Roland Veltkamp

Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke

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 (Treg cells), after experimental brain ischemia. Depletion of Treg cells profoundly increased delayed brain damage and deteriorated functional outcome. Absence of Treg cells augmented postischemic activation of resident and invading inflammatory cells including microglia and T cells, the main sources of deleterious cerebral tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), respectively. Early antagonization of TNF-α and delayed neutralization of IFN-γ prevented infarct growth in Treg cell–depleted mice. Intracerebral interleukin-10 (IL-10) substitution abrogated the cytokine overexpression after Treg cell depletion and prevented secondary infarct growth, whereas transfer of IL-10–deficient Treg cells in an adoptive transfer model was ineffective. In conclusion, Treg cells are major cerebroprotective modulators of postischemic inflammatory brain damage targeting multiple inflammatory pathways. IL-10 signaling is essential for their immunomodulatory effect.

Inhibition of lymphocyte trafficking shields the brain against deleterious neuroinflammation after stroke

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.

Infarct Volume is a Major Determiner of Post-Stroke Immune Cell Function and Susceptibility to Infection

Background and Purpose— Acute ischemic stroke in humans is associated with profound alterations in the immune system. Hallmarks of this stroke-induced immunodepression syndrome are: lymphocytopenia, impairment of T helper cell and monocyte function. We studied which stroke-specific factors predict these immunologic alterations and subsequent infections. Methods— Leukocyte/lymphocyte subsets were assessed serially by white blood cell count and fluorescence-activated cell sorter analysis in ischemic stroke patients (n=50) at baseline, day 1, and day 4 after stroke onset and compared to an age-matched control group (n=40). Concomitantly, monocytic human leukocyte antigen-DR expression and the in vitro function of blood monocytes measured by the production of tumor necrosis factor-&agr; upon stimulation with lipopolysaccharide were assessed. Associations of these immunologic parameters with stroke specific factors (National Institutes of Health Stroke Scale, infarct size) were explored. Multivariable logistic regression analysis was applied to identify early predictors for poststroke respiratory and urinary tract infections. Results— Infarct volume was the main factor associated with lymphocytopenia on day 1 and day 4 poststroke. Particularly, blood natural killer cell counts were reduced after stroke. Monocyte counts increased after ischemia paralleled by a profound deactivation predominantly after extensive infarcts. Reduced T helper cell counts, monocytic human leukocyte antigen-DR expression, and monocytic in vitro production of tumor necrosis factor-&agr; were associated with infections in univariate analyses. However, only stroke volume prevailed as independent early predictor for respiratory infections (OR 1.03; CI 1.01 to 1.04). Conclusions— Infarct volume determines the extent of lymphocytopenia, monocyte dysfunction, and is a main predictor for subsequent infections.

Hyperbaric Oxygen Reduces Blood–Brain Barrier Damage and Edema After Transient Focal Cerebral Ischemia

Background and Purpose— Hyperbaric oxygen (HBO) has been shown to protect the brain parenchyma against transient focal cerebral ischemia, but its effects on the ischemic microcirculation are largely unknown. We examined the potential of HBO to reduce postischemic blood–brain barrier (BBB) damage and edema. Methods— Wistar rats and C57/BL6 mice underwent occlusion of the middle cerebral artery (MCAO) for 2 hours. Forty minutes after filament introduction, animals breathed either 100% O2 at 3.0 atmospheres absolute (ata; HBO group) or at 1.0 ata (control) for 1 hour in an HBO chamber. In rats, MRI was performed 15 minutes after MCAO and after 15 minutes and 3, 6, 24, and 72 hours of reperfusion. In mice, BBB permeability for sodium fluorescein was measured after 24-hour reperfusion. Results— Increased BBB permeability on postcontrast T1-weighted (T1w) images had a biphasic pattern. HBO reduced volumes and intensity of enhancement. Mean abnormal enhancing volumes were 71±10 mm3 (control) versus 47±10 mm3 (HBO) at 15 minutes; 111±21 mm3 versus 69±17 mm3 3 hours; 147±44 mm3 versus 83±21 mm3 6 hours; 150±37 mm3 versus 89±14 mm3 24 hours; and 322±52 mm3 versus 215±21 mm3 72 hours (all P<0.05). Interhemispheric quotients of mean gray values on T1w were at 1.73±0.11 versus 1.57±0.07 15 minutes; 1.74±0.07 versus 1.60±0.06 at 3 hours; 1.77±0.07 versus 1.62±0.06 at 6 hours; 1.79±0.10 versus 1.60±0.05 at 24 hours; and 1.81±0.10 versus 1.62±0.07 at 72 hours (all P<0.05). HBO-treated mice had significantly lower postischemic BBB permeability than mice treated with either normobaric hyperoxia or room air. Vasogenic edema assessed on T2w images and histologic sections was significantly lower in HBO-treated rats. Conclusions— Intraischemic HBO therapy reduces early and delayed postischemic BBB damage and edema after focal ischemia in rats and mice.

