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Rationale: Arterial inflammation manifested as atherosclerosis is the leading cause of mortality worldwide. Genome-wide association studies have identified a prominent role of HDAC (histone deacetylase)-9 in atherosclerosis and its clinical complications including stroke and myocardial infarction. Objective: To determine the mechanisms linking HDAC9 to these vascular pathologies and explore its therapeutic potential for atheroprotection. Methods and Results: We studied the effects of Hdac9 on features of plaque vulnerability using bone marrow reconstitution experiments and pharmacological targeting with a small molecule inhibitor in hyperlipidemic mice. We further used 2-photon and intravital microscopy to study endothelial activation and leukocyte-endothelial interactions. We show that hematopoietic Hdac9 deficiency reduces lesional macrophage content while increasing fibrous cap thickness thus conferring plaque stability. We demonstrate that HDAC9 binds to IKK (inhibitory kappa B kinase)-α and β, resulting in their deacetylation and subsequent activation, which drives inflammatory responses in both macrophages and endothelial cells. Pharmacological inhibition of HDAC9 with the class IIa HDAC inhibitor TMP195 attenuates lesion formation by reducing endothelial activation and leukocyte recruitment along with limiting proinflammatory responses in macrophages. Transcriptional profiling using RNA sequencing revealed that TMP195 downregulates key inflammatory pathways consistent with inhibitory effects on IKKβ. TMP195 mitigates the progression of established lesions and inhibits the infiltration of inflammatory cells. Moreover, TMP195 diminishes features of plaque vulnerability and thereby enhances plaque stability in advanced lesions. Ex vivo treatment of monocytes from patients with established atherosclerosis reduced the production of inflammatory cytokines including IL (interleukin)-1β and IL-6. Conclusions: Our findings identify HDAC9 as a regulator of atherosclerotic plaque stability and IKK activation thus providing a mechanistic explanation for the prominence of HDAC9 as a vascular risk locus in genome-wide association studies. Its therapeutic inhibition may provide a potent lever to alleviate vascular inflammation. Graphical Abstract: A graphical abstract is available for this article.

Immune cell infiltration to the injured brain is a key component of the neuroinflammatory response after ischemic stroke. In contrast to the large amount of proinflammatory immune cells, regulatory T cells, are an important subgroup of T cells that are involved in maintaining immune homeostasis and suppress an overshooting immune reaction after stroke. Numerous previous reports have consistently demonstrated the beneficial role of this immunosuppressive immune cell population during the acute phase after experimental stroke by limiting inflammatory lesion progression. Two recent studies expanded now this concept and demonstrate that regulatory T cells-mediated effects also promote chronic recovery after stroke by promoting a proregenerative tissue environment. These recent findings suggest that boosting regulatory T cells could be beneficial beyond modulating the immediate neuroinflammatory response and improve chronic functional recovery.

Neuroinflammation after stroke is characterized by the activation of resident microglia and the invasion of circulating leukocytes into the brain. Although lymphocytes infiltrate the brain in small number, they have been consistently demonstrated to be the most potent leukocyte subpopulation contributing to secondary inflammatory brain injury. However, the exact mechanism how this minimal number of lymphocytes can profoundly affect stroke outcome is still largely elusive. Here, using a mouse model for ischemic stroke, we demonstrated that early activation of microglia in response to stroke is differentially regulated by distinct T cell subpopulations. Acute treatment with engineered T cells overexpressing IL-10 administered into the cisterna magna after stroke induces a switch of microglial gene expression to a profile associated with pro-regenerative functions. These findings substantiate the role of T cells in stroke with large impact on the cerebral inflammatory milieu by polarizing the microglial phenotype. Targeting T cell-microglia interactions can have direct translational relevance for further development of immune-targeted therapies for stroke and other neuroinflammatory conditions.SummaryThe crosstalk between brain infiltrating T cells and microglia in response to stroke remains elusive. Benakis et al. report that transcriptional signature of the stroke-associated microglia is reprogrammed by distinct T cell subpopulations. Engineered T cells overexpressing IL-10 administered four hours after stroke reinitiate microglial function inducing a pro-regenerative environment.

The immune system is intricately involved in brain development and physiological neuronal function. The influence of the adaptive immune system on several brain diseases has been described in great detail. In ischemic stroke, numerous studies have particularly demonstrated a key role for T cells during the acute phase after the brain injury. Recently, a critical role for T cells has also become more evident for the chronic phase after stroke in modulating delayed neuronal (dys-) function and recovery. Here, T cells may also affect various non-immunological pathways by interacting with brain-resident immune cells and modulating mechanisms such as neurogenesis and angiogenesis. This novel concept suggests T cells as potential therapeutic targets to modulate post-stroke regeneration.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called "replication crisis", detailed standardized models for all procedures are urgently needed. As an effort within the "ImmunoStroke" research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this "filament" stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with histological analysis are demonstrated.

GRN haploinsufficiency causes frontotemporal lobar degeneration and results in microglial hyperactivation, lysosomal dysfunction and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology we evaluated genetic and pharmacological approaches suppressing TREM2 dependent transition of microglia from a homeostatic to a disease associated state. Trem2 deficiency in Grn KO mice led to a reduction of microglia activation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies allowed a complete rescue of elevated levels of TREM2 together with increased shedding and reduction of TREM2 signaling. Furthermore, antibody-treated PGRN deficient hiMGL showed dampened microglial hyperactivation, reduced TREM2 signaling and phagocytic activity, however, lack of rescue of lysosomal dysfunction. Similarly, lysosomal dysfunction, lipid dysregulation and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Furthermore, NfL, a biomarker for neurodegeneration, was elevated in the Grn/Trem2 KO. These findings suggest that microglia hyperactivation is not necessarily contributing to neurotoxicity, and instead demonstrates that TREM2 exhibits neuroprotective potential in this model.

