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Surgical manipulation compromises leukocyte mobilization responses and inflammation after experimental cerebral ischemia in mice

Surgical manipulation compromises leukocyte mobilization responses and inflammation after... ORIGINAL RESEARCH ARTICLE published: 17 January 2014 doi: 10.3389/fnins.2013.00271 Surgical manipulation compromises leukocyte mobilization responses and inflammation after experimental cerebral ischemia in mice 1,2 1 1 1 1 Adam Denes *, Jesus M. Pradillo , Caroline Drake , Hannah Buggey ,Nancy J.Rothwell and Stuart M. Allan * Faculty of Life Sciences, University of Manchester, Manchester, UK Laboratory of Molecular Neuroendocrinology, Institute of Experimental Medicine, Budapest, Hungary Edited by: Acute brain injury results in peripheral inflammatory changes, although the impact of these Eric W. Roubos, Radboud University processes on neuronal death and neuroinflammation is currently unclear. To facilitate Nijmegen, Netherlands the translation of experimental studies to clinical benefit, it is vital to characterize Reviewed by: the mechanisms by which acute brain injury induces peripheral inflammatory changes, Jaleel A. Miyan, The University of and how these are affected by surgical manipulation in experimental models. Here Manchester, UK James Downing, Liverpool John we show that in mice, even mild surgical manipulation of extracranial tissues induced Moores University, UK marked granulocyte mobilization (300%) and systemic induction of cytokines. However, Eric W. Roubos, Radboud University intracranial changes induced by craniotomy, or subsequent induction of focal cerebral Nijmegen, Netherlands ischemia were required to induce egress of CXCR2-positive granulocytes from the bone *Correspondence: marrow. CXCR2 blockade resulted in reduced mobilization of granulocytes from the bone Adam Denes, Laboratory of Molecular Neuroendocrinology, marrow, caused an unexpected increase in circulating granulocytes, but failed to affect MTA KOKI, Szigony u. 43., brain injury induced by cerebral ischemia. We also demonstrate that isoflurane anaesthesia 1083 Budapest, Hungary interferes with circulating leukocyte responses, which could contribute to the reported e-mail: adam.denes@ vascular and neuroprotective effects of isoflurane. In addition, no immunosuppression manchester.ac.uk; denesa@koki.hu; Stuart M. Allan, Faculty of Life develops in the bone marrow after experimental stroke. Thus, experimental models of Sciences, University of Manchester, cerebral ischemia are compromised by surgery and anaesthesia in proportion to the AV Hill Building, Oxford Road, severity of surgical intervention and overall tissue injury. Understanding the inherent Manchester M13 9PT, UK confounding effects of surgical manipulation and development of new models of cerebral e-mail: stuart.allan@ manchester.ac.uk ischemia with minimal surgical intervention could facilitate better understanding of interactions between inflammation and brain injury. Keywords: experimental stroke, bone marrow, granulocyte, CXCR2, anaesthesia INTRODUCTION studies on systemic inflammatory mechanisms in experimental Brain injury due to cerebral ischemia, hemorrhage, or head animals remain relatively sparse. trauma results in early activation of the immune system, fol- We and others have shown that experimental stroke results lowed later by immunosuppression (Dirnagl et al., 2007; Denes in the activation of inflammatory responses in various immune et al., 2010b). Activated immune cells and inflammatory media- organs such as the spleen, blood, or bone marrow (Offner et al., tors contribute to neuroinflammation and overall outcome after 2006a,b; Chapman et al., 2009; Denes et al., 2011). Using a fila- experimental brain injury. Blockade of toll-like receptors, proin- ment model of transient, focal cerebral ischemia we also showed flammatory mediators such as interleukin-1, and immune cells that anaesthesia and surgical intervention compromise stroke- such as T cells, neutrophils, or mast cells, is protective in exper- induced inflammatory responses in the periphery (Denes et al., imental models of brain injury (Iadecola and Anrather, 2011; 2011). However, a systematic analysis of stroke-induced periph- Smith et al., 2013). However, oversuppression of the immune eral inflammatory actions, including the assessment of confound- system in response to brain injury gives rise to opportunistic ing effects of surgical manipulations has not yet been performed infections leading to impaired recovery and death of patients in experimental stroke that involves craniotomy. Such investi- and experimental animals (Dirnagl et al., 2007; Murray et al., gations could distinguish model-specific and ischemia-induced 2013). Therefore, understanding interactions between peripheral effects of experimental stroke on immune responses and yield inflammatory responses and brain injury could pave the way for important data for translation to patients. novel therapeutic interventions. Thus, we examined the effects of anaesthesia, surgical manip- Lack of translation in stroke has triggered intense discussions, ulation, and experimental stroke induced by transient, distal leading to improved preclinical guidelines on experimental mod- middle cerebral artery occlusion with craniotomy on peripheral eling (Fisher et al., 2009). Yet, compared to the large number inflammatory responses. We show that both isoflurane anaes- of clinical studies on peripheral inflammatory changes in stroke, thesia and surgical intervention induce changes in peripheral www.frontiersin.org January 2014 | Volume 7 | Article 271 | 1 Denes et al. Surgical intervention affects leukocyte responses immunecellpopulationsinthe absenceofstroke, andare likely to blood samples are presented throughout the manuscript, with contribute to systemic inflammatory responses induced by exper- the exception of Figures 1A,B. To isolate the bone marrow and imental brain injury. These effects should be accounted for in the spleen, mice were perfused transcardially with saline under experimental stroke modeling. isoflurane anaesthesia. Brains were subsequently perfused with 4% paraformaldehyde, post-fixed for 24 h, cryoprotected in 20% sucrose/PBS, and sectioned (20µm diameter) on a sledge micro- MATERIALS AND METHODS tome. Organs were homogenized as described previously (Denes MICE et al., 2010a). Male 8–12 week-old C57BL/6 mice (n = 58) were kept at 21 ± 1 C and 65% humidity with a 12 h light-dark cycle and had free CXCR2 BLOCKADE access to food and water. All animal procedures were performed To prevent CXCR2-mediated release of granulocytes from the under appropriate project license authority and adhered to the bone marrow, mice in the MCAo group were treated intraperi- UK Animals (Scientific Procedures) Act (1986) and were in accor- toneally with a selective CXCR2 antagonist (SB225002) or vehicle. dance with STAIR and ARRIVE (Fisher et al., 2009; Kilkenny SB225002 was dissolved in DMSO (stock), diluted in sterile saline et al., 2010) guidelines. 