Exhausted CD4+ T Cells during Malaria Exhibit Reduced mTORc1 Activity Correlated with Loss of T-bet Expression.

CD4+ T cell functional inhibition (exhaustion) is a hallmark of malaria and correlates with impaired parasite control and infection chronicity. However, the mechanisms of CD4+ T cell exhaustion are still poorly understood. In this study, we show that Ag-experienced (Ag-exp) CD4+ T cell exhaustion during Plasmodium yoelii nonlethal infection occurs alongside the reduction in mammalian target of rapamycin (mTOR) activity and restriction in CD4+ T cell glycolytic capacity. We demonstrate that the loss of glycolytic metabolism and mTOR activity within the exhausted Ag-expCD4+ T cell population during infection coincides with reduction in T-bet expression. T-bet was found to directly bind to and control the transcription of various mTOR and metabolism-related genes within effector CD4+ T cells. Consistent with this, Ag-expTh1 cells exhibited significantly higher and sustained mTOR activity than effector T-bet- (non-Th1) Ag-expT cells throughout the course of malaria. We identified mTOR to be redundant for sustaining T-bet expression in activated Th1 cells, whereas mTOR was necessary but not sufficient for maintaining IFN-γ production by Th1 cells. Immunotherapy targeting PD-1, CTLA-4, and IL-27 blocked CD4+ T cell exhaustion during malaria infection and was associated with elevated T-bet expression and a concomitant increased CD4+ T cell glycolytic metabolism. Collectively, our data suggest that mTOR activity is linked to T-bet in Ag-expCD4+ T cells but that reduction in mTOR activity may not directly underpin Ag-expTh1 cell loss and exhaustion during malaria infection. These data have implications for therapeutic reactivation of exhausted CD4+ T cells during malaria infection and other chronic conditions.

Memory CD8+ T cells exhibit tissue imprinting and non-stable exposure-dependent reactivation characteristics following blood-stage Plasmodium berghei ANKA infections.

Experimental cerebral malaria (ECM) is a severe complication of Plasmodium berghei ANKA (PbA) infection in mice, characterized by CD8+ T-cell accumulation within the brain. Whilst the dynamics of CD8+ T-cell activation and migration during extant primary PbA infection have been extensively researched, the fate of the parasite-specific CD8+ T cells upon resolution of ECM is not understood. In this study, we show that memory OT-I cells persist systemically within the spleen, lung and brain following recovery from ECM after primary PbA-OVA infection. Whereas memory OT-I cells within the spleen and lung exhibited canonical central memory (Tcm) and effector memory (Tem) phenotypes, respectively, memory OT-I cells within the brain post-PbA-OVA infection displayed an enriched CD69+ CD103- profile and expressed low levels of T-bet. OT-I cells within the brain were excluded from short-term intravascular antibody labelling but were targeted effectively by longer-term systemically administered antibodies. Thus, the memory OT-I cells were extravascular within the brain post-ECM but were potentially not resident memory cells. Importantly, whilst memory OT-I cells exhibited strong reactivation during secondary PbA-OVA infection, preventing activation of new primary effector T cells, they had dampened reactivation during a fourth PbA-OVA infection. Overall, our results demonstrate that memory CD8+ T cells are systemically distributed but exhibit a unique phenotype within the brain post-ECM, and that their reactivation characteristics are shaped by infection history. Our results raise important questions regarding the role of distinct memory CD8+ T-cell populations within the brain and other tissues during repeat Plasmodium infections.

The plasma membrane calcium ATPase 4 does not influence parasite levels but partially promotes experimental cerebral malaria during murine blood stage malaria.