Postischemic brain infiltration of leukocyte subpopulations differs among murine permanent and transient focal cerebral ischemia models.

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 30u2009minutes or 90u2009minutes 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 24u2009h 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 24u2009h, but permanent MCAO demonstrated much more infiltrating T cells at 5 days. Pronounced microglial activation was evident at 24u2009h 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 30u2009minutes and 90u2009minutes filament‐MCAO.

Boosting Regulatory T Cells Limits Neuroinflammation in Permanent Cortical Stroke

Inflammatory mechanisms contribute substantially to secondary tissue injury after brain ischemia. Regulatory T cells (Tregs) are key endogenous modulators of postischemic neuroinflammation. We investigated the potential of histone deacetylase inhibition (HDACi) to enhance Treg potency for experimental stroke in mice. HDACi using trichostatin A increased the number of Tregs and boosted their immunosuppressive capacity and interleukin (IL)-10 expression. In vivo treatment reduced infarct volumes and behavioral deficits after cortical brain ischemia, attenuated cerebral proinflammatory cytokine expression, and increased numbers of brain-invading Tregs. A similar effect was obtained using tubastatin, a specific inhibitor of HDAC6 and a key HDAC in Foxp3 regulation. The neuroprotective effect of HDACi depended on the presence of Foxp3+ Tregs, and in vivo and in vitro studies showed that the anti-inflammatory cytokine IL-10 was their main mediator. In summary, modulation of Treg function by HDACi is a novel and potent target to intervene at the center of neuroinflammation. Furthermore, this novel concept of modulating endogenous immune mechanisms might be translated to a broad spectrum of diseases, including primary neuroinflammatory and neurodegenerative disorders.

FTY720 Reduces Post-Ischemic Brain Lymphocyte Influx but Does Not Improve Outcome in Permanent Murine Cerebral Ischemia

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.

Oxygen therapy reduces secondary hemorrhage after thrombolysis in thromboembolic cerebral ischemia

Hyperbaric oxygen (HBO) and normobaric hyperoxia (NBO) protect the brain parenchyma and the cerebral microcirculation against ischemia. We studied their effect on secondary hemorrhage after thrombolysis in two thromboembolic middle cerebral artery occlusion (MCAO) (tMCAO) models. Beginning 60 minutes after tMCAO with either thrombin-induced thromboemboli (TT) or calcium-induced thromboemboli (CT), spontaneously hypertensive rats (n=96) breathed either air, 100% O2 (NBO), or 100% O2 at 3 bar (HBO) for 1 hour. Immediately thereafter, recombinant tissue plasminogen activator (rt-PA, 9 mg/kg) was injected. Although significant reperfusion was observed after thrombolysis in TT-tMCAO, vascular occlusion persisted in CT-tMCAO. In TT-tMCAO, NBO and HBO significantly reduced diffusion-weighted imaging–magnetic resonance imaging (MRI) lesion volume and postischemic blood–brain barrier (BBB) permeability on postcontrast T1-weighted images. NBO and, significantly more potently, HBO reduced macroscopic hemorrhage on T2* MRI and on corresponding postmortem cryosections. Oxygen therapy lowered hemoglobin content and attenuated activation of matrix metalloproteinases in the ischemic hemisphere. In contrast, NBO and HBO failed to reduce infarct size in CT but both decreased BBB damage and microscopic hemorrhagic transformation. Only HBO reduced hemoglobin extravasation in the ischemic hemisphere. In conclusion, NBO and HBO decrease infarct size after thromboembolic ischemia only if recanalization is successful. As NBO and HBO also reduce postthrombolytic intracerebral hemorrhage, combining the two with thrombolysis seems promising.