1:40 and injected as 2 mg/kg, 200µl/mouse, 20 min prior to induction of anaesthesia for MCAo surgery. MIDDLE CEREBRAL ARTERY OCCLUSION (MCAo) Distal, transient focal cerebral ischemia was induced as described In vitro STIMULATION OF BONE MARROW CELLS earlier (Pradillo et al., 2009, 2012). Briefly, mice were anaes- Bone marrow cells were isolated 72 h after 60 min MCAo or the thetized with isoflurane and ischemia was induced by a transient surgery control and stimulated in vitro with 1µg/ml bacterial ligature (60 min) of the left MCA trunk with a 10-0 suture lipopolysaccharide (LPS, E. coli O26:B6), at a density of 7.5 × 10 (Prolene, Ethicon, Somerville, NJ, USA). Occlusion and reper- cells/mL in RPMI medium, supplemented with 10% fetal calf fusion were confirmed visually under the surgical microscope. serum and penicillin/streptomycin to investigate cytokine pro- Core body temperature was maintained at 37.0 ± 0.5 Cthrough- duction. After 3 h incubation at 37 C, cells were pelleted with out the surgery by a heating blanket (Homeothermic Blanket centrifugation at 400 × g, the supernatant was collected, and cells Control Unit; Harvard Apparatus, Kent, UK) and monitored after were lysed in lysis buffer. recovery. After surgery, animals were returned to their cages and allowed free access to water and food. It was decided, apriori,to CYTOMETRIC BEAD ARRAY exclude from the study those animals that showed brain hemor- Circulating levels of IL-6 and KC (CXCL-1), and levels of IL-6 in rhage at any time of the surgery or with no reperfusion (2 mice). bone marrow cell culture supernatants and lysates were measured The survival rate was 100%. by using CBA Flex Sets (BD Biosciences, UK) according to the manufacturers protocol. The detection limit for each cytokine was SURGICAL CONTROLS 5–10 pg/ml. We investigated the effect of surgical manipulation and anaesthe- sia in the absence of MCAo on cellular and cytokine responses. FLOW CYTOMETRY To achieve this, three separate experimental conditions were used; Spleen, bone marrow, and blood cells were isolated and the first involved only anaesthesia with no surgical manipulation stained with appropriate combinations of CD45-PerCP-Cy5.5, (termed as “isoflurane”), the second exposure of the skull bone Ly6c-PerCP-Cy5.5, CD4-PE-Cy7, CD8-PE, CD3-APC, CD19-PE- but no craniotomy (termed as “sham no cran.”) and the third Cy7, MHCII-APC (eBioscience, UK), and Ly6G-PE (1A8, BD full craniotomy (termed as “sham”). Except for naive animals, Biosciences, UK) following Fc receptor blockade (eBioscience). all groups of mice were kept under isoflurane anaesthesia for the Contaminating red blood cells in bone marrow and spleen sam- same time period (75 min for control experimental conditions ples were removed by ACK solution, and FACS lysing solution and in the “MCAo” group). (BD Biosciences) was used to remove red blood cells from blood samples. Total blood cell counts were calculated by using BLOOD SAMPLING AND TISSUE PROCESSING 15µm polystyrene microbeads (Polysciences, 18328-5). Cells Blood samples were taken from the tail vein and from the right were acquired on an LSRII flow cytometer, using FACS Diva soft- cardiac ventricle, using 3.8% sodium citrate 1:10 as an anti- ware (BD Biosciences, UK). Except for the 72 h time point, flow coagulant. Repeated tail vein samples were collected at various cytometric data have been pooled from three independent experi- time points, with equal sampling times across different treat- ments, in which all experimental groups have been represented by ment groups: prior to surgery (“naïve”), 60 min after the onset at least one animal. Due to red blood cell contamination and cell of ischemia or corresponding control surgery (“0 min reperfu- labeling artifacts n = 6 blood samples, n = 3bonemarrowsam- sion”), and before transcardial perfusion (at 4 or 72 h reperfu- ples and n = 4 spleen samples have been excluded from analysis sion, not shown). Terminal cardiac blood samples were collected across experiments, pre-hoc. immediately prior to transcardial perfusion from naive mice, and from mice that had undergone surgical interventions, at 4 RANDOMIZATION, QUANTIFICATION, AND STATISTICAL ANALYSIS or 72 h reperfusion. Blood sample data from different vascular Animals were randomized for experiments and all quantita- beds were analyzed separately. To avoid potential confounding tive analyses were performed in a blinded manner. Group sizes effects of repeated tail vein samples at later time points, cardiac were determined based on our earlier in vivo experiments using Frontiers in Neuroscience | Neuroendocrine Science January 2014 | Volume 7 | Article 271 | 2 Denes et al. Surgical intervention affects leukocyte responses FIGURE 1 | Tissue injury, craniotomy, and anaesthesia result in rapid B cells (dark blue color indicates CD19+ positive cells). Proportion (D) and alteration of leukocyte responses. Extracranial tissue injury results in total cell numbers (E) of granulocytes in cardiac blood samples at 4 and 72 h increased proportion (A) and cell numbers (B) of granulocytes in the reperfusion. Proportion (F) and total cell numbers (G) of MHCII+ B cells circulation, which is indistinguishable from changes induced by sham surgery (CD19+) in cardiac blood samples at 4 and 72 h reperfusion. Sham no or MCAo. Blood samples taken from the tail vein prior to surgery and 60 min cran.—sham surgery without craniotomy. n = 5, 4, 4, 5, 6–7, 3, 3 in naïve, after the onset of MCAo/control surgery (0 min reperfusion) are shown. isoflurane, sham no cran., sham, MCAo, sham 72 h, and MCAo 72 h, ∗ ∗∗ ∗∗∗ (C) Representative dot plots showing terminal cardiac blood samples at 4 h respectively. P < 0.05, P < 0.01, P < 0.001 vs. naive. Light blue color reperfusion for graphs (D–G). P3 gate: Ly6G+ granulocytes, P4 gate: MHCII+ indicates negative (unstained) population. this experimental model. Normality of data sets was assessed RESULTS using GraphPad Prism (KS normality test). Statistical analy- SURGICAL MANIPULATION AND ANAESTHESIA ALTER LEUKOCYTE sis was performed by One-Way or Two-Way ANOVA followed RESPONSES IN THE BLOOD by Tukey’s or Bonferroni’s post-hoc multiple-comparison, using Surgical manipulation of extracranial tissues was sufficient to GraphPad Prism 5 software. In case of non-parametric data, induce rapid mobilization of granulocytes. In serial blood sam- significance was confirmed by non-parametric t-test (Mann– ples taken from the tail vein, the proportion of granulocytes high Whitney U-test) or Kruskal–Wallis test followed by Dunn’s mul- (Ly6G+ CD11b+ SSC cells) increased from 12 to 40% within tiple comparisons test. All data are expressed as mean ± standard 70–80 min (“0 min reperfusion”) in response surgery without error of the mean (s.e.m). P < 0.05 was considered statistically craniotomy (“sham no cran.,” Figure 1A), but not after isoflu- significant. rane anaesthesia alone, representing a ∼3-fold increase in total www.frontiersin.org January 2014 | Volume 7 | Article 271 | 3 Denes et al. Surgical intervention affects leukocyte responses high granulocyte numbers (Figure 1B). Granulocyte mobilization was population, CXCR2+ granulocytes (Ly6G+ CD11b+ SSC not altered further by craniotomy or MCAo (Figures 1A,B). cells) showed a decrease in the bone marrow proportional to the Granulocytosis was also evident 5–5.5 h after initiation of surgery level of surgical stress, reaching a significant 50% reduction after in cardiac, terminal blood samples (termed as “4 h reperfusion,” craniotomy and an over 60% reduction in response to MCAo Figures 1C,D), although total granulocyte numbers were lower (Figure 3B). Correspondingly, KC (CXCL-1) and IL-6 concen- compared to the earlier time point and were significantly ele- trations in the circulation increased by 20–40-fold after sham vated only in response to craniotomy or MCAo (Figure 1E). An surgery and MCAo (Figures 3C,D). Circulating KC levels showed increased proportion of granulocytes in the sham and MCAo a significant (P < 0.01) negative correlation with numbers of groups was observed up to 72 h after surgery (Figure 1D). CXCR2+ granulocytes in the bone marrow at 4 h reperfusion CD19+ MHCII+ B cells decreased profoundly (by 80–90%) (not shown). Anaesthesia alone appeared to increase B cells and T in the blood in response to anaesthesia alone, which was apparent cells in the bone marrow (Figures 3E–G) similarly to our earlier in all other groups of mice that underwent surgical manipulation findings (Denes et al., 2011). or MCAo, at 4 h reperfusion (Figures 1F,G). Similarly, anaes- thesia alone resulted in reduced circulating CD4+ and CD8+ BONE MARROW CELLS DO NOT EXHIBIT A SUPPRESSED RESPONSE T cell numbers within 4 h (Figures 2A–C). Interestingly, cran- AFTER CEREBRAL ISCHEMIA iotomy and MCAo appeared to raise T cell numbers over levels At 72 h reperfusion when systemic immunosuppression is appar- after anaesthesia alone, which was more apparent in the case ent after cerebral ischemia (Prass et al., 2003), bone marrow cells of CD4+ T cells (Figures 2B,C). Circulating B cells and CD8+ showed no difference in LPS-induced IL-6 production in vitro T cells remained significantly lower in number in surgery con- as assessed in cell lysates (Figure 4A) and cell culture super- trol groups and after MCAo compared to naïve mice at 72 h natants (Figure 4B), indicating that at this time point cells resid- reperfusion (Figures 1F,G, 2A,C). ing in the bone marrow maintain their ability to respond to endotoxin. GRANULOCYTE RELEASE FROM THE BONE MARROW INCREASES PROPORTIONALLY TO THE LEVEL OF SURGICAL STRESS AND BRAIN SURGICAL INTERVENTION AND CEREBRAL ISCHEMIA ALTER SPLENIC INJURY GRANULOCYTE RESPONSES MCAo resulted in mobilization of granulocytes from the bone Surgical manipulation of extracranial tissues with or without marrow at 4 h reperfusion (Figure 3A). Within the granulocyte craniotomy resulted in an early recruitment of granulocytes in FIGURE 2 | Isoflurane anaesthesia results in reduced circulating T cell Purple color indicates cells gated on CD3. Sham no cran.