BACKGROUND: Recent genome wide analysis studies have identified a strong association between single nucleotide variations within the human ATP2B4 gene and susceptibility to severe malaria. The ATP2B4 gene encodes the plasma membrane calcium ATPase 4 (PMCA4), which is responsible for controlling the physiological level of intracellular calcium in many cell types, including red blood cells (RBCs). It is, therefore, postulated that genetic differences in the activity or expression level of PMCA4 alters intracellular Ca2+ levels and affects RBC hydration, modulating the invasion and growth of the Plasmodium parasite within its target host cell. METHODS: In this study the course of three different Plasmodium spp. infections were examined in mice with systemic knockout of Pmca4 expression. RESULTS: Ablation of PMCA4 reduced the size of RBCs and their haemoglobin content but did not affect RBC maturation and reticulocyte count. Surprisingly, knockout of PMCA4 did not significantly alter peripheral parasite burdens or the dynamics of blood stage Plasmodium chabaudi infection or reticulocyte-restricted Plasmodium yoelii infection. Interestingly, although ablation of PMCA4 did not affect peripheral parasite levels during Plasmodium berghei infection, it did promote slight protection against experimental cerebral malaria, associated with a minor reduction in antigen-experienced T cell accumulation in the brain. CONCLUSIONS: The finding suggests that PMCA4 may play a minor role in the development of severe malarial complications, but that this appears independent of direct effects on parasite invasion, growth or survival within RBCs.

Targeting the IL33-NLRP3 axis improves therapy for experimental cerebral malaria.

Cerebral malaria (CM) is a serious neurological complication caused by Plasmodium falciparum infection. Currently, the only treatment for CM is the provision of antimalarial drugs; however, such treatment by itself often fails to prevent death or development of neurological sequelae. To identify potential improved treatments for CM, we performed a nonbiased whole-brain transcriptomic time-course analysis of antimalarial drug chemotherapy of murine experimental CM (ECM). Bioinformatics analyses revealed IL33 as a critical regulator of neuroinflammation and cerebral pathology that is down-regulated in the brain during fatal ECM and in the acute period following treatment of ECM. Consistent with this, administration of IL33 alongside antimalarial drugs significantly improved the treatment success of established ECM. Mechanistically, IL33 treatment reduced inflammasome activation and IL1ß production in microglia and intracerebral monocytes in the acute recovery period following treatment of ECM. Moreover, treatment with the NLRP3-inflammasome inhibitor MCC950 alongside antimalarial drugs phenocopied the protective effect of IL33 therapy in improving the recovery from established ECM. We further showed that IL1ß release from macrophages was stimulated by hemozoin and antimalarial drugs and that this was inhibited by MCC950. Our results therefore demonstrate that manipulation of the IL33-NLRP3 axis may be an effective therapy to suppress neuroinflammation and improve the efficacy of antimalarial drug treatment of CM.

A quantitative brain map of experimental cerebral malaria pathology.

The murine model of experimental cerebral malaria (ECM) has been utilised extensively in recent years to study the pathogenesis of human cerebral malaria (HCM). However, it has been proposed that the aetiologies of ECM and HCM are distinct, and, consequently, no useful mechanistic insights into the pathogenesis of HCM can be obtained from studying the ECM model. Therefore, in order to determine the similarities and differences in the pathology of ECM and HCM, we have performed the first spatial and quantitative histopathological assessment of the ECM syndrome. We demonstrate that the accumulation of parasitised red blood cells (pRBCs) in brain capillaries is a specific feature of ECM that is not observed during mild murine malaria infections. Critically, we show that individual pRBCs appear to occlude murine brain capillaries during ECM. As pRBC-mediated congestion of brain microvessels is a hallmark of HCM, this suggests that the impact of parasite accumulation on cerebral blood flow may ultimately be similar in mice and humans during ECM and HCM, respectively. Additionally, we demonstrate that cerebrovascular CD8+ T-cells appear to co-localise with accumulated pRBCs, an event that corresponds with development of widespread vascular leakage. As in HCM, we show that vascular leakage is not dependent on extensive vascular destruction. Instead, we show that vascular leakage is associated with alterations in transcellular and paracellular transport mechanisms. Finally, as in HCM, we observed axonal injury and demyelination in ECM adjacent to diverse vasculopathies. Collectively, our data therefore shows that, despite very different presentation, and apparently distinct mechanisms, of parasite accumulation, there appear to be a number of comparable features of cerebral pathology in mice and in humans during ECM and HCM, respectively. Thus, when used appropriately, the ECM model may be useful for studying specific pathological features of HCM.

