Fluent Essays

For this weeks case study, you will complete a reflection/discussion of the article The spleen may be an important target of stem cell therapy for stroke.  In this activity, you will read the journal article assigned for the week, summarize the purpose of the research, research methodology, and findings of the research.  You will also write about specific parts of the research work that were of interest to you, and finally explain how specific information from the article points to the designing work of a Creator.  The reflection should be at least 2 pages in length and written in APA style. REVIEW Open Access The spleen may be an important target of stem cell therapy for stroke Zhe Wang1,2, Da He1, Ya-Yue Zeng1, Li Zhu1, Chao Yang1, Yong-Juan Lu1, Jie-Qiong Huang1, Xiao-Yan Cheng1, Xiang-Hong Huang1 and Xiao-Jun Tan1* Abstract Stroke is the most common cerebrovascular disease, the second leading cause of death behind heart disease and is a major cause of long-term disability worldwide. Currently, systemic immunomodulatory therapy based on intravenous cells is attracting attention. The immune response to acute stroke is a major factor in cerebral ischaemia (CI) pathobiology and outcomes. Over the past decade, the significant contribution of the spleen to ischaemic stroke has gained considerable attention in stroke research. The changes in the spleen after stroke are mainly reflected in morphology, immune cells and cytokines, and these changes are closely related to the stroke outcomes. Autonomic nervous system (ANS) activation, release of central nervous system (CNS) antigens and chemokine/chemokine receptor interactions have been documented to be essential for efficient brain-spleen cross-talk after stroke. In various experimental models, human umbilical cord blood cells (hUCBs), haematopoietic stem cells (HSCs), bone marrow stem cells (BMSCs), human amnion epithelial cells (hAECs), neural stem cells (NSCs) and multipotent adult progenitor cells (MAPCs) have been shown to reduce the neurological damage caused by stroke. The different effects of these cell types on the interleukin (IL)-10, interferon (IFN), and cholinergic anti- inflammatory pathways in the spleen after stroke may promote the development of new cell therapy targets and strategies. The spleen will become a potential target of various stem cell therapies for stroke represented by MAPC treatment. Keywords: Stroke, Spleen, Stem cells, IL-10, Multipotent adult progenitor cells Introduction Stroke is the most common cerebrovascular disease and the second leading cause of death behind heart disease and is a major cause of long-term disability worldwide [1]. Our understanding of the pathophysiological cascade following ischaemic injury to the brain has greatly im- proved over the past few decades. Cell therapy, as a new strategy addition to traditional surgery and thrombolytic therapy, has attracted increasing attention [2]. The therapeutic options for stroke are limited, especially after the acute phase. Cell therapies offer a wider therapeutic time window, may be available for a larger number of patients and allow combinations with other rehabilitative strategies. The immune response to acute stroke is a major factor in cerebral ischaemia (CI) pathobiology and outcomes [3]. In addition to the significant increase in inflammatory levels in the brain lesion area, the immune status of other peripheral immune organs (PIOs, such as the bone mar- row, thymus, cervical lymph nodes, intestine and spleen) also change to varying degrees following CI, especially in the spleen [4]. Over the past decade, the significant contri- bution of the spleen to ischaemic stroke has gained con- siderable attention in stroke research. At present, the spleen is becoming a potential target in the field of stroke therapy for various stem cell treatments represented by multipotent adult progenitor cells (MAPCs). Two cell therapy strategies Two distinct cell therapy strategies have emerged from clinical data and animal experiments (Fig. 1). The first is the nerve repair strategy, which uses different types of stem cells with the ability to differentiate into cells that make up nerve tissue and thus can replace damaged nerves to promote recovery during the later stages after stroke [5–11]. This strategy usually involves cell delivery to the injury site by intraparenchymal brain implantation and stereotaxic injection into unaffected deep brain * Correspondence: [email protected] 1Xiangtan Central Hospital, Clinical Practice Base of Central South University, Xiangtan 411100, China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. Journal of Neuroinflammation (2019) 16:20 https://doi.org/10.1186/s12974-019-1400-0 http://crossmark.crossref.org/dialog/?doi=10.1186/s12974-019-1400-0&domain=pdf http://orcid.org/0000-0003-4913-1370 mailto:[email protected] http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/publicdomain/zero/1.0/ structures adjacent to the injury site. The main problem with this strategy is that we should not only ensure the efficient delivery of cells to the injury site but also try to reduce the invasive damage caused by the mode of deliv- ery. Moreover, evaluation of the extent to which cells survive over the long term, the differentiation fates of the surviving cells and whether survival results in func- tional engraftment is difficult. This strategy mainly in- cludes intracerebral [12–15], intrathecal [16] and intranasal administration [17] (Fig. 2). The second strategy is an immunoregulatory strategy (typically therapeutic cells are injected intravenously), Fig. 1 Two cell therapeutic strategies for stroke. Replacement of necrotic cells and immunomodulation. Therapeutic stem cells have traditionally been known to differentiate into cells that make up nerve tissue to replace necrotic cells, thereby promoting nerve regeneration and angiogenesis. Recent studies have shown that the immune regulatory capacity of stem cells provides a favourable environment for nerve and vascular regeneration Fig. 2 The main routes of administration of stem cell therapy for stroke. Although many preclinical studies and clinical applications have been carried out, the most adequate administration route for stroke is unclear. Each administration route has advantages and disadvantages for clinical translation to stroke patients. a Intranasal, b intracerebral, c intrathecal, d intra-arterial, e intraperitoneal and f intravenous Wang et al. Journal of Neuroinflammation (2019) 16:20 Page 2 of 24 which takes advantage of the release of trophic factors to promote endogenous stem cell (NSC/neural progenitor cell (NPC)) mobilisation and anti-apoptotic effects in addition to the anti-inflammatory and immunomodula- tory effects encountered after systemic cell delivery. The mechanism of action appears to be reliant on “by- stander” effects; these effects are likely to include immu- nomodulatory and anti-inflammatory effects mediated by the systemic release of trophic factors [18, 19], since neither animal nor human data have found any signs of actual engraftment of intravenously delivered cells in the brain [20–22]. In addition, many therapeutic stem cells have been found to migrate to PIOs, such as the spleen, following brain injury to play an immunoregulatory role, thus providing a good environment for nerve and vascu- lar regeneration in vivo. This strategy mainly includes intra-arterial [23–26], intraperitoneal [27] and intraven- ous administration [28–31] (Fig. 2). Currently, systemic immunomodulatory therapy based on intravenous cells is attracting increasing attention [29]. Immunoregulation may be a better strategy Further insight into the role of the two strategies has been provided by studies using cellular therapies in ex- perimental models of brain ischaemia. All cells are more efficacious when administered systemically than when delivered via intracerebral administration [32–37], prob- ably because intracerebral administration does not guar- antee the extent to which cells can migrate from their implantation site in human subjects. Placing cells within the cystic space left as a long-term consequence of is- chaemic damage in the absence of some type of bio-scaffold will be unlikely to promote cell adherence or persistence. Moreover, gliosis on the margins of the damaged region may impede cell migration or axonal outgrowth in the same manner as encountered after spinal cord injury. The pathological progression of stroke is a complex systemic process, and changes in state occur in tissues besides intracranial tissues. Studies have shown that the immune response/regulation after stroke plays an im- portant role in the pathological progression of stroke. The immune response is an important endogenous mechanism of post-stroke activation. Although the im- mune response following stroke, including cytokine pro- duction and inflammatory cell infiltration into damaged brain tissues, has been known for many years [38–40], the complexity of the mechanisms involved in post-stroke immune activation, inflammatory damage and tissue repair are unknown. In the future, immuno- modulation will be an important potential therapeutic strategy for stroke. Moreover, finding the most appropri- ate therapeutic target for therapeutic cells may further improve the effectiveness of immunomodulatory treatment. Stem cells and immunoregulation after stroke At present, most cells used for immunoregulation ther- apy after stroke are various types of stem cells. However, animal experiments have shown that anti-inflammatory immune cells (such as regulatory T cells (Tregs), helper T (Th)-2 cells and regulatory B cells (Bregs)) can also al- leviate brain damage [41–44]. In addition, some immune cells are activated after stroke, such as monocytes in some