—sham surgery numbers. CD8+ T cells (A) and CD4+ T cells (B) are reduced in number without craniotomy. n = 4, 4, 4, 5, 6, 3, 3 in naïve, isoflurane, sham no in cardiac blood samples within 4 h following isoflurane anaesthesia. cran., sham, MCAo, sham 72 h, and MCAo 72 h, respectively. P < 0.05, ∗∗ (C) Representative dot blots showing CD8+ and CD4+ T cells in cardiac P < 0.01 vs. naive. Light blue color indicates negative (unstained) blood samples 4 h after reperfusion, control surgeries, or anaesthesia. population. Frontiers in Neuroscience | Neuroendocrine Science January 2014 | Volume 7 | Article 271 | 4 Denes et al. Surgical intervention affects leukocyte responses FIGURE 3 | Surgical intervention and anaesthesia result in altered amount of surgical stress. Anaesthesia results in increased MHCII+ B leukocyte responses in the bone marrow. (A) Bone marrow cells (E) CD4+ T cells (F) and CD8+ T cells (G) in the bone marrow high granulocytes (Ly6G+,CD11b+, SSC cells) are reduced within 4 h after within 4 h. Sham no cran.—sham surgery without craniotomy. n = 7, 5, MCAo. (B) Both MCAo and craniotomy result in reduced CXCR2+ 6, 7, 7–8 in naïve, isoflurane, sham no cran., sham, and MCAo, ∗ ∗∗ ∗∗∗ granulocytes in the bone marrow at 4 h reperfusion. KC (CXCL1, C)and respectively. P < 0.05, P < 0.01, P < 0.001 vs. naïve, unless IL-6 (D) levels increase in the circulation within 4 h proportionally to the indicated otherwise on the graphs; P < 0.05 vs. isoflurane. CXCR2 BLOCKADE PREVENTS GRANULOCYTE MOBILIZATION FROM THE BONE MARROW BUT DOES NOT ALTER ISCHEMIC BRAIN INJURY The selective CXCR2 inhibitor SB225002 (White et al., 1998)pre- vented granulocyte release from the bone marrow as confirmed by a significant reduction of granulocytes in the bone marrow in vehicle-treated mice 4 h after MCAo compared to mice treated with SB225002 (Figures 6A,B). Similarly to our earlier experi- ments using the intraluminal filament method to induce cerebral ischemia (Denes et al., 2011) bone marrow granulocyte numbers were stabilized by 24 h post MCAo (Figure 6A). To our surprise, CXCR2 blockade although reducing granulocyte release from the FIGURE 4 | Bone marrow cells do not exhibit a suppressed response to bone marrow, resulted in an increase in circulating granulocytes endotoxin after distal tMCAo. Bone marrow cells were isolated 72 h after at 4 h reperfusion compared to vehicle (Figure 6C). We investi- MCAo and stimulated in vitro with bacterial lipopolysaccharide (LPS) to gated whether CXCR2 blockade resulted in any changes in infarct investigate cytokine production. Cell lysates (A) and supernatants (B) were measured for IL-6 production and release. n.s., not significant. n = 3. size, but no difference between SB225002 treatment and vehicle ∗ ∗∗ P < 0.05, P < 0.01 vs. control. was observed (Figure 6D), indicating that CXCR2-mediated sig- nals do not contribute substantially to brain injury in the current experimental model of cerebral ischemia. thespleen(Figure 5A). This effect was not apparent in the DISCUSSION CXCR2+ granulocyte population (Figure 5B). MCAo signifi- cantly reduced splenic total granulocyte- and CXCR2+ gran- Here we present evidence that even mild surgical manipulation ulocyte numbers (Figures 5A,B)comparedtoshamsurgery at results in marked systemic granulocyte mobilization responses. 4 h reperfusion. No changes were observed in B cell or T Granulocyte mobilization is proportional to the level of surgi- cell populations in the spleen in any of the surgical groups cal stress and is further augmented by brain injury. Granulocyte (Figures 5C–E). responses to cranial injury or cerebral ischemia involve primarily www.frontiersin.org January 2014 | Volume 7 | Article 271 | 5 Denes et al. Surgical intervention affects leukocyte responses FIGURE 5 | Leukocyte responses in the spleen. Changes in total CD8+ T cells (E) at 4 h reperfusion. Sham no cran.—sham surgery without high granulocytes (Ly6G+,CD11b+, SSC cells, A) and CXCR2+ granulocytes craniotomy. n = 6, 6, 6, 5, 6 in naïve, isoflurane, sham no cran., sham, and ∗ ∗∗∗ ∗∗ (B) in the spleen 4 h following reperfusion or control surgical interventions. MCAo, respectively. P < 0.05 and P < 0.001 vs. naïve; P < 0.01 (A) No changes are observed in splenic MHCII+ B cells (C) CD4+ T cells (D) and and P < 0.05 (B) sham vs. MCAo. the mobilization of CXCR2-positive granulocytes, which is not which only extracranial tissues were manipulated. Extracranial seen after manipulation of extracranial tissues. In addition, we manipulation consisting of an incision on the skin and tempo- show that anaesthesia profoundly alters leukocyte responses, an rary dislocation of small pericranial muscles was sufficient to effect that lasts for several hours or even days. Collectively, these generate a systemic inflammatory response evidenced by granulo- data highlight the role of anaesthesia and surgical intervention in cyte mobilization and a 10-fold increase in circulating KC levels. systemic inflammatory responses, which could have an impact on Since these changes did not involve major alterations in CXCR2+ experimental models of cerebral ischemia, but could also serve granulocytes, identification of the mechanisms involved (contri- important information for the management of patients subjected bution of bone marrow-derived cells, signals that initiate and to various surgeries in the clinic. maintain systemic granulocyte responses, etc.) warrant further In our previous study we revealed changes in the bone mar- investigation. It is likely that activation of the HPA axis and the row in response to transient, focal cerebral ischemia induced by autonomic nervous system (Elenkov et al., 2000) could contribute an intraluminal filament that is advanced through the internal to surgery-induced peripheral changes, however the exact signals carotid artery to occlude the MCA (Denes et al., 2011). Although mediating leukocyte responses need to be identified. In fact, we we demonstrated changes induced by brain injury itself, we also confirmed here our earlier findings showing an increased mobi- showed that anaesthesia and surgical intervention could impact lization of CXCR2-positive granulocytes in response to brain on bone marrow leukocyte responses (Denes et al., 2011). Since injury, compared with other surgical manipulations (Denes et al., the filament MCAo model involves surgical manipulation around 2011). the neck including exposure of the salivary glands, the vagus nerve We found that isoflurane anaesthesia alone resulted in a pro- and other tissues around the trachea, in the present study we per- found reduction in circulating lymphocytes within hours. Some formed a systematic analysis of bone marrow, spleen, and blood effects of isoflurane on leukocyte activation have been reported responses, using a distal MCAo model, to reveal stroke-specific earlier (Yuki et al., 2012; Carbo et al., 2013), and a decrease in changes in leukocyte mobilization as well as the impact of surgi- Th1/Th2 ratio in the blood has been observed in patients under- cal intervention and anaesthesia. Since the present model includes going craniotomy in response to isoflurane (Inada et al., 2004). craniotomy, a potential confounder that disturbs the internal However, the present study demonstrates a rapid and sustained milieu of the brain, we also included control surgeries during reduction in circulating T cell numbers induced by isoflurane Frontiers in Neuroscience | Neuroendocrine Science January 2014 | Volume 7 | Article 271 | 6 Denes et al. Surgical intervention affects leukocyte responses FIGURE 6 | Blockade of CXCR2-mediated granulocyte release from Ly6G+, blue) in the bone marrow at 4 h