Prolonged diet-induced obesity in mice modifies the inflammatory response and leads to worse outcome after stroke.

BACKGROUND: Obesity increases the risk for ischaemic stroke and is associated with worse outcome clinically and experimentally. Most experimental studies have used genetic models of obesity. Here, a more clinically relevant model, diet-induced obesity, was used to study the impact of obesity over time on the outcome and inflammatory response after stroke. METHODS: Male C57BL/6 mice were maintained on a high-fat (60% fat) or control (12% fat) diet for 2, 3, 4 and 6 months when experimental stroke was induced by transient occlusion of the middle cerebral artery (MCAo) for either 20 (6-month diet) or 30 min (2-, 3-, 4- and 6-month diet). Ischaemic damage, blood-brain barrier (BBB) integrity, neutrophil number and chemokine expression in the brain were assessed at 24 h. Plasma chemokine levels (at 4 and 24 h) and neutrophil number in the liver (at 24 h) were measured. Physiological parameters (body weight and blood glucose) were measured in naïve control- and high-fat-fed mice at all time points and blood pressure at 3 and 6 months. Blood cell counts were also assessed in naïve 6-month control- and high-fat-fed mice. RESULTS: Mice fed a high-fat diet for 6 months had greater body weight, blood glucose and white and red blood cell count but no change in systolic blood pressure. After 4 and 6 months of high-fat feeding, and in the latter group with a 30-min (but not 20-min) occlusion of the MCA, obese mice had greater ischaemic brain damage. An increase in blood-brain barrier permeability, chemokine expression (CXCL-1 and CCL3), neutrophil number and microglia/macrophage cells was observed in the brains of 6-month high-fat-fed mice after 30-min MCAo. In response to stroke, chemokine (CXCL-1) expression in the plasma and liver was significantly different in obese mice (6-month high-fat fed), and a greater number of neutrophils were detected in the liver of control but not obese mice. CONCLUSIONS: The detrimental effects of diet-induced obesity on stroke were therefore dependent on the severity of obesity and length of ischaemic challenge. The altered inflammatory response in obese mice may play a key role in its negative impact on stroke.

Lymphatic network drainage resolves cerebral edema and facilitates recovery from experimental cerebral malaria.

While brain swelling, associated with fluid accumulation, is a known feature of pediatric cerebral malaria (CM), how fluid and macromolecules are drained from the brain during recovery from CM is unknown. Using the experimental CM (ECM) model, we show that fluid accumulation in the brain during CM is driven by vasogenic edema and not by perivascular cerebrospinal fluid (CSF) influx. We identify that fluid and molecules are removed from the brain extremely quickly in mice with ECM to the deep cervical lymph nodes (dcLNs), predominantly through basal routes and across the cribriform plate and the nasal lymphatics. In agreement, we demonstrate that ligation of the afferent lymphatic vessels draining to the dcLNs significantly impairs fluid drainage from the brain and lowers anti-malarial drug recovery from the ECM syndrome. Collectively, our results provide insight into the pathways that coordinate recovery from CM.

Hypoxia coordinates the spatial landscape of myeloid cells within glioblastoma to affect survival.

