Level in Chronic Hepatitis C Viral Infection Is Reduced by Anti-Viral Treatments. PLoS One. 2017;12:e0170028. doi: 10.1371/journal.pone.0170028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Nowicki KW, Hosaka K, Walch FJ, Scott EW, Hoh BL. M1 macrophages are required for murine cerebral aneurysm formation. J Neurointerv Surg. 2017 doi: 10.1136/neurintsurg-2016-012911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Offner H, Subramanian S, Parker SM, Afentoulis ME, Vandenbark AA, Hurn PD. Experimental stroke induces massive, rapid activation of the peripheral immune system. J Cereb Blood Flow Metab. 2006a;26:654–665. doi: 10.1038/sj.jcbfm.9600217. [DOI] [PubMed] [Google Scholar]
  49. Offner H, Subramanian S, Parker SM, Wang C, Afentoulis ME, Lewis A, Vandenbark AA, Hurn PD. Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circulating macrophages. J Immunol. 2006b;176:6523–6531. doi: 10.4049/jimmunol.176.11.6523. [DOI] [PubMed] [Google Scholar]
  50. Ostrowski RP, Schulte RW, Nie Y, Ling T, Lee T, Manaenko A, Gridley DS, Zhang JH. Acute splenic irradiation reduces brain injury in the rat focal ischemic stroke model. Transl Stroke Res. 2012;3:473–481. doi: 10.1007/s12975-012-0206-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Park J, Miyakawa T, Shiokawa A, Nakajima-Adachi H, Tanokura M, Hachimura S. Splenic stromal cells from aged mice produce higher levels of IL-6 compared to young mice. Mediators Inflamm. 2014;2014:826987. doi: 10.1155/2014/826987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Pennypacker KR, Offner H. The role of the spleen in ischemic stroke. J Cereb Blood Flow Metab. 2015;35:186–187. doi: 10.1038/jcbfm.2014.212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Planas AM, Gomez-Choco M, Urra X, Gorina R, Caballero M, Chamorro A. Brain-derived antigens in lymphoid tissue of patients with acute stroke. J Immunol. 2012;188:2156–2163. doi: 10.4049/jimmunol.1102289. [DOI] [PubMed] [Google Scholar]
  54. Pommerening MJ, Rahbar E, Minei K, Holcomb JB, Wade CE, Schreiber MA, Cohen MJ, Underwood SJ, Nelson M, Cotton BA. Splenectomy is associated with hypercoagulable thrombelastography values and increased risk of thromboembolism. Surgery. 2015;158:618–626. doi: 10.1016/j.surg.2015.06.014. [DOI] [PubMed] [Google Scholar]
  55. Popa-Wagner A, Carmichael ST, Kokaia Z, Kessler C, Walker LC. The response of the aged brain to stroke: too much, too soon? Curr Neurovasc Res. 2007;4:216–227. doi: 10.2174/156720207781387213. [DOI] [PubMed] [Google Scholar]
  56. Pradillo JM, Denes A, Greenhalgh AD, Boutin H, Drake C, McColl BW, Barton E, Proctor SD, Russell JC, Rothwell NJ, Allan SM. Delayed administration of interleukin-1 receptor antagonist reduces ischemic brain damage and inflammation in comorbid rats. J Cereb Blood Flow Metab. 2012;32:1810–1819. doi: 10.1038/jcbfm.2012.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Reeves MJ, Bushnell CD, Howard G, Gargano JW, Duncan PW, Lynch G, Khatiwoda A, Lisabeth L. Sex differences in stroke: epidemiology, clinical presentation, medical care, and outcomes. Lancet Neurol. 2008;7:915–926. doi: 10.1016/S1474-4422(08)70193-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Ritzel RM, Crapser J, Patel AR, Verma R, Grenier JM, Chauhan A, Jellison ER, McCullough LD. Age-Associated Resident Memory CD8 T Cells in the Central Nervous System Are Primed To Potentiate Inflammation after Ischemic Brain Injury. J Immunol. 2016;196:3318–3330. doi: 10.4049/jimmunol.1502021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Ritzel RM, Patel AR, Pan S, Crapser J, Hammond M, Jellison E, McCullough LD. Age- and location-related changes in microglial function. Neurobiol Aging. 2015;36:2153–2163. doi: 10.1016/j.neurobiolaging.2015.02.016. [DOI] [PubMed] [Google Scholar]
  60. Rojas JI, Zurru MC, Romano M, Patrucco L, Cristiano E. Acute ischemic stroke and transient ischemic attack in the very old–risk factor profile and stroke subtype between patients older than 80 years and patients aged less than 80 years. Eur J Neurol. 2007;14:895–899. doi: 10.1111/j.1468-1331.2007.01841.x. [DOI] [PubMed] [Google Scholar]
  61. Rubin LG, Schaffner W. Clinical practice. Care of the asplenic patient. N Engl J Med. 2014;371:349–356. doi: 10.1056/NEJMcp1314291. [DOI] [PubMed] [Google Scholar]
  62. Schumann RR. Old and new findings on lipopolysaccharide-binding protein: a soluble pattern-recognition molecule. Biochem Soc Trans. 2011;39:989–993. doi: 10.1042/BST0390989. [DOI] [PubMed] [Google Scholar]