PIOs or astrocytes and have been shown to have protective effects in experimental animals [45–47]. Stem cell therapy has received considerable attention and application because of the easy access, strong prolif- eration and low immunogenicity of the cells. Treatments based on different types of stem cells have been studied for years and even decades in animal models of stroke. Included in the following subsections are specific exam- ples of cell therapies that have been extensively studied in animal models and taken forward to clinical trials. For instance, human umbilical cord blood cells (hUCBs) [32–34], haematopoietic stem cells (HSCs) [35], bone marrow stem cells (BMSCs) [36], human amnion epithe- lial cells (hAECs) [48] and neural stem cells (NSCs) [37] have all been shown to reduce neural injury in experi- mental models of stroke. Interestingly, almost all studies have found that when administered systemically, stem cells migrate to the in- jured brain and PIOs and in some cases have been shown to modulate the immune response to stroke [32, 35–37, 48], which may be one reason that this injection route is more efficacious. Studies have also shown that only a small number of stem cells injected intravenously after a stroke can be transported through the blood-brain barrier to damaged brain tissue [31]. This finding suggests that regulation of the peripheral im- mune status after stroke may be a potentially important therapeutic strategy, especially for improvement of the long-term prognosis in stroke patients. In addition to stem cells themselves, exosomes derived from some stem cells have been found to have thera- peutic effects on haemorrhagic stroke [49]. For instance, transplantation of pluripotent mesenchymal stem cell (MSC)-derived exosomes promoted functional recovery in an experimental intracerebral haemorrhage (ICH) rat model [50]. MSC-derived exosomes can amplify en- dogenous brain repair mechanisms and induce neurores- torative effects after CI [51]. Exosomes carry a concentrated group of functional molecules (DNA, ribo- somal RNA, circular RNA, long noncoding RNA, micro- RNA, proteins and lipids) that serve as intercellular communicators not only locally but also systemically. These molecules may be part of the long-distance Wang et al. Journal of Neuroinflammation (2019) 16:20 Page 3 of 24 cell-to-cell communication that operates by paracrine function through secretory factors in the extracellular environment and is responsible for the long-distance ef- fects during cell therapy. Stroke and inflammation The pathophysiological process of stroke is very complex and involves energy metabolism disorders, acidosis, loss of cellular homeostasis, excitotoxicity, activation of neu- rons and glial cells, blood-brain barrier (BBB) destruc- tion and accompanying leukocyte infiltration [52]. Evidence suggests that the immune system is involved in the various pathological stages of stroke [53]. CI initiates an inhibitory effect on lymphatic organs through the autonomic nervous system (ANS), which increases the risk of infection after stroke. Infection after stroke is a major cause of disability and death after stroke [54]. On the other hand, the innate immune system also contrib- utes to repair of brain tissue [55] (Fig. 3). Inflammatory cell infiltration and tissue damage The inflammatory response to stroke starts immediately in the lacuna after arterial occlusion, and production of reactive oxygen species (ROS) increases rapidly in the coagulation-promoting state, accompanied by activation of complement, platelets and endothelial cells [56, 57]. Increased cyclooxygenase-2 (COX-2) activity in inflam- matory cells and neurons may lead to increased ROS production in the injured tissues and severe prostaglan- din toxicity [58, 59]. ROS also help reduce nitric oxide (NO) activity, leading to platelet aggregation and leukocyte adhesion and thus aggravation of ischaemic in- jury [60]. After a few minutes of arterial occlusion, the relevant intracellular and extracellular regulation begins immediately. Acute local injury is sensed by pattern rec- ognition receptors (PRRs) by interaction with pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [61– 63]. These factors are released by stressed cells in the blood cascade, and PRRs in neurons and glial cells can activate intracellular signal transduction pathways to in- crease the expression of different pro-inflammatory genes [64, 65]. This mechanism activates immune sys- tem factors that cause mast cells to release vasoactive mediators, such as histamine, protease and tumour ne- crosis factor (TNF), whereas macrophages release pro-inflammatory factors [66]. After the rapid produc- tion of inflammatory signals, the interaction between ad- hesion molecules and integrins is mediated by adhesion receptors to facilitate leukocyte infiltration into the brain parenchyma [67, 68]. After