Myeloid cells are highly prevalent in glioblastoma (GBM), existing in a spectrum of phenotypic and activation states. We now have limited knowledge of the tumor microenvironment (TME) determinants that influence the localization and the functions of the diverse myeloid cell populations in GBM. Here, we have utilized orthogonal imaging mass cytometry with single-cell and spatial transcriptomic approaches to identify and map the various myeloid populations in the human GBM tumor microenvironment (TME). Our results show that different myeloid populations have distinct and reproducible compartmentalization patterns in the GBM TME that is driven by tissue hypoxia, regional chemokine signaling, and varied homotypic and heterotypic cellular interactions. We subsequently identified specific tumor subregions in GBM, based on composition of identified myeloid cell populations, that were linked to patient survival. Our results provide insight into the spatial organization of myeloid cell subpopulations in GBM, and how this is predictive of clinical outcome.

Plasmodium falciparum-infected erythrocyte co-culture with the monocyte cell line THP-1 does not trigger production of soluble factors reducing brain microvascular barrier function.

Monocytes contribute to the pro-inflammatory immune response during the blood stage of a Plasmodium falciparum infection, but their precise role in malaria pathology is not clear. Besides phagocytosis, monocytes are activated by products from P. falciparum infected erythrocytes (IE) and one of the activation pathways is potentially the NLR family pyrin domain containing 3 (NLRP3) inflammasome, a multi-protein complex that leads to the production of interleukin (IL)-1ß. In cerebral malaria cases, monocytes accumulate at IE sequestration sites in the brain microvascular and the locally produced IL-1ß, or other secreted molecules, could contribute to leakage of the blood-brain barrier. To study the activation of monocytes by IE within the brain microvasculature in an in vitro model, we co-cultured IT4var14 IE and the monocyte cell line THP-1 for 24 hours and determined whether generated soluble molecules affect barrier function of human brain microvascular endothelial cells, measured by real time trans-endothelial electrical resistance. The medium produced after co-culture did not affect endothelial barrier function and similarly no effect was measured after inducing oxidative stress by adding xanthine oxidase to the co-culture. While IL-1ß does decrease barrier function, barely any IL-1ß was produced in the co- cultures, indicative of a lack of or incomplete THP-1 activation by IE in this co-culture model.

The comparable tumour microenvironment in sporadic and NF2-related schwannomatosis vestibular schwannoma.

Bilateral vestibular schwannoma is the hallmark of NF2-related schwannomatosis, a rare tumour predisposition syndrome associated with a lifetime of surgical interventions, radiotherapy and off-label use of the anti-angiogenic drug bevacizumab. Unilateral vestibular schwannoma develops sporadically in non-NF2-related schwannomatosis patients for which there are no drug treatment options available. Tumour-infiltrating immune cells such as macrophages and T-cells correlate with increased vestibular schwannoma growth, which is suggested to be similar in sporadic and NF2-related schwannomatosis tumours. However, differences between NF2-related schwannomatosis and the more common sporadic disease include NF2-related schwannomatosis patients presenting an increased number of tumours, multiple tumour types and younger age at diagnosis. A comparison of the tumour microenvironment in sporadic and NF2-related schwannomatosis tumours is therefore required to underpin the development of immunotherapeutic targets, identify the possibility of extrapolating ex vivo data from sporadic vestibular schwannoma to NF2-related schwannomatosis and help inform clinical trial design with the feasibility of co-recruiting sporadic and NF2-related schwannomatosis patients. This study drew together bulk transcriptomic data from three published Affymetrix microarray datasets to compare the gene expression profiles of sporadic and NF2-related schwannomatosis vestibular schwannoma and subsequently deconvolved to predict the abundances of distinct tumour immune microenvironment populations. Data were validated using quantitative PCR and Hyperion imaging mass cytometry. Comparative bioinformatic analyses revealed close similarities in NF2-related schwannomatosis and sporadic vestibular schwannoma tumours across the three datasets. Significant inflammatory markers and signalling pathways were closely matched in NF2-related schwannomatosis and sporadic vestibular schwannoma, relating to the proliferation of macrophages, angiogenesis and inflammation. Bulk transcriptomic and imaging mass cytometry data identified macrophages as the most abundant immune population in vestibular schwannoma, comprising one-third of the cell mass in both NF2-related schwannomatosis and sporadic tumours. Importantly, there were no robust significant differences in signalling pathways, gene expression, cell type abundance or imaging mass cytometry staining between NF2-related schwannomatosis and sporadic vestibular schwannoma. These data indicate strong similarities in the tumour immune microenvironment of NF2-related schwannomatosis and sporadic vestibular schwannoma.