Heindl et al. describe the local proliferation and clustering of T cells in the brain of mice and humans after stroke. This previously unrecognized phenomenon could explain why blocking cerebral leukocyte invasion might fail to improve long-term stroke recovery., Neuroinflammation is an emerging focus of translational stroke research. Preclinical studies have demonstrated a critical role for brain-invading lymphocytes in post-stroke pathophysiology. Reducing cerebral lymphocyte invasion by anti-CD49d antibodies consistently improves outcome in the acute phase after experimental stroke models. However, clinical trials testing this approach failed to show efficacy in stroke patients for the chronic outcome 3 mo after stroke. Here, we identify a potential mechanistic reason for this phenomenon by detecting chronic T cell accumulation—evading the systemic therapy—in the post-ischemic brain. We observed a persistent accumulation of T cells in mice and human autopsy samples for more than 1 mo after stroke. Cerebral T cell accumulation in the post-ischemic brain was driven by increased local T cell proliferation rather than by T cell invasion. This observation urges re-evaluation of current immunotherapeutic approaches, which target circulating lymphocytes for promoting recovery after stroke., Graphical Abstract

Stroke causes severe and long-lasting symptoms in patients. Besides focal deficits such as speech impairment and limb weakness, stroke also results in neuropsychiatric symptoms, including fatigue, anxiety, and depression, which are debilitating and often impair post-stroke rehabilitation. However, in experimental stroke research, the study of neuropsychiatric symptoms and their therapeutic targeting has so far been largely neglected, which can be mainly attributed to the lack of appropriate tools to investigate such deficits in mice. Here, we report that neuropsychiatric symptoms can be differentiated from focal deficits and specifically modulated independent of treating the primary lesion. In order to achieve this, we developed a novel behavior analysis tool by assessing test performance of various tests, combining outcome parameters to cover functional domains of focal and neuropsychiatric symptoms, and finally weighted results into a time point-specific score. This weighted score enabled us to clearly differentiate focal deficits and neuropsychiatric symptoms and detect these until the chronic phase after stroke. Using this analysis tool, we detected that neutralizing systemic cytokines (TNF-α, IL-1β and IL-6) specifically ameliorated neuropsychiatric symptoms but did not affect focal deficits or lesion volume. Hence, most conventional studies analyzing only focal deficits and lesion volume as primary outcome measures would have missed these significant and translationally relevant therapeutic effects. We anticipate that these findings will encourage more detailed analyses of neuropsychiatric symptoms particularly for anti-inflammatory therapies in stroke and that the presented weighted composite score will facilitate this development.

BACKGROUND CCL23 role in the inflammatory response after acute brain injuries remains elusive. Here, we evaluated whether CCL23 blood levels associate with acquired cerebral lesions and determined CCL23 predictive capacity for assessing stroke prognosis. We used preclinical models to study the CCL23 homologous chemokines in rodents, CCL9 and CCL6. METHODS Baseline CCL23 blood levels were determined on 245 individuals, including ischaemic strokes (IS), stroke mimics and controls. Temporal profile of circulating CCL23 was explored from baseline to 24 h in 20 of the IS. In an independent cohort of 120 IS with a 3-month follow-up, CCL23 blood levels were included in logistic regression models to predict IS outcome. CCL9/CCL6 cerebral expression was evaluated in rodent models of brain damage. Both chemokines were also profiled in circulation and histologically located on brain following ischaemia. RESULTS Baseline CCL23 blood levels did not discriminate IS, but permitted an accurate discrimination of patients presenting acute brain lesions (P = 0.003). IS exhibited a continuous increase from baseline to 24 h in circulating CCL23 (P

After traumatic brain injury (TBI), neurons surviving the initial insult can undergo chronic (secondary) degeneration via poorly understood mechanisms, resulting in long-term cognitive impairment. Although a neuroinflammatory response is promptly activated after TBI, it is unknown whether it has a significant role in chronic phases of TBI (>1 year after injury). Using a closed-head injury model of TBI in mice, we showed by MRI scans that TBI caused substantial degeneration at the lesion site within a few weeks and these did not expand significantly thereafter. However, chronic alterations in neurons were observed, with reduced dendritic spine density lasting >1 year after injury. In parallel, we found a long-lasting inflammatory response throughout the entire brain. Deletion of one allele of CX3CR1, a chemokine receptor, limited infiltration of peripheral immune cells and largely prevented the chronic degeneration of the injured brain and provided a better functional recovery in female, but not male, mice. Therefore, targeting persistent neuroinflammation presents a new therapeutic option to reduce chronic neurodegeneration.SIGNIFICANCE STATEMENTTraumatic brain injury (TBI) often causes chronic neurological problems including epilepsy, neuropsychiatric disorders, and dementia through unknown mechanisms. Our study demonstrates that inflammatory cells invading the brain lead to secondary brain damage. Sex-specific amelioration of chronic neuroinflammation rescues the brain degeneration and results in improved motor functions. Therefore, this study pinpoints an effective therapeutic approach to preventing secondary complications after TBI.

Experimental stroke models producing clinically relevant functional deficits are often associated with high mortality. Because the mechanisms that underlie post-stroke mortality are largely unknown, results obtained using these models are often difficult to interpret, thereby limiting their translational potential. Given that specific forms of post-stroke care reduce mortality in patients, we hypothesized that inadequate food and water intake may underlie mortality following experimental stroke. C57BL/6 mice were subjected to 1 h of intraluminal filament middle cerebral artery occlusion. Nutritional support beginning on the second day after filament middle cerebral artery occlusion reduced the 14-day mortality rate from 59% to 15%. The surviving mice in the post-stroke support group had the same infarct size as non-surviving control mice, suggesting that post-stroke care was not neuroprotective and that inadequate food and/or water intake are the main reasons for filament middle cerebral artery occlusion–induced mortality. This notion was supported by the presence of significant hypoglycemia, ketonemia, and dehydration in control mice. Taken together, these data suggest that post–filament middle cerebral artery occlusion mortality in mice is not primarily caused by ischemic brain damage, but secondarily by inadequate food and/or water intake. Thus, providing nutritional support following filament middle cerebral artery occlusion greatly minimizes mortality bias and allows the study of long-term morphological and functional sequelae of stroke in mice.

Stroke is a major health burden as it is a leading cause of morbidity and mortality worldwide. Blood flow restoration, through thrombolysis or endovascular thrombectomy, is the only effective treatment but is restricted to a limited proportion of patients due to time window constraint and accessibility to technology. Over the past two decades, research has investigated the basic mechanisms that lead to neuronal death following cerebral ischemia. However, the use of neuroprotective paradigms in stroke has been marked by failure in translation from experimental research to clinical practice. In the past few years, much attention has focused on the immune response to acute cerebral ischemia as a major factor to the development of brain lesions and neurological deficits. Key inflammatory processes after stroke include the activation of resident glial cells as well as the invasion of circulating leukocytes. Recent research on anti-inflammatory strategies for stroke has focused on limiting the transendothelial migration of peripheral immune cells from the compromised vasculature into the brain parenchyma. However, recent trials testing the blockage of cerebral leukocyte infiltration in patients reported inconsistent results. This emphasizes the need to better scrutinize how immune cells are regulated at the blood–brain interface and enter the brain parenchyma, and particularly to also consider alternative cerebral infiltration routes for leukocytes, including the meninges and the choroid plexus. Understanding how immune cells migrate to the brain via these alternative pathways has the potential to develop more effective approaches for anti-inflammatory stroke therapies.