reperfusion. (C) SB225002 the bone marrow does not alter infarct size after experimental increases granulocyte numbers in the cardiac blood at 4 h reperfusion stroke. (A) Treatment with the selective CXCR2 antagonist SB225002 after MCAo. (D) Infarct size is not altered in response to SB225002 prevents granulocyte release from the bone marrow after MCAo treatment at 24 h reperfusion, after MCAo. (A–C) n = 3, 3, 5, 5 in compared to vehicle at 4 h reperfusion. Total granulocyte numbers vehicle 4 h, SB225002 4 h, vehicle 24 h, and SB225002 24 h samples, ∗ # recovered from 1 femur and 1 tibia are shown. (B) Representative respectively, (D) n = 5–6. P < 0.05 vs. vehicle 4 h, P < 0.05 vs. high density plots showing CD11b+ SSC granulocytes (gated also on 24 h time points. anaesthesia, which could have important implications clinically CXCR2 is the best characterized cell surface receptor on gran- and also in models of experimental stroke. The contribution of ulocytes involved in cell mobilization to stimuli mediated by T cells to the ischemic brain injury is well documented (Iadecola CXCL1 (KC) and CXCL2/3 (MIP-2) (Matzer et al., 2004; Eash and Anrather, 2011). Since volatile anaesthetics can exert neuro- et al., 2010; Veenstra and Ransohoff, 2012). Although brain protective properties (Kawaguchi et al., 2005), it is possible that injury-induced granulocyte mobilization from the bone mar- at least in part, these actions could be mediated via blunted T cell row was prevented by CXCR2 blockade, circulating granulocytes responses. increased in response to SB225002 treatment. CXCR2 inhibition The spleen is profoundly affected by brain injury (Offner et al., was found effective to reduce IL-8- or KC-induced granulocyte 2006b), leading to loss of B cells and development of immuno- mobilization in the blood and block granulocyte egress from suppression, however, much less is known about how cerebral the bone marrow (White et al., 1998; Martin et al., 2003; Eash ischemia contributes to responses of myeloid cells in the spleen. et al., 2010). We show here that very early granulocyte mobiliza- In fact, a population of splenic monocytes are released rapidly tion responses in response to brain injury are not dependent on upon activation and contributes to ischemic processes in the heart CXCR2, and might not (or only in part) require the contribu- (Swirski et al., 2009; Leuschner et al., 2012). We found that sur- tion of the bone marrow in the current experimental model. This gical manipulation results in increased granulocyte recruitment could explain the lack of an effect by CXCR2 blockade on the to the spleen whilst cerebral ischemia reduced surgery-induced size of ischemic brain injury, which has also been confirmed by increases in splenic granulocytes. Although the role and mecha- another study (Brait et al., 2011). Brain injury in different experi- nisms of surgery-induced splenic granulocytosis requires further mental models might not be influenced by peripheral leukocyte investigations, this scenario indicates that the effect of surgical actions to the same extent. For example, blockade of granulo- manipulation on leukocyte responses in peripheral organs is likely cyte responses or hematopoietic MyD88-dependent actions is not to be a confounder in current experimental stroke models. associated with reduced central inflammation or brain damage www.frontiersin.org January 2014 | Volume 7 | Article 271 | 7 Denes et al. Surgical intervention affects leukocyte responses after cold-induced cortical injury (Koedel et al., 2007). In con- Dirnagl, U., Klehmet, J., Braun, J. S., Harms, H., Meisel, C., Ziemssen, T., et al. (2007). Stroke-induced immunodepression: experimental evidence and trast, neutrophils contribute to brain injury when experimental clinical relevance. Stroke 38, 770–773. doi: 10.1161/01.STR.0000251441.89 stroke is preceded by systemic inflammation (McColl et al., 2007). 665.bc Therefore, the source of neutrophils mobilized after brain injury Eash, K. J., Greenbaum, A. M., Gopalan, P. K., and Link, D. C. (2010). CXCR2 and their contribution to the population that is recruited into and CXCR4 antagonistically regulate neutrophil trafficking from murine bone the brain need to be investigated in further studies. Stroke in marrow. J. Clin. Invest. 120, 2423–2431. doi: 10.1172/JCI41649 Elenkov, I. J., Wilder, R. L., Chrousos, G. P., and Vizi, E. S. (2000). The sympa- patients results in increased circulating granulocyte numbers and thetic nerve–an integrative interface between two supersystems: the brain and loss of T cells (Vogelgesang et al., 2008), which corresponds to the immune system. Pharmacol. Rev. 52, 595–638. the findings presented in our study. Surgical interventions and Fisher, M., Feuerstein, G., Howells, D. W., Hurn, P. 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Since circulating white 2044.2004.03837.x blood cell- and blood cytokine data from stroke patients and from Kawaguchi, M., Furuya, H., and Patel, P. M. (2005). Neuroprotective effects of those undergoing anaesthesia or surgical interventions are widely anesthetic agents. J. Anesth. 19, 150–156. doi: 10.1007/s00540-005-0305-5 available, blood cell responses and mechanisms of brain injury Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., and Altman, D. G. (2010). in experimental models should be investigated in a translational Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 8:e1000412. doi: 10.1371/journal.pbio.1000412 context. Koedel, U., Merbt, U. M., Schmidt, C., Angele, B., Popp, B., Wagner, H., In conclusion, our data indicate that anaesthesia- and surgery- et al. (2007). Acute brain injury triggers MyD88-dependent, TLR2/4- induced leukocyte responses interact with those induced by independent inflammatory responses. 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(2009). Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science 325, 612–616. doi: 10.1126/sci- ence.1175202 Received: 30 September 2013; accepted: 21 December 2013; published online: 17 Veenstra, M., and Ransohoff, R. M. (2012). Chemokine receptor CXCR2: physiol- January 2014. ogy regulator and neuroinflammation controller? J. Neuroimmunol. 246, 1–9. Citation: Denes A, Pradillo JM, Drake C, Buggey H, Rothwell NJ and Allan SM (2014) doi: 10.1016/j.jneuroim.2012.02.016 Surgical manipulation compromises leukocyte mobilization responses and inflamma- Vogelgesang, A., Grunwald, U., Langner, S., Jack, R., Broker, B. M., Kessler, tion after experimental cerebral ischemia in mice. Front. Neurosci. 7:271. doi: 10.3389/ C., et al. (2008). Analysis of lymphocyte subsets in patients with stroke fnins.2013.00271 and their influence on infection after stroke. Stroke 39, 237–241. doi: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers 10.1161/STROKEAHA.107.493635 in Neuroscience. White, J. R.,Lee,J.M., Young,P.R., Hertzberg, R. P.,Jurewicz, A. J.,Chaikin,M. Copyright © 2014 Denes, Pradillo, Drake, Buggey, Rothwell and Allan. This is an A., et al. (1998). Identification of a potent, selective non-peptide CXCR2 antag- open-access article distributed under the terms of the Creative Commons Attribution onist that inhibits interleukin-8-induced neutrophil migration. J. Biol. Chem. License (CC BY). The use, distribution or reproduction in other forums is permit- 273, 10095–10098. doi: 10.1074/jbc.273.17.10095 ted, provided the original author(s) or licensor are credited and that the original Yuki, K., Bu, W., Xi, J., Sen, M., Shimaoka, M., and Eckenhoff, R. G. (2012). publication in this journal is cited, in accordance with accepted academic prac- Isoflurane binds and stabilizes a closed conformation of the leukocyte function- tice. No use, distribution or reproduction is permitted which does not comply with associated antigen-1. FASEB J. 26, 4408–4417. doi: 10.1096/fj.12-212746 these terms. www.frontiersin.org January 2014 | Volume 7 | Article 271 | 9 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Neuroscience Pubmed Central

Surgical manipulation compromises leukocyte mobilization responses and inflammation after experimental cerebral ischemia in mice

Frontiers in Neuroscience , Volume 7 – Jan 17, 2014

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Copyright © 2014 Denes, Pradillo, Drake, Buggey, Rothwell and Allan.