Ligature-induced periodontitis induces systemic inflammation but does not alter acute outcome after stroke in mice.

BACKGROUND: Stroke is a major cause of disability and mortality. Poorer outcome after stroke is associated with concomitant inflammatory and infectious disease. Periodontitis is a chronic inflammatory disease of the dental supporting structures and is a prominent risk factor for many systemic disorders, including cardiovascular disease and stroke. While epidemiological studies suggest that periodontitis increases the likelihood of stroke, its impact on stroke severity is poorly understood. Here, we sought to determine the contribution of periodontitis to acute stroke pathology. METHODS: We characterized a murine ligature model of periodontitis for inflammatory responses that could potentially impact stroke outcome. We applied this model and then subjected mice to either transient or permanent middle cerebral artery occlusion. We also enhanced the periodontitis model with repeated intravenous administration of a periodontal-specific lipopolysaccharide to better mimic the clinical condition. RESULTS: Ligature-induced periodontitis caused bone loss, bacterial growth, and increased local inflammatory cell trafficking. Systemically, periodontitis increased circulating levels of pro-inflammatory cytokines, and primed bone marrow monocytes to produce elevated tumour necrosis factor-alpha (TNFα). Despite these changes, periodontitis alone or in tandem with repeated lipopolysaccharide challenge did not alter infarct volume, blood-brain barrier breakdown, or systemic inflammation after experimental stroke. CONCLUSIONS: Our data show that despite elevated systemic inflammation in periodontitis, oral inflammatory disease does not impact acute stroke pathology in terms of severity, determined primarily by infarct volume. This indicates that, at least in this experimental paradigm, periodontitis alone does not alter acute outcome after cerebral ischemia.

Stroke Induces Prolonged Changes in Lipid Metabolism, the Liver and Body Composition in Mice.

During recovery, stroke patients are at risk of developing long-term complications that impact quality of life, including changes in body weight and composition, depression and anxiety, as well as an increased risk of subsequent vascular events. The aetiologies and time-course of these post-stroke complications have not been extensively studied and are poorly understood. Therefore, we assessed long-term changes in body composition, metabolic markers and behaviour after middle cerebral artery occlusion in mice. These outcomes were also studied in the context of obesity, a common stroke co-morbidity proposed to protect against post-stroke weight loss in patients. We found that stroke induced long-term changes in body composition, characterised by a sustained loss of fat mass with a recovery of lean weight loss. These global changes in response to stroke were accompanied by an altered lipid profile (increased plasma free fatty acids and triglycerides) and increased adipokine release at 60 days. After stroke, the liver also showed histological changes indicative of liver damage and a decrease in plasma alanine aminotransferase (ALT) was observed. Stroke induced depression and anxiety-like behaviours in mice, illustrated by deficits in exploration, nest building and burrowing behaviours. When initial infarct volumes were matched between mice with and without comorbid obesity, these outcomes were not drastically altered. Overall, we found that stroke induced long-term changes in depressive/anxiety-like behaviours, and changes in plasma lipids, adipokines and the liver that may impact negatively on future vascular health.

Microglial Priming as Trained Immunity in the Brain.

In this review we discuss the possibility that the phenomenon of microglial priming can be explained by the mechanisms that underlie trained immunity. The latter involves the enhancement of inflammatory responses by epigenetic mechanisms that are mobilized after first exposure to an inflammatory stimulus. These mechanisms include long-lasting histone modifications, including H3K4me1 deposition at latent enhancer regions. Although such changes may be beneficial in peripheral infectious disease, in the context of microglial priming they may drive increased microglia reactivity that is damaging in diseases of brain aging.