Microbiome alterations have been shown to affect stroke outcome. However, to what extent the presence of a gut microbiome per se is affecting post-stroke neuroinflammation has not been tested. By comparing germfree mice with recolonized (Ex-GF) and conventional SPF mice, we were able to demonstrate that bacterial colonization reduces stroke volumes. Bacterial colonization increased cerebral expression of cytokines as well as microglia/macrophage cell counts in contrast to improved stroke outcome. Interestingly, the microbiome-mediated brain protection was absent in lymphocyte-deficient mice. These findings support the concept of lymphocyte-driven protective neuroinflammation after stroke under control of the microbiome.

Introduction: The gut microbiota influences the severity of brain injury through regulation of intestinal immune homeostasis. However, molecular cues involved in microbiota-immune interaction after stroke are unknown. Metabolites have a pivotal role in connecting commensals to the immune response. Specifically, bacteria-derived metabolites of tryptophan catabolism activate the transcription factor aryl hydrocarbon receptor (AhR) which regulates T cell polarization directly or via tolerogenic function of dendritic cells (DCs). Hypothesis: Since adaptive immune cells are involved in stroke, we hypothesize that modulation of tryptophan metabolism–orchestrated by bacteria–modifies T cell differentiation and influences stroke outcome. Methods: Stroke is induced by the occlusion of the middle cerebral artery (MCAO) in C57BL6 mice. Blood and the distal small intestine are sampled for the detection of tryptophan metabolites and quantified by metagenomics or mRNA expression. Immune cells are isolated from the intestine and analyzed by flow cytometry. Stroke outcome is measured by infarct volumetry and behaviour tests. Results: Tryptophan catabolism was augmented in mice subjected to stroke-induced dysbiosis–shown by a) an increase of the most proximal metabolite kynurenine and a concomitant decrease of tryptophan and b) an upregulation of mRNA expression of the rate-limiting enzyme involved in tryptophan’s breakdown. This was paralleled by an increase of AhR mRNA in the ileum after MCAO. To test the implication of AhR on the tolerogenic function of DCs, we used AhR-DCs knock-out mice. We found that CD103+CD11b+ DCs lacking AhR were downregulated after stroke compared to wild-type (WT) mice. Interestingly, polarization state of T cells was modified in DCs lacking AhR (regulatory versus Th17 cell ratio). This could influence the central nervous system immune response and induce protection after stroke, as suggested by a reduction of infarct volume observed 3 days after stroke in AhR-DCs knock-out mice compared to WT mice. Conclusion: These findings suggest that stroke-induced dysbiosis leads to an increase of tryptophan catabolism, and that AhR depletion in DCs influences immune cell polarization and stroke outcome.

Background and purpose Acute brain injuries induce a systemic immune depression syndrome (SIDS) that predisposes patients to bacterial infections. While cellular compartments of this syndrome have been well characterized, the contribution of humoral immune mechanisms and particularly immunoglobulins to SIDS has not been investigated so far. Methods We determined serum immunoglobulin levels and infectious complications at several time points in 159 ischemic and hemorrhagic stroke patients. Additionally, findings were verified in a transient middle cerebral artery occlusion model. A novel immunoassay was established to analyze the IgG excretion ratio in mice. Results We identified a transient IgG reduction in patients suffering from substantial ischemic or hemorrhagic brain injuries. The IgG-reduction was associated with subsequent bacterial infections. Similarly, transient hypogammaglobulinemia was detected in a murine stroke model. We then used this animal model to further distinguish the mechanism of the IgG reduction by an IgG transfer paradigm. Excretional loss rather than deficient production of IgG was demonstrated to underlay hypogammaglobulinemia. Conclusions This is the first report of transient hypogammaglobulinemia after ischemic and hemorrhagic stroke suggesting involvement in infectious complications. These findings pave the road for further studies investigating post-stroke hypogammaglobulinemia as a druggable target for stroke-induced complications.

Background and Purpose— Neuroinflammation plays an important role in ischemic brain injury. Regulatory T cells (Treg) are important endogenous immune modulators. We tested the hypothesis that Treg amplification with a CD28 superagonistic monoclonal antibody (CD28SA) reduces brain damage in murine cerebral ischemia. Methods— Cerebral ischemia was induced by coagulation of the distal middle cerebral artery or by 60 minutes filament occlusion of the proximal middle cerebral artery in C57BL6 mice. 150 μg CD28SA was injected intraperitoneally 3 or 6 hours after ischemia onset. Outcome was determined by infarct volumetry and behavioral testing. Brain-infiltrating leukocyte subpopulations were analyzed by flow cytometry and immunohistochemistry 3 and 7 days after middle cerebral artery occlusion. Results— CD28SA reduced infarct size in both models and attenuated functional deficit 7 days after stroke induction. Mice treated with CD28SA increased numbers of Treg in spleen and brain. Tregs were functionally active and migrated into the brain where they accumulated and proliferated in the peri-infarct area. More than 60% of brain infiltrating Treg produced interleukin-10 in CD28SA compared with 30% in control. Conclusions— In vivo expansion and amplification of Treg by CD28SA attenuates the inflammatory response and improves outcome after experimental stroke.

The interaction of the immune system with the brain is necessary for development and surveillance of the healthy brain. The influence of the adaptive immune system on several brain diseases has been described in great detail. In ischemic stroke, a growing body of evidence has demonstrated a key role for T cells in the acute phase after stroke. Pro- and anti-inflammatory T cell subpopulations impact in this early phase the inflammatory milieu and directly affect secondary lesion progression and neuronal injury. Recently, a functional role for T cells has also become more evident also in delayed neuronal (dys-)function and late-phase recovery after stroke. Here, T cells may also affect various non-immunological pathways involved in tissue repair, neuronal plasticity and functional recovery. These pleiotropic effects of T cells on mechanisms such as neurogenesis and angiogenesis suggest T cells as potential therapeutic target to modulate post-stroke regeneration. This chapter will provide a comprehensive overview of the current knowledge about the role of T cells in stroke with a particular focus on regenerative processes in the chronic phase.