ischaemia, these cells rapidly release pro-inflammatory mediators into the area, and Fig. 3 Inflammation after stroke. DAMPs released from necrotic neurons activate macrophages through PRRs and the inflammasome. Activated macrophages enhance inflammation by releasing pro-inflammatory cytokines and recruiting T cells, which contribute to maintenance of inflammation through IL-17. DCs also activate and enhance antigen presentation to T cells. Gelatinase released by activated mast cells and MPP-9 produced by infiltrating neutrophils destroy the function of the BBB, resulting in brain oedema. Then, under the action of chemokines, leukocytes infiltrate into the damaged brain tissue, thereby expanding inflammation and injury. Several days after acute stroke, the cytokines produced by the innate immune system change to an anti-inflammatory phenotype, which contributes to inhibition of inflammation. The ratio and biodistribution of M1 and M2 microglia also changes, with anti-inflammatory M2 microglia becoming dominant again. Debris is cleaned up by microglia and macrophages. NSCs/ NPCs are mobilised and migrate to the lesion. The environment becomes conducive to nerve regeneration, angiogenesis and BBB restructuring Wang et al. Journal of Neuroinflammation (2019) 16:20 Page 4 of 24 these cytokines contribute to leukocyte infiltration into damaged tissues and activate antigen presentation be- tween dendritic cells (DCs) and T cells [69, 70]. T cells cause tissue damage through IFN-γ and ROS. IL-23 re- leased by microglia and macrophages activates T cells to produce IL-17, which aggravates the acute ischaemic brain injury [71]. Ultimately, this neuroimmune imbal- ance leads to an early downregulation of systemic cellu- lar immune responses, resulting in functional deactivation of monocytes, Helper T (Th) cells and in- variable natural killer T cells (iNKTs) [72]. This stage is often accompanied by increased lymphocyte apoptosis, inhibition of peripheral cytokine release and helper Th1 cells and changes in the Th1/Th2 ratio. Stroke-induced immunosuppression helps to increase the risk of infec- tion, leading to adverse functional outcomes [73]. Phenotypic and spatial distributions of microglia Microglia can be regarded as resident immune cells in the central nervous system (CNS) that are activated by local and systemic infections, neurodegenerative diseases and tissue damage. Microglia respond quickly to stroke injury. Microglia enter the ischaemic centre within 60 min after focal ischaemia, and the number of acti- vated microglia increases significantly for up to 24 h. Pro-inflammatory M1 microglia (which release TNF-α, IL-1β, IL-6 and IL-18) [74] can be observed in the is- chaemic core within 24 h after CI, and the number of M1 microglia increases gradually within 2 weeks of CI [75]. Inhibitory M2 microglia (which participate in neu- roprotection and promote repair of damaged cells through production of transforming growth factor (TGF)-β, nerve growth factor (NGF) and IL-4) [74] begin to appear 24 h after injury, and their number grad- ually increases over time for up to 1 week after ischae- mia [38]. The phenotypes and spatial distributions of microglia change with the expansion of damaged brain tissue [76, 77]. Astrocytic proliferation and activation Astrocytes are the most abundant cell type in the CNS and perform multiple functions that are both detrimen- tal and beneficial for neuronal survival from the acute phase to the recovery phase after ischaemic stroke [78]. IL-15 expression is increased in astrocytes in mouse and human brains after CI, which elevates the level and acti- vation of CD8+ T cells and natural killer cells (NKs), resulting in aggravation of brain tissue damage [79, 80]. IL-15 blockade reduces the effects of NKs, CD8+ T cells and CD4+ T cells in the brains of mice after ischaemia/ reperfusion (I/R), resulting in a reduction of the infarct size and improvement in motor and locomotor activity [80]. During the recovery phase, IFN-α is mainly in- volved in regulation of astrocytic proliferation through blocking and activation [29]. Astrocytes regulate the for- mation and maintenance of synapses, cerebral blood flow and BBB integrity [81]. Astrocytes also indirectly regulate inflammation by affecting neuronal survival during acute injury and axonal regrowth [81]. Activated astrocytes are beneficial for the recovery of neurological function after stroke [82]. Recent studies have suggested that this endogenous protective mechanism may involve mitochondrial transport from astrocytes to neurons after brain injury, which is mediated by a calcium-dependent mechanism involving CD38 and cyclic ADP ribose sig- nalling [83]. Mast cells and BBB breakdown Mast cells, which are located in the perivascular space surrounding the brain parenchymal vessels and in the dura mater of the meninges, are activated during the early stage after stroke and