Detection of Paroxysmal Atrial Fibrillation in Acute Stroke Patients

Background: Atrial fibrillation (AF) is a frequent cause of stroke, but detecting paroxysmal AF (pAF) poses a challenge. We investigated whether continuous bedside ECG monitoring in a stroke unit detects pAF more sensitively than 24-hour Holter ECG, and tested whether examining RR interval dynamics on short-term ECG recordings using an automated screening algorithm (ASA) for pAF detection is a useful tool to predict the risk of pAF outside periods of manifest AF. Methods: Patients >60 years with acute ischemic stroke or transient ischemic attacks (TIA) were prospectively enrolled unless initial ECG revealed AF or they had a history of paroxysmal or persistent AF. ASA was performed on 1- to 2-hour ECG recordings in the emergency room and patients were classified into 5 risk categories for pAF. All patients underwent continuous bedside ECG monitoring for >48 h. Additionally, 24-hour Holter ECG was performed. Results: 136 patients were enrolled (median age: 72 years, male: 58.8%). In 29 (21.3%), pAF was newly diagnosed by continuous bedside ECG monitoring. pAF increased with age (p = 0.031). Median time to first pAF detection on continuous bedside ECG monitoring was 36 h. In 16 patients, pAF was detected by continuous bedside ECG monitoring prior to the performance of 24-hour Holter ECG. Thirteen of the remaining patients were pAF positive on continuous bedside ECG monitoring, but 24-hour Holter detected only 3 patients. Accordingly, the sensitivity of 24-hour Holter was 0.23. Sensitivity of higher-risk categories of ASA compared to continuous bedside ECG monitoring was 0.72, and specificity 0.63. Conclusion: Continuous bedside ECG monitoring is more sensitive than 24-hour Holter ECG for pAF detection in acute stroke/TIA patients. Screening patients for pAF outside AF episodes using ASA requires further development.

Paroxysmal Atrial Fibrillation Is More Prevalent than Persistent Atrial Fibrillation in Acute Stroke and Transient Ischemic Attack Patients

Background: Atrial fibrillation (AF) is a common cause of ischemic stroke and transient ischemic attack (TIA). More extensive diagnostic effort is required to detect paroxysmal AF (pxAF) than persistent AF (pAF); the prevalence of pxAF in stroke patients is unknown. We evaluated the prevalence of pAF and pxAF in ischemic stroke and TIA patients. Methods: Consecutive patients with acute ischemic stroke/TIA were enrolled prospectively. We aimed to detect patients with a history of AF, with AF newly diagnosed in the emergency room (ER), or with newly diagnosed AF during a 3-month period following the event. Differences in the frequency of AF diagnosis with respect to the disposition of patients after ER work-up were assessed. Results: A total of 692 patients were enrolled (male: 52.2%; ischemic stroke: 69.1%; TIA: 30.9%). A previously documented history of AF was present in 19.7% (pAF: 47.1%, pxAF: 52.9%). In 3.8% of patients, AF was newly diagnosed in the ER (pxAF: 61.5%) and in 5.2% during the 3-month follow-up period. The overall prevalence of AF was 28.6% (pxAF: 62.6%). Previously documented pxAF evaded diagnosis at ER presentation in 48.6%. The prevalence of AF increased with age (p < 0.001). Patients with pxAF were younger than those with pAF (p = 0.004) and more often female (p = 0.05). The presence of any AF was associated with higher initial NIHSS scores (p < 0.001) and higher modified Rankin scores after 3 months (p < 0.001). Conclusion: pxAF occurs more often than pAF in stroke/TIA patients. As effective stroke prevention is available for AF, it is important to develop and evaluate sensitive methods for detecting pxAF.