Rationale: Currently, there are no blood-based biomarkers with clinical utility for acute ischemic stroke (IS). MicroRNAs show promise as disease markers because of their cell type–specific expression patterns and stability in peripheral blood. Objective: To identify circulating microRNAs associated with acute IS, determine their temporal course up to 90 days post-stroke, and explore their utility as an early diagnostic marker. Methods and Results: We used RNA sequencing to study expression changes of circulating microRNAs in a discovery sample of 20 patients with IS and 20 matched healthy control subjects. We further applied quantitative real-time polymerase chain reaction in independent samples for validation (40 patients with IS and 40 matched controls), replication (200 patients with IS, 100 healthy control subjects), and in 72 patients with transient ischemic attacks. Sampling of patient plasma was done immediately upon hospital arrival. We identified, validated, and replicated 3 differentially expressed microRNAs, which were upregulated in patients with IS compared with both healthy control subjects (miR-125a-5p [1.8-fold; P =1.5×10 −6 ], miR-125b-5p [2.5-fold; P =5.6×10 −6 ], and miR-143-3p [4.8-fold; P =7.8×10 −9 ]) and patients with transient ischemic attack (miR-125a-5p: P =0.003; miR-125b-5p: P =0.003; miR-143-3p: P =0.005). Longitudinal analysis of expression levels up to 90 days after stroke revealed a normalization to control levels for miR-125b-5p and miR-143-3p starting at day 2 while miR-125a-5p remained elevated. Levels of all 3 microRNAs depended on platelet numbers in a platelet spike-in experiment but were unaffected by chemical hypoxia in Neuro2a cells and in experimental stroke models. In a random forest classification, miR-125a-5p, miR-125b-5p, and miR-143-3p differentiated between healthy control subjects and patients with IS with an area under the curve of 0.90 (sensitivity: 85.6%; specificity: 76.3%), which was superior to multimodal cranial computed tomography obtained for routine diagnostics (sensitivity: 72.5%) and previously reported biomarkers of acute IS (neuron-specific enolase: area under the curve=0.69; interleukin 6: area under the curve=0.82). Conclusions: A set of circulating microRNAs (miR-125a-5p, miR-125b-5p, and miR-143-3p) associates with acute IS and might have clinical utility as an early diagnostic marker.

Stroke induces a profound neuroinflammatory reaction that leads to secondary cerebral tissue injury. Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that is endogenously produced by immune cells and limits this inflammatory reaction. Previously, therapeutic administration of IL-10 has been shown to be neuroprotective in experimental stroke. However, the signaling pathways affected by this approach are largely unknown. The aim of this study was to verify the neuroprotective effects of IL-10 in an experimental mouse stroke model and to analyze the pathways modulated by this approach. Therefore, we performed a whole genome microarray analysis comparing the cerebral gene expression profile at two time points after cortical stroke in IL-10-treated and control C57Bl/6J mice. We administered IL-10 locally by intracerebroventricular injection. We were able to validate a reduction of infarct volume by IL-10 administration and characterized the kinetics of endogenous cerebral IL-10 expression after stroke. The microarray analysis revealed that IL-10 treatment effectively downregulated pro-inflammatory signaling cascades which were upregulated by the ischemic lesion in the acute phase after the stroke. This is the first study characterizing the global gene regulation profile of IL-10 immunotherapy for ischemic stroke. Our results emphasize the key role of IL-10 as a neuroprotective cytokine and suggest several novel downstream pathways for further investigation to better understand the mechanisms of post-stroke neuroinflammation.

Background and Purpose— Neuroinflammatory processes contribute to secondary neuronal damage after intracerebral hemorrhage. We aimed to characterize the time course of brain immigration of different leukocyte subsets after striatal injection of either autologous blood or collagenase in mice. Methods— Intracerebral hemorrhage was induced by injection of either autologous blood (20 μL) or collagenase (0.03 U) in C57Bl/6J mice. Hematoma volumetry was performed on cryosections. Blood volume was measured by hemoglobin spectrophotometry. Leukocytes were isolated from hemorrhagic hemisphere 1, 3, 5, and 14 days after intracerebral hemorrhage, stained for leukocyte markers, and measured by flow cytometry. Heterologous blood injection from CD45.1 mice was used to investigate the origin of brain-invading leukocytes. Results— Collagenase injection induced a larger hematoma volume but a similar blood content compared with blood injection. Cerebral leukocyte infiltration in the hemorrhagic hemisphere was similar in both models. The majority of leukocytes isolated from the brain originated from the circulation. CD4 + T lymphocytes were the predominant brain leukocyte population in both models. However, cerebral granulocyte counts were higher after collagenase compared with blood injection. Conclusions— Brain infiltration of systemic immune cells is similar in both murine intracerebral hemorrhage models. The pathophysiological impact of invading leukocytes and, in particular, of T cells requires further investigation.

Stroke causes 5.7 million deaths annually. This ranks stroke as the second most common cause of death and, additionally, it is a major cause of disability. Because of an ageing population, stroke incidence and costs will greatly increase in the future. This makes stroke an ongoing social and economic burden, in contrast to the only very limited therapeutic options. In the last decade vast sums were spent on translational research focused on neuroprotective strategies in the acute phase of ischaemic stroke. A plethora of candidate agents were tested in experimental models and preclinical studies, but none was proven effective in clinical trials. This gave rise to discussions about the possible reasons for this failure, ending up mainly with criticism of methodological aspects of the preclinical and clinical studies, or of the relevance of animal studies in drug development. Indeed, the question could rather be whether neuroprotection is the right target for successful stroke treatment. In this context, a paradigm change can currently be observed: the focus of experimental and translational stroke research is shifting from early neuroprotection to delayed mechanisms such as stroke-associated comorbidities, regeneration and plasticity. In this review we highlight a few recently emerging fields in translational stroke research. One such topic is the crosstalk between immunity and the injured brain as key pathomechanism in stroke. On one hand, innate and adaptive immune cells play an important role in the fate of injured brain tissue after stroke; on the other, peripheral immune alterations are critically involved in post-stroke comorbidities. Another emerging research area is the analysis of mechanisms involved in regeneration and neuronal plasticity after stroke. Here, we discuss the current understanding of basic mechanisms involved after brain injury, clinical imaging approaches and therapeutic strategies to promote regeneration in stroke patients.

Inflammation is a hallmark in the pathogenesis of acute stroke and contributes substantially to secondary neuronal degeneration. Circulating leukocytes—particularly pro-inflammatory T cells—invading the ischemic brain contribute to post-stroke neuroinflammation and exacerbation of stroke outcome. However, a subpopulation of T cells, regulatory T cells (Treg), are potent immunosuppressant cells involved in resolution of tissue inflammation and have a key role in inflammatory diseases. Recent studies have identified a prominent role of Foxp3+ Treg cells in modulating the inflammatory response to acute brain ischemia and thereby contributing to stroke outcome. Most studies observed a neuroprotective function of Treg cells by inhibiting an overshooting immunological “collateral damage” after stroke and first Treg-targeted therapeutic strategies have been tested. This chapter provides a comprehensive overview of the current knowledge on Treg cells in experimental and clinical stroke.