Acute high-fat feeding leads to disruptions in glucose homeostasis and worsens stroke outcome.

Chronic consumption of diets high in fat leads to obesity and can negatively affect brain function. Rodents made obese by long-term maintenance on a high-fat diet have worse outcome after experimental stroke. High-fat consumption for only three days does not induce obesity but has rapid effects on the brain including memory impairment. However, the effect of brief periods of high-fat feeding or high-fat consumption in the absence of obesity on stroke is unknown. We therefore tested the effect of an acute period of high-fat feeding (three days) in C57B/6 mice on outcome after middle cerebral artery occlusion (MCAo). In contrast to a chronic high-fat diet (7.5 months), an acute high-fat diet had no effect on body weight, adipose tissue, lipid profile or inflammatory markers (in periphery and the brain). Three days of high-fat feeding impaired glucose tolerance, increased plasma glucose and insulin and brain expression of the glucose transporter GLUT-1. Ischaemic damage was increased (48%) in mice fed an acute high-fat diet, and was associated with a further reduction in GLUT-1 in the ischaemic hemisphere. These data demonstrate that only a brief period of high-fat consumption has a negative effect on glucose homeostasis and worsens outcome after ischaemic stroke.

Comparison of CD8+ T Cell Accumulation in the Brain During Human and Murine Cerebral Malaria.

CD8+ T cells have been shown to play a critical role in the pathogenesis of experimental cerebral malaria (ECM) in mice, but their role in development of human cerebral malaria (HCM) remains unclear. Thus, in this study we have provided the first direct contrast of the accumulation of CD8+ T cells in the brain during HCM and ECM. HCM cases were from children who died of Plasmodium falciparum cerebral malaria at Queen Elizabeth Central Hospital (Malawi) between 2003 and 2010. ECM was induced by infecting C57BL/6J mice with P. berghei ANKA. We demonstrate similarities in the intracerebral CD8+ T cell responses in ECM and HCM, in particular an apparent shared choroid plexus-meningeal route of CD8+ T cell accumulation in the brain. Nevertheless, we also reveal some potentially important differences in compartmentalization of CD8+ T cells within the cerebrovascular bed in HCM and ECM.

Development of a characterised tool kit for the interrogation of NLRP3 inflammasome-dependent responses.

Inflammation is an established contributor to disease and the NLRP3 inflammasome is emerging as a potential therapeutic target. A number of small molecule inhibitors of the NLRP3 pathway have been described. Here we analysed the most promising of these inhibitor classes side by side to assess relative potency and selectivity for their respective putative targets. Assessed using ASC inflammasome-speck formation, and release of IL-1ß, in both human monocyte/macrophage THP1 cells and in primary mouse microglia, we compared the relative potency and selectivity of P2X7 inhibitors, inflammasome inhibitors (diarylsulfonylurea vs. the NBC series), and caspase-1 inhibitors. In doing so we are now able to provide a well characterised small molecule tool kit for interrogating and validating inflammasome-dependent responses with a range of nanomolar potency inhibitors against established points in the inflammasome pathway.

The blood-brain barrier after stroke: Structural studies and the role of transcytotic vesicles.

Blood-brain barrier breakdown worsens ischaemic damage, but it is unclear how molecules breach the blood-brain barrier in vivo. Using the obese ob/ob mouse as a model of enhanced blood-brain barrier breakdown, we investigated how stroke-induced structural changes to the microvasculature related to blood-brain barrier permeability. Ob/ob mice underwent middle cerebral artery occlusion, followed by 4 or 24 h reperfusion. Blood-brain barrier integrity was assessed using IgG and horseradish peroxidase staining, and blood-brain barrier structure by two-dimensional and three-dimensional electron microscopy. At 4 and 24 h post-stroke, ob/ob mice had increased ischaemic damage and blood-brain barrier breakdown compared to ob/- controls, and vessels from both genotypes showed astrocyte end-foot swelling and increased endothelial vesicles. Ob/ob mice had significantly more endothelial vesicles at 4 h in the striatum, where blood-brain barrier breakdown was most severe. Both stroke and genotype had no effect on tight junction structure visualised by electron microscopy, or protein expression in isolated microvessels. Astrocyte swelling severity did not correlate with tissue outcome, being unaffected by genotype or reperfusion times. However, the rare instances of vessel lumen collapse were always associated with severe astrocyte swelling in two-dimensional and three-dimensional electron microscopy. Endothelial vesicles were therefore the best spatial and temporal indicators of blood-brain barrier breakdown after cerebral ischaemia.