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10.3389/fnins.2013.00271
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Abstract

ORIGINAL RESEARCH ARTICLE published: 17 January 2014 doi: 10.3389/fnins.2013.00271 Surgical manipulation compromises leukocyte mobilization responses and inflammation after experimental cerebral ischemia in mice 1,2 1 1 1 1 Adam Denes *, Jesus M. Pradillo , Caroline Drake , Hannah Buggey ,Nancy J.Rothwell and Stuart M. Allan * Faculty of Life Sciences, University of Manchester, Manchester, UK Laboratory of Molecular Neuroendocrinology, Institute of Experimental Medicine, Budapest, Hungary Edited by: Acute brain injury results in peripheral inflammatory changes, although the impact of these Eric W. Roubos, Radboud University processes on neuronal death and neuroinflammation is currently unclear. To facilitate Nijmegen, Netherlands the translation of experimental studies to clinical benefit, it is vital to characterize Reviewed by: the mechanisms by which acute brain injury induces peripheral inflammatory changes, Jaleel A. Miyan, The University of and how these are affected by surgical manipulation in experimental models. Here Manchester, UK James Downing, Liverpool John we show that in mice, even mild surgical manipulation of extracranial tissues induced Moores University, UK marked granulocyte mobilization (300%) and systemic induction of cytokines. However, Eric W. Roubos, Radboud University intracranial changes induced by craniotomy, or subsequent induction of focal cerebral Nijmegen, Netherlands ischemia were required to induce egress of CXCR2-positive granulocytes from the bone *Correspondence: marrow. CXCR2 blockade resulted in reduced mobilization of granulocytes from the bone Adam Denes, Laboratory of Molecular Neuroendocrinology, marrow, caused an unexpected increase in circulating granulocytes, but failed to affect MTA KOKI, Szigony u. 43., brain injury induced by cerebral ischemia. We also demonstrate that isoflurane anaesthesia 1083 Budapest, Hungary interferes with circulating leukocyte responses, which could contribute to the reported e-mail: adam.denes@ vascular and neuroprotective effects of isoflurane. In addition, no immunosuppression manchester.ac.uk; denesa@koki.hu; Stuart M. Allan, Faculty of Life develops in the bone marrow after experimental stroke. Thus, experimental models of Sciences, University of Manchester, cerebral ischemia are compromised by surgery and anaesthesia in proportion to the AV Hill Building, Oxford Road, severity of surgical intervention and overall tissue injury. Understanding the inherent Manchester M13 9PT, UK confounding effects of surgical manipulation and development of new models of cerebral e-mail: stuart.allan@ manchester.ac.uk ischemia with minimal surgical intervention could facilitate better understanding of interactions between inflammation and brain injury. Keywords: experimental stroke, bone marrow, granulocyte, CXCR2, anaesthesia INTRODUCTION studies on systemic inflammatory mechanisms in experimental Brain injury due to cerebral ischemia, hemorrhage, or head animals remain relatively sparse. trauma results in early activation of the immune system, fol- We and others have shown that experimental stroke results lowed later by immunosuppression (Dirnagl et al., 2007; Denes in the activation of inflammatory responses in various immune et al., 2010b). Activated immune cells and inflammatory media- organs such as the spleen, blood, or bone marrow (Offner et al., tors contribute to neuroinflammation and overall outcome after 2006a,b; Chapman et al., 2009; Denes et al., 2011). Using a fila- experimental brain injury. Blockade of toll-like receptors, proin- ment model of transient, focal cerebral ischemia we also showed flammatory mediators such as interleukin-1, and immune cells that anaesthesia and surgical intervention compromise stroke- such as T cells, neutrophils, or mast cells, is protective in exper- induced inflammatory responses in the periphery (Denes et al., imental models of brain injury (Iadecola and Anrather, 2011; 2011). However, a systematic analysis of stroke-induced periph- Smith et al., 2013). However, oversuppression of the immune eral inflammatory actions, including the assessment of confound- system in response to brain injury gives rise to opportunistic ing effects of surgical manipulations has not yet been performed infections leading to impaired recovery and death of patients in experimental stroke that involves craniotomy. Such investi- and experimental animals (Dirnagl et al., 2007; Murray et al., gations could distinguish model-specific and ischemia-induced 2013). Therefore, understanding interactions between peripheral effects of experimental stroke on immune responses and yield inflammatory responses and brain injury could pave the way for important data for translation to patients. novel therapeutic interventions. Thus, we examined the effects of anaesthesia, surgical manip- Lack of translation in stroke has triggered intense discussions, ulation, and experimental stroke induced by transient, distal leading to improved preclinical guidelines on experimental mod- middle cerebral artery occlusion with craniotomy on peripheral eling (Fisher et al., 2009). Yet, compared to the large number inflammatory responses. We show that both isoflurane anaes- of clinical studies on peripheral inflammatory changes in stroke, thesia and surgical intervention induce changes in peripheral www.frontiersin.org January 2014 | Volume 7 | Article 271 | 1 Denes et al. Surgical intervention affects leukocyte responses immunecellpopulationsinthe absenceofstroke, andare likely to blood samples are presented throughout the manuscript, with contribute to systemic inflammatory responses induced by exper- the exception of Figures 1A,B. To isolate the bone marrow and imental brain injury. These effects should be accounted for in the spleen, mice were perfused transcardially with saline under experimental stroke modeling. isoflurane anaesthesia. Brains were subsequently perfused with 4% paraformaldehyde, post-fixed for 24 h, cryoprotected in 20% sucrose/PBS, and sectioned (20µm diameter) on a sledge micro- MATERIALS AND METHODS tome. Organs were homogenized as described previously (Denes MICE et al., 2010a). Male 8–12 week-old C57BL/6 mice (n = 58) were kept at 21 ± 1 C and 65% humidity with a 12 h light-dark cycle and had free CXCR2 BLOCKADE access to food and water. All animal procedures were performed To prevent CXCR2-mediated release of granulocytes from the under appropriate project license authority and adhered to the bone marrow, mice in the MCAo group were treated intraperi- UK Animals (Scientific Procedures) Act (1986) and were in accor- toneally with a selective CXCR2 antagonist (SB225002) or vehicle. dance with STAIR and ARRIVE (Fisher et al., 2009; Kilkenny SB225002 was dissolved in DMSO (stock), diluted in sterile saline et al., 2010) guidelines. 1:40 and injected as 2 mg/kg, 200µl/mouse, 20 min prior to induction of anaesthesia for MCAo surgery. MIDDLE CEREBRAL ARTERY OCCLUSION (MCAo) Distal, transient focal cerebral ischemia was induced as described In vitro STIMULATION OF BONE MARROW CELLS earlier (Pradillo et al., 2009, 2012). Briefly, mice were anaes- Bone marrow cells were isolated 72 h after 60 min MCAo or the thetized with isoflurane and ischemia was induced by a transient surgery control and stimulated in vitro with 1µg/ml bacterial ligature (60 min) of the left MCA trunk with a 10-0 suture lipopolysaccharide (LPS, E. coli O26:B6), at a density of 7.5 × 10 (Prolene, Ethicon, Somerville, NJ, USA). Occlusion and reper- cells/mL in RPMI medium, supplemented with 10% fetal calf fusion were confirmed visually under the surgical microscope. serum and penicillin/streptomycin to investigate cytokine pro- Core body temperature was maintained at 37.0 ± 0.5 Cthrough- duction. After 3 h incubation at 37 C, cells were pelleted with out the surgery by a heating blanket (Homeothermic Blanket centrifugation at 400 × g, the supernatant was collected, and cells Control Unit; Harvard Apparatus, Kent, UK) and monitored after were lysed in lysis buffer. recovery. After surgery, animals