Despite the efficacy of neuroprotective approaches in animal models of stroke, their translation has so far failed from bench to bedside. One reason is presumed to be a low quality of preclinical study design, leading to bias and a low a priori power. In this study, we propose that the key read-out of experimental stroke studies, the volume of the ischemic damage as commonly measured by free-handed planimetry of TTC-stained brain sections, is subject to an unrecognized low inter-rater and test-retest reliability with strong implications for statistical power and bias. As an alternative approach, we suggest a simple, open-source, software-assisted method, taking advantage of automatic-thresholding techniques. The validity and the improvement of reliability by an automated method to tMCAO infarct volumetry are demonstrated. In addition, we show the probable consequences of increased reliability for precision, p-values, effect inflation, and power calculation, exemplified by a systematic analysis of experimental stroke studies published in the year 2015. Our study reveals an underappreciated quality problem in translational stroke research and suggests that software-assisted infarct volumetry might help to improve reproducibility and therefore the robustness of bench to bedside translation.

The secondary neuroinflammatory response has come into focus of experimental stroke research. Immunological mechanisms after acute stroke are being investigated in the hope to identify novel and druggable pathways that contribute to secondary infarct growth after stroke. Among a variety of neuroimmunological events after acute brain ischemia, including microglial activation, brain leukocyte invasion, and secretion of pro-inflammatory factors, lymphocytes have been identified as the key leukocyte subpopulation driving the neuroinflammatory response and contributing to stroke outcome. Several studies have shown that pro-inflammatory lymphocyte subpopulations worsen stroke outcome and that inhibiting their invasion to the injured brain is neuroprotective. In contrast to the effector functions of pro-inflammatory lymphocytes, regulatory T cells (Treg) are critically involved in maintaining immune homeostasis and have been characterized as disease-limiting protective cells in several inflammatory conditions, particularly in primary inflammatory diseases of the central nervous system (CNS). However, due to the complex function of regulatory cells in immune homeostasis and disease, divergent findings have been described for the role of Treg in stroke models. Emerging evidence suggests that this discrepancy arises from potentially differing functions of Treg depending on the predominant site of action within the neurovascular unit and the surrounding inflammatory milieu. This article will provide a comprehensive review of current findings on Treg in brain ischemia models and discuss potential reasons for the observed discrepancies.

Cellular and humoral inflammations play important roles in ischemic brain injury. The effectiveness of immunomodulatory therapies may critically depend on the chosen experimental model. Our purpose was to compare the post-ischemic neuroinflammation among murine permanent and transient middle cerebral artery occlusion (MCAO) models. Permanent MCAO was induced by transtemporal electrocoagulation and 30 minutes or 90 minutes transient MCAO was induced by intraluminal filament in C57BL/6 mice. Infiltration of leukocyte subpopulations was quantified by immunohistochemistry and fluorescence-activated cell sorting. Cerebral cytokine and adhesion molecule expression was measured by real-time polymerase chain reaction (RT-PCR). Neutrophil infiltration was noted at 24 h after transient MCAO, but did not further increase until 5 days in the permanent MCAO model. Few T cells were observed in both MCAO models at 24 h, but permanent MCAO demonstrated much more infiltrating T cells at 5 days. Pronounced microglial activation was evident at 24 h and 5 days after permanent but not after transient MCAO. The number of invading NK cells and expression of MHCII on CD11b+ cells did not differ among the three groups. Five days after MCAO, the expression of IL-1, TNF-α and IFN-γ and of the adhesion molecules ICAM-1 and VCAM-1 was significantly higher in the permanent than in the transient MCAO groups. Cellular and humoral inflammation differs substantially among commonly used MCAO models. Neuroinflammation is more pronounced after permanent electrocoagulatory MCAO compared with 30 minutes and 90 minutes filament-MCAO.

Background and Purpose— Cerebral ischemia is ensued by a cellular immune depression syndrome. The postischemic functional capacity of T lymphocytes is controversial, and interactions between leukocyte subsets are largely unknown. Understanding the immunologic interplay between antigen-presenting cells and lymphocytes as well as between distinct lymphocyte subsets after stroke might be of clinical/therapeutic significance because animal data argue for a cerebroprotective effect of, for example, CD4+CD25+ regulatory T cells. Methods— Ex vivo CD4+ T cell proliferation was analyzed in experimental and human stroke using fluorescence activated cell sorter analysis. To investigate suppressive effects of CD4+CD25+ regulatory T cells as well as the influence of costimulatory cells on CD4+ T cell proliferation, subsets were magnetically sorted before proliferation assay setup. Results— After stroke: (1) proliferation of mouse and human CD4+ T cells on T cell receptor stimulation was unaltered; (2) the suppressive effect of CD4+CD25+ regulatory T cells in mouse and man was unaltered; and (3) efficacy of circulating costimulatory cells from stroke animals was reduced by a mean of 0.6 (SEM 0.1, P =0.001) CD4+ T cell division numbers compared with sham-treated animals. Conclusions— Reduced costimulatory efficacy of circulating costimulatory cells in mice is an important feature of stroke-induced immunodepression. Understanding the interplay of costimulatory cells and responder T cells (eg, CD4+ T cells or CD4+CD25+ regulatory T cells) after stroke may offer new insights into the prevention of secondary inflammatory damage to the brain and help to guide new therapeutic strategies.

Inflammatory cascades are increasingly recognized as an important pathophysiological mechanism in intracerebral hemorrhage (ICH). In contrast, the effect of ICH on the systemic immune system has barely been investigated. We examined the effects of different hematoma volumes on immune cell subpopulations in experimental murine ICH. In C57BL/6 mice, ICH was induced by striatal injection of autologous blood (10, 30 or 50 μL). Control animals received the respective sham operation. Three days after ICH induction, differential blood leukocyte counting was performed. Lymphocyte subpopulations were further characterized by flow cytometry in blood, spleen, lymph node and thymus. Infectious complications were studied using microbiological cultures of blood and lungs. Only after large ICH a marked decrease of leukocyte counts and most lymphocyte subsets was observed in all organs. Despite this general leukocytopenia, a significant, up to 10-fold increase, was detected in the monocyte population after extensive hemorrhage. After moderate ICH induction, only specific lymphocyte subpopulations were differentially affected. Mature thymic cells were unaffected while immature CD4+CD8+ cells were depleted by over 90% after large ICH. A significant proportion of mice with extensive ICH (36.4%) developed spontaneous pneumonia and/or bacteremia while none of the sham operated mice had infectious complications. The ICH size determines the extent of systemic immunomodulation. Large ICH predisposes animals to infections.