contribute to BBB break- down and brain oedema by releasing gelatinase [84, 85]. Pharmacological mast cell stabilisation with cromogly- cate reduces haemorrhage formation and mortality after administration of thrombolytics in experimental ischae- mic stroke [86], which may involve promotion of BBB breakdown and neutrophil infiltration by mast cells [87]. Inflammasome activation Inflammatory reactions lead to the production of inflam- matory cytokines and the death of neurons and glial cells, which are regulated by a multiprotein complex called the inflammasome [67]. Nod-like receptors (NLR) in neurons and glial cells may mediate production of the inflammasome, which participates in the inflammatory response to aseptic tissue damage during CI [64]. The inflammasome in damaged brain tissue produces IL-1β and IL-18 after activation, which can cause specific cell death called inflammatory necrosis [88]. In this way, the inflammasome not only helps activate and support in- nate immunity but also aggravates tissue damage. Inflammation relief and tissue repair Inflammation after stroke is also inhibited by auto-suppression, and its remission is regulated by many immunosuppressive factors. The termination of inflam- mation also triggers structural and functional remodel- ling of damaged brain tissue. The first mechanism involved in this stage is the clearance of dead cells and is accomplished by microglia and infiltrating macrophages, which are mainly composed of phagocytes [76, 89]. Im- munoglobulins targeting CNS antigens may promote the release of IL-10 and TGF-β, thereby inhibiting the im- mune response and the production of adhesion mole- cules and inflammatory cytokines [90]. These multipotent immunoregulatory factors can inhibit in- flammation and contribute to tissue repair, and their Wang et al. Journal of Neuroinflammation (2019) 16:20 Page 5 of 24 protective effects are conducive to cell survival in ischae- mic areas [60]. These growth factors are released by in- flammatory cells, neurons and astrocytes and support cell budding, neuronal growth, angiogenesis and even tissue remodelling after ischaemic injury [91]. Insulin-like growth factor (IGF)-1 plays a key role in the neurogenesis process after ischaemic injury, and the astrocyte response is also necessary for the functional re- covery of damaged tissues [92]. The roles of vascular endothelial growth factor (VEGF) and neutrophil metal- loproteinase are also required for angiogenesis; together, they support the joint activity of inflammatory cells and astrocytes [93]. Changes in peripheral immune organs after stroke The pathological process after stroke is a complex sys- temic immune state change. In addition to severe in- flammation in brain tissues (including inflammatory chemokine production, inflammatory cell infiltration, microglial activation and inflammasome production) [94], the state of PIOs also changes significantly after stroke [95] (Fig. 4). Bone marrow CD34+ HSCs/ haematopoietic progenitor cells (HPCs) in bone marrow are mobilised rapidly into the peripheral blood circulation under post-stroke pathological stress and play an important protective role in the pathological process of CI [96]. The prognosis can be effectively im- proved by accelerating the mobilisation of protective cells in bone marrow or increasing their levels in periph- eral circulation after stroke [97, 98]. Clinical trials have also shown that intra-arterial injection of bone marrow-derived CD34+ haematopoietic stem cells/pro- genitor cells can significantly improve the prognosis of acute ischaemic stroke patients and greatly reduce their mortality and disability rates [26]. In addition, CI regu- lates the elevation of CD4+CD25+FoxP3+ Tregs from bone marrow via the sympathetic nervous system (SNS) [95]. Stroke reduces C-X-C chemokine ligand (CXCL) 12 expression in bone marrow but increases C-X-C che- mokine receptor (CXCR) 4 expression in Tregs and other bone marrow cells. Destruction of the CXCR4-CXCL12 axis in bone marrow promotes mobil- isation of Tregs and other CXCR4+ cells into peripheral circulation and eventually migration to damaged brain tissues to facilitate tissue repair [95]. Thymus Animal data have shown that the thymus exhibits loss of a large number of lymphocytes within 12 h after ischae- mia/reperfusion (I/R). Cytokine production also changes from the Th1 to the Th2 phenotype [99, 100]. Lympho- cytes, such as B cells, T cells and natural killer cells (NKs), were found to be highly apoptotic [101], and the thymic morphology of the tested mice exhibited signifi- cant atrophy after I/R [102]. The non-toxic apoptosis in- hibitor Q-VD-OPH significantly reduced programmed death of thymocytes after I/R, effectively reducing the Fig. 4 Changes in PIOs after stroke. Morphological and biochemical changes occur in the bone marrow, thymus, cervical lymph nodes and intestine after stroke