were returned to their cages and allowed free access to water and food. It was decided, apriori,to CYTOMETRIC BEAD ARRAY exclude from the study those animals that showed brain hemor- Circulating levels of IL-6 and KC (CXCL-1), and levels of IL-6 in rhage at any time of the surgery or with no reperfusion (2 mice). bone marrow cell culture supernatants and lysates were measured The survival rate was 100%. by using CBA Flex Sets (BD Biosciences, UK) according to the manufacturers protocol. The detection limit for each cytokine was SURGICAL CONTROLS 5–10 pg/ml. We investigated the effect of surgical manipulation and anaesthe- sia in the absence of MCAo on cellular and cytokine responses. FLOW CYTOMETRY To achieve this, three separate experimental conditions were used; Spleen, bone marrow, and blood cells were isolated and the first involved only anaesthesia with no surgical manipulation stained with appropriate combinations of CD45-PerCP-Cy5.5, (termed as “isoflurane”), the second exposure of the skull bone Ly6c-PerCP-Cy5.5, CD4-PE-Cy7, CD8-PE, CD3-APC, CD19-PE- but no craniotomy (termed as “sham no cran.”) and the third Cy7, MHCII-APC (eBioscience, UK), and Ly6G-PE (1A8, BD full craniotomy (termed as “sham”). Except for naive animals, Biosciences, UK) following Fc receptor blockade (eBioscience). all groups of mice were kept under isoflurane anaesthesia for the Contaminating red blood cells in bone marrow and spleen sam- same time period (75 min for control experimental conditions ples were removed by ACK solution, and FACS lysing solution and in the “MCAo” group). (BD Biosciences) was used to remove red blood cells from blood samples. Total blood cell counts were calculated by using BLOOD SAMPLING AND TISSUE PROCESSING 15µm polystyrene microbeads (Polysciences, 18328-5). Cells Blood samples were taken from the tail vein and from the right were acquired on an LSRII flow cytometer, using FACS Diva soft- cardiac ventricle, using 3.8% sodium citrate 1:10 as an anti- ware (BD Biosciences, UK). Except for the 72 h time point, flow coagulant. Repeated tail vein samples were collected at various cytometric data have been pooled from three independent experi- time points, with equal sampling times across different treat- ments, in which all experimental groups have been represented by ment groups: prior to surgery (“naïve”), 60 min after the onset at least one animal. Due to red blood cell contamination and cell of ischemia or corresponding control surgery (“0 min reperfu- labeling artifacts n = 6 blood samples, n = 3bonemarrowsam- sion”), and before transcardial perfusion (at 4 or 72 h reperfu- ples and n = 4 spleen samples have been excluded from analysis sion, not shown). Terminal cardiac blood samples were collected across experiments, pre-hoc. immediately prior to transcardial perfusion from naive mice, and from mice that had undergone surgical interventions, at 4 RANDOMIZATION, QUANTIFICATION, AND STATISTICAL ANALYSIS or 72 h reperfusion. Blood sample data from different vascular Animals were randomized for experiments and all quantita- beds were analyzed separately. To avoid potential confounding tive analyses were performed in a blinded manner. Group sizes effects of repeated tail vein samples at later time points, cardiac were determined based on our earlier in vivo experiments using Frontiers in Neuroscience | Neuroendocrine Science January 2014 | Volume 7 | Article 271 | 2 Denes et al. Surgical intervention affects leukocyte responses FIGURE 1 | Tissue injury, craniotomy, and anaesthesia result in rapid B cells (dark blue color indicates CD19+ positive cells). Proportion (D) and alteration of leukocyte responses. Extracranial tissue injury results in total cell numbers (E) of granulocytes in cardiac blood samples at 4 and 72 h increased proportion (A) and cell numbers (B) of granulocytes in the reperfusion. Proportion (F) and total cell numbers (G) of MHCII+ B cells circulation, which is indistinguishable from changes induced by sham surgery (CD19+) in cardiac blood samples at 4 and 72 h reperfusion. Sham no or MCAo. Blood samples taken from the tail vein prior to surgery and 60 min cran.—sham surgery without craniotomy. n = 5, 4, 4, 5, 6–7, 3, 3 in naïve, after the onset of MCAo/control surgery (0 min reperfusion) are shown. isoflurane, sham no cran., sham, MCAo, sham 72 h, and MCAo 72 h, ∗ ∗∗ ∗∗∗ (C) Representative dot plots showing terminal cardiac blood samples at 4 h respectively. P < 0.05, P < 0.01, P < 0.001 vs. naive. Light blue color reperfusion for graphs (D–G). P3 gate: Ly6G+ granulocytes, P4 gate: MHCII+ indicates negative (unstained) population. this experimental model. Normality of data sets was assessed RESULTS using GraphPad Prism (KS normality test). Statistical analy- SURGICAL MANIPULATION AND ANAESTHESIA ALTER LEUKOCYTE sis was performed by One-Way or Two-Way ANOVA followed RESPONSES IN THE BLOOD by Tukey’s or Bonferroni’s post-hoc multiple-comparison, using Surgical manipulation of extracranial tissues was sufficient to GraphPad Prism 5 software. In case of non-parametric data, induce rapid mobilization of granulocytes. In serial blood sam- significance was confirmed by non-parametric t-test (Mann– ples taken from the tail vein, the proportion of granulocytes high Whitney U-test) or Kruskal–Wallis test followed by Dunn’s mul- (Ly6G+ CD11b+ SSC cells) increased from 12 to 40% within tiple comparisons test. All data are expressed as mean ± standard 70–80 min (“0 min reperfusion”) in response surgery without error of the mean (s.e.m). P < 0.05 was considered statistically craniotomy (“sham no cran.,” Figure 1A), but not after isoflu- significant. rane anaesthesia alone, representing a ∼3-fold increase in total www.frontiersin.org January 2014 | Volume 7 | Article 271 | 3 Denes et al. Surgical intervention affects leukocyte responses high granulocyte numbers (Figure 1B). Granulocyte mobilization was population, CXCR2+ granulocytes (Ly6G+ CD11b+ SSC not altered further by craniotomy or MCAo (Figures 1A,B). cells) showed a decrease in the bone marrow proportional to the Granulocytosis was also evident 5–5.5 h after initiation of surgery level of surgical stress, reaching a significant 50% reduction after in cardiac, terminal blood samples (termed as “4 h reperfusion,” craniotomy and an over 60% reduction in response to MCAo Figures 1C,D), although total granulocyte numbers were lower (Figure 3B). Correspondingly, KC (CXCL-1) and IL-6 concen- compared to the earlier time point and were significantly ele- trations in the circulation increased by 20–40-fold after sham vated only in response to craniotomy or MCAo (Figure 1E). An surgery and MCAo (Figures 3C,D). Circulating KC levels showed increased proportion of granulocytes in the sham and MCAo a significant (P < 0.01) negative correlation with numbers of groups was observed up to 72 h after surgery (Figure 1D). CXCR2+ granulocytes in the bone marrow at 4 h reperfusion CD19+ MHCII+ B cells decreased profoundly (by 80–90%) (not shown). Anaesthesia alone appeared to increase B cells and T in the blood in response to anaesthesia alone, which was apparent cells in the bone marrow (Figures 3E–G) similarly to our earlier in all other groups of mice that underwent surgical manipulation findings (Denes et al., 2011). or MCAo, at 4 h reperfusion (Figures 1F,G). Similarly, anaes- thesia alone resulted in reduced circulating CD4+ and CD8+ BONE MARROW CELLS DO NOT EXHIBIT A SUPPRESSED RESPONSE T cell numbers within 4 h (Figures 2A–C). Interestingly, cran- AFTER CEREBRAL ISCHEMIA iotomy and MCAo appeared to raise T cell numbers over levels At 72 h reperfusion when systemic immunosuppression is appar- after anaesthesia alone, which was more apparent in the case ent after cerebral ischemia (Prass et al., 2003), bone marrow cells of CD4+ T cells (Figures 2B,C). Circulating B cells and CD8+ showed no difference in LPS-induced IL-6 production in vitro T cells remained significantly lower in number in surgery con- as assessed in cell lysates (Figure 4A) and cell culture super- trol groups and after MCAo compared to naïve mice at 72 h natants (Figure 4B), indicating that at this time point cells resid- reperfusion (Figures 1F,G, 2A,C). ing in the bone marrow maintain their ability to respond to endotoxin. GRANULOCYTE RELEASE FROM THE BONE MARROW INCREASES PROPORTIONALLY TO THE LEVEL OF SURGICAL STRESS AND BRAIN SURGICAL INTERVENTION AND CEREBRAL ISCHEMIA ALTER SPLENIC INJURY GRANULOCYTE RESPONSES MCAo resulted in mobilization of granulocytes from the bone Surgical manipulation of extracranial tissues with or without marrow at 4 h reperfusion (Figure 3A). Within the granulocyte craniotomy resulted in an early recruitment of granulocytes in FIGURE 2 | Isoflurane anaesthesia results in reduced circulating T cell Purple color indicates cells gated on CD3. Sham no cran.