T lymphocytes are increasingly recognized as key modulators of detrimental inflammatory cascades in acute ischaemic stroke, but the potential of T cell-targeted therapy in brain ischaemia is largely unexplored. Here, we characterize the effect of inhibiting leukocyte very late antigen-4 and endothelial vascular cell adhesion molecule-1-mediated brain invasion-currently the most effective strategy in primary neuroinflammatory brain disease in murine ischaemic stroke models. Very late antigen-4 blockade by monoclonal antibodies improved outcome in models of moderate stroke lesions by inhibiting cerebral leukocyte invasion and neurotoxic cytokine production without increasing the susceptibility to bacterial infections. Gene silencing of the endothelial very late antigen-4 counterpart vascular cell adhesion molecule-1 by in vivo small interfering RNA injection resulted in an equally potent reduction of infarct volume and post-ischaemic neuroinflammation. Furthermore, very late antigen-4-inhibition effectively reduced the post-ischaemic vascular cell adhesion molecule-1 upregulation, suggesting an additional cross-signalling between invading leukocytes and the cerebral endothelium. Dissecting the specific impact of leukocyte subpopulations showed that invading T cells, via their humoral secretion (interferon-γ) and immediate cytotoxic mechanisms (perforin), were the principal pathways for delayed post-ischaemic tissue injury. Thus, targeting T lymphocyte-migration represents a promising therapeutic approach for ischaemic stroke.

Patients with extensive brain infarcts are at increased risk for stroke-associated respiratory tract infections (SARTI), which cause worse outcome. The benefit of general antibiotic prophylaxis is controversial. Early diagnosis of SARTI may improve patient selection for antimicrobial therapy. Procalcitonin (PCT) is widely recognized as serum marker for bacterial infections. Its diagnostic value with respect to SARTI has not been assessed systematically. Serum PCT levels were analyzed in ischemic stroke patients (n = 50) at day 1 (d1) and day 4 (d4) after stroke onset. PCT test performance was assessed by receiver operator characteristics (ROC) curve analysis. Multivariable logistic regression analysis was applied to identify early predictors for SARTI. Higher d4 serum PCT levels were associated with SARTI; ROC curve analysis revealed an area under the curve (AUC) of 0.79 (95%-confidence interval (CI) 0.61–0.96). A 0.25-ng/ml cutoff resulted in a test sensitivity and specificity of 42 and 96%, respectively. Positive (LR+) and negative (LR−) likelihood ratios were 10.8 and 0.6, respectively. In predicting SARTI, multivariable logistic regression analysis controlling for infarct volume ruled out an independent explanatory effect of serum PCT. Greater infarct volume (odds ratio (OR) 1.06, 95%-CI 1.02–1.1) prevailed as independent SARTI-predictor. In the absence of clinical signs, post-stroke screening for SARTI using serum PCT levels is not useful since test sensitivity is low. If the clinical suspicion for SARTI is strong, serum PCT-testing (>0.25 ng/ml) may improve diagnostic accuracy by improving specificity.

Systemic and local inflammatory processes have a key, mainly detrimental role in the pathophysiology of ischemic stroke. Currently, little is known about endogenous counterregulatory immune mechanisms. We examined the role of the key immunomodulators CD4(+)CD25(+) forkhead box P3 (Foxp3)(+) regulatory T lymphocytes (T(reg) cells), after experimental brain ischemia. Depletion of T(reg) cells profoundly increased delayed brain damage and deteriorated functional outcome. Absence of T(reg) cells augmented postischemic activation of resident and invading inflammatory cells including microglia and T cells, the main sources of deleterious cerebral tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), respectively. Early antagonization of TNF-alpha and delayed neutralization of IFN-gamma prevented infarct growth in T(reg) cell-depleted mice. Intracerebral interleukin-10 (IL-10) substitution abrogated the cytokine overexpression after T(reg) cell depletion and prevented secondary infarct growth, whereas transfer of IL-10-deficient T(reg) cells in an adoptive transfer model was ineffective. In conclusion, T(reg) cells are major cerebroprotective modulators of postischemic inflammatory brain damage targeting multiple inflammatory pathways. IL-10 signaling is essential for their immunomodulatory effect.

For years, low reproducibility of preclinical trials and poor translation of promising preclinical therapies to the clinic have posed major challenges to translational research in most biomedical fields. To overcome the limitations that stand between experimental and clinical research, international consortia have attempted to establish standardized guidelines for study design and for reporting the resulting data. In addition, multicenter preclinical randomized controlled trials (pRCTs) have been proposed as a suitable tool for ‘bridging the gap’ between experimental research and clinical trials. We recently reported the design and results of the first such pRCT in which we confirmed the feasibility of using a coordinated approach with standardized protocols in collaboration with independent multinational research centers. However, despite its successes, this first pRCT also had several difficulties, particularly with respect to following the protocols established in the study design and analyzing the data. Here, we review our experiences performing the study, and we analyze and discuss the lessons learned from performing the first pRCT. Moreover, we provide suggestions regarding how obstacles can be overcome to improve the performance and outcome of future pRCT studies. Translational research is hampered by low reproducibility of preclinical studies and countless failed clinical trials. International consortia have proposed preclinical multicenter trials as an intermediate step to overcome this ‘translational roadblock’. We have recently performed the first such preclinical randomized controlled trial (pRCT) by adopting key elements of clinical study design to preclinical research. In this review, we discuss the lessons learned from this trial and provide suggestions how to optimize future pRCTs. This article is part of the 60th Anniversary special issue.

Stroke is the leading cause of morbidity and mortality worldwide. Inflammatory cascades have a major impact on outcome and regeneration after ischemic stroke. High-mobility group box 1 (HMGB1) has come into the focus of experimental and clinical stroke research because it is released from necrotic brain tissue and its differential redox forms attract and activate immune cells after ischemic brain injury. HMGB1 is a potent inducer of inflammatory cascades, and thereby, secondary deterioration of neurological outcome.The role of HMGB1 in sterile inflammation is well established. Emerging evidence suggests that HMGB1 modulates neuroinflammation after experimental brain ischemia and that it may be a useful prognostic biomarker for stroke patients.HMGB1 is instantly released from necrotic cells in the ischemic core and activates an early inflammatory response. In addition, brain-released HMGB1 can be redox modified in the circulation and activate peripheral immune cells. HMGB1 concentrations correlate with disease severity and outcome after brain injury. This is the first review depicting the crucial role of HMGB1 in the initiation and perpetuation of secondary immune alterations after experimental and clinical stroke.HMGB1-dependent signaling pathways are on the verge and have the potential to become a central topic in experimental stroke research. Current and upcoming projects in this field will be paving the way for future translational approaches targeting the center of poststroke inflammation to improve stroke recovery and long-term outcome.