Adipose tissue and metabolic and inflammatory responses to stroke are altered in obese mice.

Obesity is an independent risk factor for stroke, although several clinical studies have reported that obesity improves stroke outcome. Obesity is hypothesised to aid recovery by protecting against post-stroke catabolism. We therefore assessed whether obese mice had an altered metabolic and inflammatory response to stroke. Obese ob/ob mice underwent a 20-min middle cerebral artery occlusion and 24-h reperfusion. Lipid metabolism and expression of inflammatory cytokines were assessed in the plasma, liver and adipose tissue. The obese-specific metabolic response to stroke was assessed in plasma using non-targeted ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics coupled with univariate and multivariate analysis. Obesity had no effect on the extent of weight loss 24 h after stroke but affected the metabolic and inflammatory responses to stroke, predominantly affecting lipid metabolism. Specifically, obese mice had increases in plasma free fatty acids and expression of adipose lipolytic enzymes. Metabolomics identified several classes of metabolites affected by stroke in obese mice, including fatty acids and membrane lipids (glycerophospholipids, lysophospholipids and sphingolipids). Obesity also featured increases in inflammatory cytokines in the plasma and adipose tissue. Overall, these results demonstrate that obesity affected the acute metabolic and inflammatory response to stroke and suggest a potential role for adipose tissue in this effect. These findings could have implications for longer-term recovery and also further highlight the importance of considering comorbidities in preclinical stroke research, especially when identifying biomarkers for stroke. However, further work is required to assess whether these changes translate into long-term effects on recovery.

The IMPROVE Guidelines (Ischaemia Models: Procedural Refinements Of in Vivo Experiments).

Most in vivo models of ischaemic stroke target the middle cerebral artery and a spectrum of stroke severities, from mild to substantial, can be achieved. This review describes opportunities to improve the in vivo modelling of ischaemic stroke and animal welfare. It provides a number of recommendations to minimise the level of severity in the most common rodent models of middle cerebral artery occlusion, while sustaining or improving the scientific outcomes. The recommendations cover basic requirements pre-surgery, selecting the most appropriate anaesthetic and analgesic regimen, as well as intraoperative and post-operative care. The aim is to provide support for researchers and animal care staff to refine their procedures and practices, and implement small incremental changes to improve the welfare of the animals used and to answer the scientific question under investigation. All recommendations are recapitulated in a summary poster (see supplementary information).

Obesity and stroke: Can we translate from rodents to patients?

Obesity is a risk factor for stroke and is consequently one of the most common co-morbidities found in patients. There is therefore an identified need to model co-morbidities preclinically to allow better translation from bench to bedside. In preclinical studies, both diet-induced and genetically obese rodents have worse stroke outcome, characterised by increased ischaemic damage and an altered inflammatory response. However, clinical studies have reported an 'obesity paradox' in stroke, characterised by reduced mortality and morbidity in obese patients. We discuss the potential reasons why the preclinical and clinical studies may not agree, and review the mechanisms identified in preclinical studies through which obesity may affects stroke outcome. We suggest inflammation plays a central role in this relationship, as obesity features increases in inflammatory mediators such as C-reactive protein and interleukin-6, and chronic inflammation has been linked to worse stroke risk and outcome.