—sham surgery numbers. CD8+ T cells (A) and CD4+ T cells (B) are reduced in number without craniotomy. n = 4, 4, 4, 5, 6, 3, 3 in naïve, isoflurane, sham no in cardiac blood samples within 4 h following isoflurane anaesthesia. cran., sham, MCAo, sham 72 h, and MCAo 72 h, respectively. P < 0.05, ∗∗ (C) Representative dot blots showing CD8+ and CD4+ T cells in cardiac P < 0.01 vs. naive. Light blue color indicates negative (unstained) blood samples 4 h after reperfusion, control surgeries, or anaesthesia. population. Frontiers in Neuroscience | Neuroendocrine Science January 2014 | Volume 7 | Article 271 | 4 Denes et al. Surgical intervention affects leukocyte responses FIGURE 3 | Surgical intervention and anaesthesia result in altered amount of surgical stress. Anaesthesia results in increased MHCII+ B leukocyte responses in the bone marrow. (A) Bone marrow cells (E) CD4+ T cells (F) and CD8+ T cells (G) in the bone marrow high granulocytes (Ly6G+,CD11b+, SSC cells) are reduced within 4 h after within 4 h. Sham no cran.—sham surgery without craniotomy. n = 7, 5, MCAo. (B) Both MCAo and craniotomy result in reduced CXCR2+ 6, 7, 7–8 in naïve, isoflurane, sham no cran., sham, and MCAo, ∗ ∗∗ ∗∗∗ granulocytes in the bone marrow at 4 h reperfusion. KC (CXCL1, C)and respectively. P < 0.05, P < 0.01, P < 0.001 vs. naïve, unless IL-6 (D) levels increase in the circulation within 4 h proportionally to the indicated otherwise on the graphs; P < 0.05 vs. isoflurane. CXCR2 BLOCKADE PREVENTS GRANULOCYTE MOBILIZATION FROM THE BONE MARROW BUT DOES NOT ALTER ISCHEMIC BRAIN INJURY The selective CXCR2 inhibitor SB225002 (White et al., 1998)pre- vented granulocyte release from the bone marrow as confirmed by a significant reduction of granulocytes in the bone marrow in vehicle-treated mice 4 h after MCAo compared to mice treated with SB225002 (Figures 6A,B). Similarly to our earlier experi- ments using the intraluminal filament method to induce cerebral ischemia (Denes et al., 2011) bone marrow granulocyte numbers were stabilized by 24 h post MCAo (Figure 6A). To our surprise, CXCR2 blockade although reducing granulocyte release from the FIGURE 4 | Bone marrow cells do not exhibit a suppressed response to bone marrow, resulted in an increase in circulating granulocytes endotoxin after distal tMCAo. Bone marrow cells were isolated 72 h after at 4 h reperfusion compared to vehicle (Figure 6C). We investi- MCAo and stimulated in vitro with bacterial lipopolysaccharide (LPS) to gated whether CXCR2 blockade resulted in any changes in infarct investigate cytokine production. Cell lysates (A) and supernatants (B) were measured for IL-6 production and release. n.s., not significant. n = 3. size, but no difference between SB225002 treatment and vehicle ∗ ∗∗ P < 0.05, P < 0.01 vs. control. was observed (Figure 6D), indicating that CXCR2-mediated sig- nals do not contribute substantially to brain injury in the current experimental model of cerebral ischemia. thespleen(Figure 5A). This effect was not apparent in the DISCUSSION CXCR2+ granulocyte population (Figure 5B). MCAo signifi- cantly reduced splenic total granulocyte- and CXCR2+ gran- Here we present evidence that even mild surgical manipulation ulocyte numbers (Figures 5A,B)comparedtoshamsurgery at results in marked systemic granulocyte mobilization responses. 4 h reperfusion. No changes were observed in B cell or T Granulocyte mobilization is proportional to the level of surgi- cell populations in the spleen in any of the surgical groups cal stress and is further augmented by brain injury. Granulocyte (Figures 5C–E). responses to cranial injury or cerebral ischemia involve primarily www.frontiersin.org January 2014 | Volume 7 | Article 271 | 5 Denes et al. Surgical intervention affects leukocyte responses FIGURE 5 | Leukocyte responses in the spleen. Changes in total CD8+ T cells (E) at 4 h reperfusion. Sham no cran.—sham surgery without high granulocytes (Ly6G+,CD11b+, SSC cells, A) and CXCR2+ granulocytes craniotomy. n = 6, 6, 6, 5, 6 in naïve, isoflurane, sham no cran., sham, and ∗ ∗∗∗ ∗∗ (B) in the spleen 4 h following reperfusion or control surgical interventions. MCAo, respectively. P < 0.05 and P < 0.001 vs. naïve; P < 0.01 (A) No changes are observed in splenic MHCII+ B cells (C) CD4+ T cells (D) and and P < 0.05 (B) sham vs. MCAo. the mobilization of CXCR2-positive granulocytes, which is not which only extracranial tissues were manipulated. Extracranial seen after manipulation of extracranial tissues. In addition, we manipulation consisting of an incision on the skin and tempo- show that anaesthesia profoundly alters leukocyte responses, an rary dislocation of small pericranial muscles was sufficient to effect that lasts for several hours or even days. Collectively, these generate a systemic inflammatory response evidenced by granulo- data highlight the role of anaesthesia and surgical intervention in cyte mobilization and a 10-fold increase in circulating KC levels. systemic inflammatory responses, which could have an impact on Since these changes did not involve major alterations in CXCR2+ experimental models of cerebral ischemia, but could also serve granulocytes, identification of the mechanisms involved (contri- important information for the management of patients subjected bution of bone marrow-derived cells, signals that initiate and to various surgeries in the clinic. maintain systemic granulocyte responses, etc.) warrant further In our previous study we revealed changes in the bone mar- investigation. It is likely that activation of the HPA axis and the row in response to transient, focal cerebral ischemia induced by autonomic nervous system (Elenkov et al., 2000) could contribute an intraluminal filament that is advanced through the internal to surgery-induced peripheral changes, however the exact signals carotid artery to occlude the MCA (Denes et al., 2011). Although mediating leukocyte responses need to be identified. In fact, we we demonstrated changes induced by brain injury itself, we also confirmed here our earlier findings showing an increased mobi- showed that anaesthesia and surgical intervention could impact lization of CXCR2-positive granulocytes in response to brain on bone marrow leukocyte responses (Denes et al., 2011). Since injury, compared with other surgical manipulations (Denes et al., the filament MCAo model involves surgical manipulation around 2011). the neck including exposure of the salivary glands, the vagus nerve We found that isoflurane anaesthesia alone resulted in a pro- and other tissues around the trachea, in the present study we per- found reduction in circulating lymphocytes within hours. Some formed a systematic analysis of bone marrow, spleen, and blood effects of isoflurane on leukocyte activation have been reported responses, using a distal MCAo model, to reveal stroke-specific earlier (Yuki et al., 2012; Carbo et al., 2013), and a decrease in changes in leukocyte mobilization as well as the impact of surgi- Th1/Th2 ratio in the blood has been observed in patients under- cal intervention and anaesthesia. Since the present model includes going craniotomy in response to isoflurane (Inada et al., 2004). craniotomy, a potential confounder that disturbs the internal However, the present study demonstrates a rapid and sustained milieu of the brain, we also included control surgeries during reduction in circulating T cell numbers induced by isoflurane Frontiers in Neuroscience | Neuroendocrine Science January 2014 | Volume 7 | Article 271 | 6 