Gemma Llovera et al., Numerous treatments have been reported to provide a beneficial outcome in experimental animal stroke models; however, these treatments (with the exception of tissue plasminogen activator) have failed in clinical trials. To improve the translation of treatment efficacy from bench to bedside, we have performed a preclinical randomized controlled multicenter trial (pRCT) to test a potential stroke therapy under circumstances closer to the design and rigor of a clinical randomized control trial. Anti-CD49d antibodies,which inhibit the migration of leukocytes into the brain, were previously investigated in experimental strokemodels by individual laboratories. Despite the conflicting results from four positive and one inconclusive preclinical studies, a clinical trial was initiated. To confirm the preclinical results and to test the feasibility of conducting a pRCT, six independent European research centers investigated the efficacy of anti-CD49d antibodies in two distinct mouse models of stroke in a centrally coordinated, randomized, and blinded approach. The results pooled from all research centers revealed that treatment with CD49d-specific antibodies significantly reduced both leukocyte invasion and infarct volume after the permanent distal occlusion of the middle cerebral artery, which causes a small cortical infarction. In contrast, anti-CD49d treatment did not reduce lesion size or affect leukocyte invasion after transient proximal occlusion of the middle cerebral artery, which induces large lesions. These results suggest that the benefits of immune-targeted approachesmay depend on infarct severity and localization. This study supports the feasibility of performing pRCTs.

The current research topic and eBook “Mechanisms of neuroinflammation and inflammatory neurodegeneration in acute brain injury” was initiated as a reaction to the rapidly expanding literature on inflammatory mechanisms in the pathophysiology of acute brain injuries. The scope of this compilation of reviews, opinion, and original research articles was to give a broad overview of the diverse cellular compartments and mechanisms involved in the inflammatory response to brain tissue injury. Although a specific aspect of the pathophysiology of acute brain injuries, the immune system interacts in highly complex as well as diverse mechanisms with the damaged brain. On one side acute brain lesions, such as brain ischemia, hemorrhage or traumatic injury, induce a local neuroinflammatory reaction, wherein microglial cells represent the local immune cells (Benakis et al., 2015; Lourbopoulos et al., 2015). This local inflammatory response has a major impact on outcome with differential effects during the phases of post-stroke lesion evolution and recovery (Shichita et al., 2014). Intriguingly, besides abundant evidence on post-stroke neuroinflammation immunological mechanisms similar mechanisms are also observed in traumatic brain injuries (Schwarzmaier and Plesnila, 2014), intracerebral hemorrhage (Mracsko and Veltkamp, 2014) and even ethanol-induced neurotoxicity (Alfonso-Loeches et al., 2014; Sokolowski et al., 2014) or direct application of exogenous pathogens (Gullo et al., 2014) with functional consequences for neuronal outcome. Moreover, Gauberti et al. (2014) present an overview on state-of-the-art molecular magnetic resonance imaging of neuroinflammatory markers. In recent years also the molecular pathways and effector molecules of inflammation-induced neurotoxicity after acute injuries have been investigated in great detail: Murray et al. (2015) describe in their review the prominent role of the pro-inflammatory cytokine IL-1, Orsini et al. (2014) give an overview on the complement system in neuroinflammation, while Albert-Weissenberger et al. (2014a) focus on the contribution of the kallikrein-kinin system in traumatic brain injury and Zhao et al. (2014) review the current knowledge on programed death-1/programed death ligand signaling. In addition to the activation of local inflammatory pathways in the injured brain, invasion of peripheral immune cells to the brain is a critical step in secondary neuroinflammation. Gelderblom et al. (2014) review the role of gdT cells as a pro-inflammatory invariant T cell subpopulation recruited to the injured brain. In contrast, Urra et al. (2014) discuss potential mechanisms of antigen-specific autoimmunity after acute brain injury. In addition, the original research article by Kim et al. (2014) underlines that the cellular immune response to ischemic brain injury might differ substantially between commonly used mouse strains. In addition to an overview and discussion of basic mechanisms and involved pathways in secondary neuroinflammation after acute brain injury, our research topic also contains several reviews and original articles on novel therapeutic approaches to modulate the immune response. Rissiek et al. (2014) introduce nanobodies as a novel tool for targeting neuroinflammation. Brunkhorst et al. (2014) provide an overview on the promising approach of blocking cellular neuroinflammation with Fingolimod. Bodhankar et al. (2014) review the current literature on targeting the PD-L1 and PD-L2 pathways. The original article by Mouihate (2014) reports a novel role for hormonal replacement therapy in neuroinflammation and the original article by Albert-Weissenberger et al. (2014b) the use of C1-inhibitors in a cortical cryolesion model. Dotson et al. (2014) have tested the use of recombinant TCR ligand with differential effects in young and old mice (see also commentary by Pennypacker, 2014). In summary this research topic gathered contributions from the leading laboratories working in the field of secondary neuroinflammation after brain injury with nearly 100 authors from 4 continents. We are confident that this compilation covers most established and emerging research questions in this specific research field and presents an up-to-date overview on inflammatory mechanisms and drug targets in acute brain injuries.

The secondary neuroinflammatory response has attracted increasing attention in experimental stroke research.1 Analyses of immunologic mechanisms after acute stroke have been performed in the hope of identifying key pathomechanisms that contribute to secondary infarct growth and can be modulated to benefit a large proportion of patients with stroke. Among a variety of pathophysiologic mechanisms, including microglial activation, brain leukocyte invasion, and secretion of proinflammatory factors, lymphocytes have been uncovered as the key leukocyte subpopulation that determines the neuroinflammatory outcome. Several studies have shown that proinflammatory lymphocytes, such as TH1, TH17, and γδ T-cells, worsen stroke outcome, and that blocking of their brain invasion is neuroprotective.2–4 Contrary to proinflammatory lymphocytes, regulatory T-cells (Treg) and B-cells (Breg) have been characterized as disease-limiting protective cells5; in particular, in primary inflammatory diseases of the central nervous system. The discovery of the important role of Treg in other T-cell–driven pathologies has initiated productive research efforts on the role of Treg, including in ischemic brain injury, over the past years. However, because of the complex function of regulatory cells in immune homeostasis and disease, as well as partially divergent findings using different stroke models, uncertainty has emerged about the pathophysiologic function of regulatory lymphocytes in stroke.6–8 This article will comprehensively review current findings on regulatory lymphocytes in brain ischemia models and discuss potential reasons for the observed discrepancies. The immune system has evolved several regulatory mechanisms, including cell depletion, anergy, and unresponsiveness to autoantigens, to prevent damage of endogenous tissues because of an overshooting immune reaction. The presence of Treg actively suppressing autoimmune reactivity is one of the key mechanisms preserving immune homeostasis and limiting inflammatory collateral damage.9 Depletion of CD25+CD4+ Treg naturally arising in the immune system induces autoimmune diseases, and reconstitution …

In their insightful and stimulating comment on our article1 and on the persistent controversy over the pathophysiological role of regulatory T cells in general, Gauberti and Vivien address perspectives and potential pitfalls of experimental stroke research in the field of neuroinflammation. Obviously, we share their overall view that there is potential for immunomodulatory therapies, including amplification of regulatory T cells, that limit early inflammatory cytoxicity after stroke. Specifically, Gauberti and Vivien make the convincing argument that the most widely used stroke model, transient mechanical vascular occlusion of the middle cerebal artery (TMVO), may induce pathophysiological processes, including thromboinflammation, that are unlikely to play a role in other models and in the majority of strokes in patients. General scepticism toward the translational validity of the …