and play respective roles in the stroke outcome Wang et al. Journal of Neuroinflammation (2019) 16:20 Page 6 of 24 incidence of bacteraemia after CI injury and improving the survival rate of the mice [103]. Cervical lymph nodes Treg levels in brain tissue and cervical lymph nodes in- crease significantly after CI [104]. This increase may be due to changes in BBB permeability after stroke as well as other pathological causes, resulting in a large number of efflux cells and soluble proteins migrating from the brain tissue to the cervical lymph nodes. These cells and proteins migrate to the cervical lymph nodes and play an important role in regulating the pathological immune response after stroke [105, 106]. Many brain-derived an- tigens that migrate to the cervical lymph nodes after stroke may promote autoimmunity and Treg-based immunomodulation [107]. In addition, antigen-specific T lymphocytes may circulate from other parts of the body to the cervical lymph nodes, where they enter tar- geted cells via integrin expression on their surfaces and are transported to the damaged hemisphere [108]. Intestine Experimental and clinical evidence has shown that tem- porary impairment of the immune response is an im- portant factor in the high post-stroke infection rate [53, 109]. The intestine is often exposed to a large number of microorganisms and thus provides potential access to pathogens. Therefore, intestinal barrier dysfunction may be an important risk factor for bacterial translocation and endogenous infection. The numbers of T and B cells in aggregated lymph nodes have been shown to decrease significantly after CI, whereas the numbers of NKs and macrophages do not differ significantly. Compared with that of the control group, no significant change occurred in the lymphocyte subsets of the intestinal epithelium and lamina propria in rats with CI [110]. Stroke may have different effects on the immune cell composition in the intestinal lymphoid tissue, and this change may in- crease the susceptibility to infection after stroke [110]. In addition to the immune cell structure, the intestinal flora also plays an important role in the stroke progno- sis. The interaction between the immune system and in- testinal epithelial surface symbiotic microorganisms is essential for the development, maintenance and functio- nalization of immune cells [111, 112]. Intestinal symbi- otic microorganisms are the most abundant symbiotic chambers in the human body and have potential as a method to regulate the levels of lymphocytes, including Tregs and γδT cells, which play key roles in the patho- logical process of stroke [67]. Altering the intestinal symbiotic microbial structure of mice using amoxicillin-clavulanic acid compound antibiotics in- duces tolerance and protection of the mice against I/R injury [112]. This protective effect can be transferred directly between mice through faecal feeding behaviour. Other antibiotics, such as vancomycin, can play a similar role in altering the structure of the intestinal flora and inducing tolerance to I/R in mice [112]. This protective mechanism may be due to alteration of the intestinal symbiotic microflora structure, resulting in the produc- tion of Tregs in intestinal lymph nodes derived from the small intestine. Treg homing in the intestine inhibits the differentiation of IL-17+ γδT cells via IL-10 secretion. After stroke, effector T cells migrate from the intestine to the meninges, because the decrease in IL-17+ γδT cells reduces CXCL1 and CXCL2 expression in ischae- mic brain tissue, thereby reducing the migration and in- filtration of leukocytes into the ischaemic brain tissue and the resulting brain tissue damage [112]. The role of the spleen in stroke Splenectomy has been shown to play a protective role in various brain injury models, including permanent/tem- porary middle cerebral artery occlusion (p/t MCAO), ICH and traumatic brain injury (TBI) [113–118]. Splen- ectomy before pMCAO significantly reduces the infarct size, numbers of neutrophils and activated microglia in the damaged brain tissue [113], IFN-γ level and number of infiltrating immune cells [119]. Splenectomy before tMCAO results in a significantly lower cerebral infarc- tion volume and IFN-γ level after ischaemia and does not increase the risk of post-stroke infection [114]. Splenectomy immediately after different TBI injury models can also reduce nerve injury. For instance, vascu- lar injury and brain oedema in the cerebral ischaemic re- gion were significantly reduced in the splenectomy group [116–118]. Similar protective effects were ob- served in aged rats either before tMCAO or immediately after reperfusion with splenectomy [120]. However, splenectomy fails to provide long-term protection against I/R. In one study, splenectomy was performed 3 days after reperfusion, and the infarct volume, nerve function and peripheral blood immune cell …