Denes et al. Surgical intervention affects leukocyte responses FIGURE 6 | Blockade of CXCR2-mediated granulocyte release from Ly6G+, blue) in the bone marrow at 4 h reperfusion. (C) SB225002 the bone marrow does not alter infarct size after experimental increases granulocyte numbers in the cardiac blood at 4 h reperfusion stroke. (A) Treatment with the selective CXCR2 antagonist SB225002 after MCAo. (D) Infarct size is not altered in response to SB225002 prevents granulocyte release from the bone marrow after MCAo treatment at 24 h reperfusion, after MCAo. (A–C) n = 3, 3, 5, 5 in compared to vehicle at 4 h reperfusion. Total granulocyte numbers vehicle 4 h, SB225002 4 h, vehicle 24 h, and SB225002 24 h samples, ∗ # recovered from 1 femur and 1 tibia are shown. (B) Representative respectively, (D) n = 5–6. P < 0.05 vs. vehicle 4 h, P < 0.05 vs. high density plots showing CD11b+ SSC granulocytes (gated also on 24 h time points. anaesthesia, which could have important implications clinically CXCR2 is the best characterized cell surface receptor on gran- and also in models of experimental stroke. The contribution of ulocytes involved in cell mobilization to stimuli mediated by T cells to the ischemic brain injury is well documented (Iadecola CXCL1 (KC) and CXCL2/3 (MIP-2) (Matzer et al., 2004; Eash and Anrather, 2011). Since volatile anaesthetics can exert neuro- et al., 2010; Veenstra and Ransohoff, 2012). Although brain protective properties (Kawaguchi et al., 2005), it is possible that injury-induced granulocyte mobilization from the bone mar- at least in part, these actions could be mediated via blunted T cell row was prevented by CXCR2 blockade, circulating granulocytes responses. increased in response to SB225002 treatment. CXCR2 inhibition The spleen is profoundly affected by brain injury (Offner et al., was found effective to reduce IL-8- or KC-induced granulocyte 2006b), leading to loss of B cells and development of immuno- mobilization in the blood and block granulocyte egress from suppression, however, much less is known about how cerebral the bone marrow (White et al., 1998; Martin et al., 2003; Eash ischemia contributes to responses of myeloid cells in the spleen. et al., 2010). We show here that very early granulocyte mobiliza- In fact, a population of splenic monocytes are released rapidly tion responses in response to brain injury are not dependent on upon activation and contributes to ischemic processes in the heart CXCR2, and might not (or only in part) require the contribu- (Swirski et al., 2009; Leuschner et al., 2012). We found that sur- tion of the bone marrow in the current experimental model. This gical manipulation results in increased granulocyte recruitment could explain the lack of an effect by CXCR2 blockade on the to the spleen whilst cerebral ischemia reduced surgery-induced size of ischemic brain injury, which has also been confirmed by increases in splenic granulocytes. Although the role and mecha- another study (Brait et al., 2011). Brain injury in different experi- nisms of surgery-induced splenic granulocytosis requires further mental models might not be influenced by peripheral leukocyte investigations, this scenario indicates that the effect of surgical actions to the same extent. For example, blockade of granulo- manipulation on leukocyte responses in peripheral organs is likely cyte responses or hematopoietic MyD88-dependent actions is not to be a confounder in current experimental stroke models. associated with reduced central inflammation or brain damage www.frontiersin.org January 2014 | Volume 7 | Article 271 | 7 Denes et al. Surgical intervention affects leukocyte responses after cold-induced cortical injury (Koedel et al., 2007). In con- Dirnagl, U., Klehmet, J., Braun, J. S., Harms, H., Meisel, C., Ziemssen, T., et al. (2007). Stroke-induced immunodepression: experimental evidence and trast, neutrophils contribute to brain injury when experimental clinical relevance. Stroke 38, 770–773. doi: 10.1161/01.STR.0000251441.89 stroke is preceded by systemic inflammation (McColl et al., 2007). 665.bc Therefore, the source of neutrophils mobilized after brain injury Eash, K. J., Greenbaum, A. M., Gopalan, P. K., and Link, D. C. (2010). CXCR2 and their contribution to the population that is recruited into and CXCR4 antagonistically regulate neutrophil trafficking from murine bone the brain need to be investigated in further studies. Stroke in marrow. J. Clin. Invest. 120, 2423–2431. doi: 10.1172/JCI41649 Elenkov, I. J., Wilder, R. L., Chrousos, G. P., and Vizi, E. S. (2000). The sympa- patients results in increased circulating granulocyte numbers and thetic nerve–an integrative interface between two supersystems: the brain and loss of T cells (Vogelgesang et al., 2008), which corresponds to the immune system. Pharmacol. Rev. 52, 595–638. the findings presented in our study. Surgical interventions and Fisher, M., Feuerstein, G., Howells, D. W., Hurn, P. D., Kent, T. A., anaesthesia also influence leukocyte responses in patients (Inada Savitz, S. I., et al. (2009). Update of the stroke therapy academic indus- try roundtable preclinical recommendations. Stroke 40, 2244–2250. doi: et al., 2004). However, similarly to most experimental stroke 10.1161/STROKEAHA.108.541128 models, the MCAo model is inherently confounded by effects of Iadecola, C., and Anrather, J. (2011). The immunology of stroke: from mechanisms anaesthesia and surgical intervention, and we show that surgical to translation. Nat. Med. 17, 796–808. doi: 10.1038/nm.2399 interventions alone are capable of inducing profound changes in Inada, T., Yamanouchi, Y., Jomura, S., Sakamoto, S., Takahashi, M., Kambara, leukocyte responses. These changes might contribute to and/or T., et al. (2004). Effect of propofol and isoflurane anaesthesia on the immune response to surgery. Anaesthesia 59, 954–959. doi: 10.1111/j.1365- alter brain injury in experimental models. Since circulating white 2044.2004.03837.x blood cell- and blood cytokine data from stroke patients and from Kawaguchi, M., Furuya, H., and Patel, P. M. (2005). Neuroprotective effects of those undergoing anaesthesia or surgical interventions are widely anesthetic agents. J. Anesth. 19, 150–156. doi: 10.1007/s00540-005-0305-5 available, blood cell responses and mechanisms of brain injury Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., and Altman, D. G. (2010). in experimental models should be investigated in a translational Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 8:e1000412. doi: 10.1371/journal.pbio.1000412 context. Koedel, U., Merbt, U. M., Schmidt, C., Angele, B., Popp, B., Wagner, H., In conclusion, our data indicate that anaesthesia- and surgery- et al. (2007). Acute brain injury triggers MyD88-dependent, TLR2/4- induced leukocyte responses interact with those induced by independent inflammatory responses. 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Stroke 39, 237–241. doi: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers 10.1161/STROKEAHA.107.493635 in Neuroscience. White, J. R.,Lee,J.M., Young,P.R., Hertzberg, R. P.,Jurewicz, A. J.,Chaikin,M. Copyright © 2014 Denes, Pradillo, Drake, Buggey, Rothwell and Allan. This is an A., et al. (1998). Identification of a potent, selective non-peptide CXCR2 antag- open-access article distributed under the terms of the Creative Commons Attribution onist that inhibits interleukin-8-induced neutrophil migration. J. Biol. Chem. License (CC BY). The use, distribution or reproduction in other forums is permit- 273, 10095–10098. doi: 10.1074/jbc.273.17.10095 ted, provided the original author(s) or licensor are credited and that the original Yuki, K., Bu, W., Xi, J., Sen, M., Shimaoka, M., and Eckenhoff, R. G. (2012). publication in this journal is cited, in accordance with accepted academic prac- Isoflurane binds and stabilizes a closed conformation of the leukocyte function- tice. No use, distribution or reproduction is permitted which does not comply with associated antigen-1. FASEB J. 26, 4408–4417. doi: 10.1096/fj.12-212746 these terms. www.frontiersin.org January 2014 | Volume 7 | Article 271 | 9

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