Background Post-stroke immune depression contributes to the development of infections which are major complications after stroke. Previous experimental and clinical studies suggested that humoral stress mediators induce immune dysfunction. However, prospective clinical studies testing this concept are missing and no data exists for other cerebrovascular diseases including intracerebral hemorrhage (ICH) and TIA. Methods We performed a prospective clinical study investigating 166 patients with TIA, ischemic and hemorrhagic stroke. We measured a broad panel of stress mediators, leukocyte subpopulations, cytokines and infection markers from hospital admission to day 7 and on follow-up after 2–3 months. Multivariate regression analyses detected independent predictors of immune dysfunction and bacterial infections. ROC curves were used to test the diagnostic value of these parameters. Results Only severe ischemic strokes and ICH increased some catecholamine metabolites, ACTH and cortisol levels. Immunodysfunction was eminent already on hospital admission after large brain lesions with lymphocytopenia as a key feature. None of the stress mediators was an independent predictor of lymphocytopenia or infections. However, lymphocytopenia on hospital admission was detected as an independent explanatory variable of later infections. NIHSSS and lymphocytopenia on admission were excellent predictors of infection. Conclusions Our results question the present pathophysiological concept of stress-hormone mediated immunodysfunction after stroke. Early lymphocytopenia was identified as an early independent predictor of post-stroke infections. Absence of lymphocytopenia may serve as a negative predictive marker for stratification for early antibiotic treatment.

Response: We appreciate the interest of Dr Moll1 in our experimental study in which we examined the effect of different hemostatic approaches on early hematoma growth in mice treated with dabigatran etexilate. In contrast to another recently published article2 using a similar experimental model, treatment with high doses of dabigatran in our study induced an excess early hematoma growth compared with nonanticoagulated mice, which we documented by serial MRI. Moreover, we showed that prothrombin complex (PCC) was the most consistently effective agent preventing the excess hematoma growth in mice treated with either 4.5 mg/kg or 9 mg/kg of dabigatran etexilate intraperitoneally. Finally, we tested the effect of 3 different doses of PCC on …

Background and Purpose— Dabigatran-etexilate (DE) recently has been approved for stroke prevention in atrial fibrillation. However, lack of effective antagonists represents a major concern in the event of intracerebral hemorrhage (ICH). The aims of the present study were to establish a murine model of ICH associated with dabigatran, and to test the efficacy of different hemostatic factors in preventing hematoma growth. Methods— In C57BL/6 mice receiving DE (4.5 or 9.0 mg/kg), in vivo and in vitro coagulation assays and dabigatran plasma levels were measured repeatedly. Thirty minutes after inducing ICH by striatal collagenase injection, mice received an intravenous injection of saline, prothrombin complex concentrate (PCC; 100 U/kg), murine fresh-frozen plasma (200 μL), or recombinant human factor VIIa (8.0 mg/kg). ICH volume was quantified on brain cryosections 24 hours later. Results— DE substantially prolonged tail vein bleeding time and ecarin clotting time for 4 hours corresponding to dabigatran plasma levels. Intracerebral hematoma expansion was observed mainly during the first 3 hours on serial T2* MRI. Anticoagulation with high doses of DE increased the hematoma volume significantly. PCC and, less consistently, fresh-frozen plasma prevented excess hematoma expansion caused by DE, whereas recombinant human factor VIIa was ineffective. Prevention of hematoma growth and reversal of tail vein bleeding time by PCC were dose-dependent. Conclusions— The study provides strong evidence that PCC and, less consistently, fresh-frozen plasma prevent excess intracerebral hematoma expansion in a murine ICH model associated with dabigatran. The efficacy and safety of this strategy must be further evaluated in clinical studies.

Background Inflammatory cascades contribute to secondary injury after intracerebral hemorrhage (ICH) via humoral factors and cell-mediated cytotoxicity. Several experimental models were previously developed to analyze post-hemorrhagic neuroinflammation. However, neuroinflammatory markers have not been compared face-to-face between these models so far, and therefore, pathophysiological conclusions drawn from only one individual model may not be valid. Methods We compared neuroinflammatory pathways in the two most common murine models: striatal injection of autologous blood or collagenase. Expression of pro- and anti-inflammatory cytokines (IL-1, TNF-α, IFN-γ, IL-6, TGF-β and IL-10) as well adhesion molecule expression (VCAM-1, ICAM-1) was analyzed by RT-PCR at several time points after ICH induction. Outcome and physiological parameters were compared between the models. Results Both models induced a profound and dynamic increase in the expression of pro-inflammatory cytokines and adhesion molecules. However, blood injection resulted in significantly more pronounced alteration of these markers than collagenase injection. This difference was associated with worse outcome after blood injection compared to the collagenase model despite equal ICH volumes. Conclusions This is the first study performing a face-to-face comparison of neuroinflammatory pathways in the two most widely used murine ICH models, revealing substantial differences between the models. This discrepancies need to be taken into account in designing future studies employing experimental ICH models, especially when analyzing neuroinflammatory pathways and therapies.

Background The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms. Methodology/Principal Findings FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls. Conclusions/Significance In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.

Background and Purpose— Therapeutic modification of the postischemic immune processes is a key target of current experimental stroke research. For successful translation into the clinical setting, experimental studies must account for the impact of different strokes on the immune system including susceptibility to infection. Herein, we characterize the impact of 3 ischemia models on systemic immunological and microbiological parameters. Methods— In C57Bl/6 mice (n=235), the middle cerebral artery was occluded (MCAO) either permanently by distal coagulation or transiently by an intraluminal filament for 30 minutes or 90 minutes. Differential leukocyte counts were performed in blood and lymphatic organs. Lymphocyte subpopulations and apoptotic cells were characterized by flow cytometry. Blood cytokine concentrations were measured by ELISA. Microbiological cultures were grown from blood and lung samples. Results— Only extensive infarcts induced leukopenia 24 hours, 3 days and 7 days after MCAO and decreased lymphocyte counts in spleen, lymph nodes and thymus. In contrast, small infarcts led to no significant changes in differential blood count or reduction of overall cell counts in lymphatic organs. Splenic lymphocyte apoptosis and blood cytokine production was significantly increased after extensive lesions compared to mild ischemia. Hypothermia and weight loss occurred only in mice with large infarcts which also suffered from pneumonia and sepsis. In contrast to infarct size, location and side of the infarct did not affect physiological parameters and immune cell alterations. Conclusions— Postischemic systemic immunomodulation and infectious complications differ substantially among stroke models. Translational studies of immunomodulatory therapies for stroke must account for this heterogeneity.