  63. Seifert HA, Hall AA, Chapman CB, Collier LA, Willing AE, Pennypacker KR. A transient decrease in spleen size following stroke corresponds to splenocyte release into systemic circulation. J Neuroimmune Pharmacol. 2012a;7:1017–1024. doi: 10.1007/s11481-012-9406-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Seifert HA, Leonardo CC, Hall AA, Rowe DD, Collier LA, Benkovic SA, Willing AE, Pennypacker KR. The spleen contributes to stroke induced neurodegeneration through interferon gamma signaling. Metab Brain Dis. 2012b;27:131–141. doi: 10.1007/s11011-012-9283-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Seifert HA, Pennypacker KR. Molecular and cellular immune responses to ischemic brain injury. Transl Stroke Res. 2014;5:543–553. doi: 10.1007/s12975-014-0349-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Shao H, Xu Q, Wu Q, Ma Q, Salgueiro L, Wang J, Eton D, Webster KA, Yu H. Defective CXCR4 expression in aged bone marrow cells impairs vascular regeneration. J Cell Mol Med. 2011;15:2046–2056. doi: 10.1111/j.1582-4934.2010.01231.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest. 2012;122:787–795. doi: 10.1172/JCI59643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Stanimirovic D, Satoh K. Inflammatory mediators of cerebral endothelium: a role in ischemic brain inflammation. Brain Pathol. 2000;10:113–126. doi: 10.1111/j.1750-3639.2000.tb00248.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Stichel CC, Luebbert H. Inflammatory processes in the aging mouse brain: participation of dendritic cells and T-cells. Neurobiol Aging. 2007;28:1507–1521. doi: 10.1016/j.neurobiolaging.2006.07.022. [DOI] [PubMed] [Google Scholar]
  70. Tracey KJ. The inflammatory reflex. Nature. 2002;420:853–859. doi: 10.1038/nature01321. [DOI] [PubMed] [Google Scholar]
  71. Tschaikowsky K, Hedwig-Geissing M, Schmidt J, Braun GG. Lipopolysaccharide-binding protein for monitoring of postoperative sepsis: complemental to C-reactive protein or redundant? PLoS One. 2011;6:e23615. doi: 10.1371/journal.pone.0023615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Tuttolomondo A, Pecoraro R, Pinto A. Studies of selective TNF inhibitors in the treatment of brain injury from stroke and trauma: a review of the evidence to date. Drug Des Devel Ther. 2014;8:2221–2238. doi: 10.2147/DDDT.S67655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. van Deventer SJ, Buller HR, ten Cate JW, Aarden LA, Hack CE, Sturk A. Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood. 1990;76:2520–2526. [PubMed] [Google Scholar]
  74. Venna VR, Weston G, Benashski SE, Tarabishy S, Liu F, Li J, Conti LH, McCullough LD. NF-kappaB contributes to the detrimental effects of social isolation after experimental stroke. Acta Neuropathol. 2012;124:425–438. doi: 10.1007/s00401-012-0990-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Verma R, Harris NM, Friedler BD, Crapser J, Patel AR, Venna V, McCullough LD. Reversal of the Detrimental Effects of Post-Stroke Social Isolation by Pair-Housing is Mediated by Activation of BDNF-MAPK/ERK in Aged Mice. Sci Rep. 2016;6:25176. doi: 10.1038/srep25176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Villeda SA, Luo J, Mosher KI, Zou B, Britschgi M, Bieri G, Stan TM, Fainberg N, Ding Z, Eggel A, Lucin KM, Czirr E, Park JS, Couillard-Despres S, Aigner L, Li G, Peskind ER, Kaye JA, Quinn JF, Galasko DR, Xie XS, Rando TA, Wyss-Coray T. The ageing systemic milieu negatively regulates neurogenesis and cognitive function. Nature. 2011;477:90–94. doi: 10.1038/nature10357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Watkins LR, Maier SF, Goehler LE. Cytokine-to-brain communication: a review & analysis of alternative mechanisms. Life Sci. 1995;57:1011–1026. doi: 10.1016/0024-3205(95)02047-m. [DOI] [PubMed] [Google Scholar]
  78. Westendorp WF, Nederkoorn PJ, Vermeij JD, Dijkgraaf MG, van de Beek D. Post-stroke infection: a systematic review and meta-analysis. BMC Neurol. 2011;11:110. doi: 10.1186/1471-2377-11-110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Whiteley W, Chong WL, Sengupta A, Sandercock P. Blood markers for the prognosis of ischemic stroke: a systematic review. Stroke. 2009;40:e380–389. doi: 10.1161/STROKEAHA.108.528752. [DOI] [PubMed] [Google Scholar]
  80. Yan FL, Zhang JH. Role of the sympathetic nervous system and spleen in experimental stroke-induced immunodepression. Med Sci Monit. 2014;20:2489–2496. doi: 10.12659/MSM.890844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Zhang BJ, Men XJ, Lu ZQ, Li HY, Qiu W, Hu XQ. Splenectomy protects experimental rats from cerebral damage after stroke due to anti-inflammatory effects. Chin Med J (Engl) 2013;126:2354–2360. [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

1

Fig S1: Gating strategy for immune cells in the brain.

Supplementary Table 1: Circulating pro-inflammatory cytokines levels in sham and MCAo aged mice at 96 hours stroke. Data is presented as mean± SEM and analyzed by Wilcoxon rank sum test.

NIHMS909269-supplement-1.docx (397.5KB, docx)
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NIHMS909269-supplement-2.docx (12.2KB, docx)
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NIHMS909269-supplement-3.docx (12.5KB, docx)

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