L-selectin inhibition does not reduce injury in a rabbit model of transient focal cerebral ischemia

Neutrophils are known to mediate injury in acute ischemic stroke especially during reperfusion. Migration of neutrophils into regions of ischemic injury involves binding to the endothelial cells via interactions with various adhesion molecules. One adhesion molecule, L-selectin, is found on the surface of leukocytes, and is shed prior to leukocyte infiltration. We studied whether a humanized antibody to L-selectin (HuDREG200) might limit ischemic injury in an experimental stroke model. New Zealand White rabbits underwent transorbital occlusion of the left middle cerebral, anterior cerebral and internal carotid arteries using aneurysm clips for 2 h followed by 6 h of reperfusion. Treatment with a saturating dose (4 mg kg-1) of HuDREG200 (n = 8) or vehicle (n = 8) was administered 20 min after occlusion and given as a single i.v. bolus. Hemispheric ischemic neuronal damage (IND) as seen on hematoxylin and eosin stained sections was no different between groups (HuDREG200, 23.3% +/- 6%; vehicle, 19.6% +/- 6%; mean +/- SEM, n.s., t-test). Immunohistochemical staining with neutrophil elastase confirmed the presence of neutrophils within regions of IND in control brains, but treatment did not alter their numbers within ischemic tissue. We conclude that antagonism of neutrophil adhesion at the level of L-selectin does not alter ischemic injury in experimental stroke.     

白介素17、白介素33在颅内动脉粥样硬化斑块中的作用及在缺血性卒中的展望

颅内血管粥样硬化会导致脑血管病,在颅内血管斑块的形成过程中以及缺血后的脑损伤 中,炎症反应都起到至关重要的作用。近年来研究证实,白介素-17（interleukin-17,IL-17）与受体结合 激活通路促进动脉粥样硬化斑块的形成,并且加重脑缺血组织的损伤,而白介素-33（interleukin-33, IL-33）在缺血性卒中通过I L-33/ST2L信号通路参与了颅内动脉硬化斑块的形成过程,且被证实是一 种保护因素。本文总结了在颅内大血管硬化斑块的形成过程中IL-17与IL-33之间的相互影响。其各自 水平的高低可反映疾病的严重程度,提出其对缺血性卒中的临床治疗及预示预后转归具有重要意义。     

Inducible nitric oxide synthase expression in human cerebral infarcts

The inducible or “immunological” isoform of nitric oxide synthase (iNOS) is induced in many cell types by inflammatory stimuli and synthesizes toxic amounts of NO. In rodent models of focal cerebral ischemia, iNOS is expressed in neutrophils invading the injured brain and in local blood vessels. Studies with iNOS inhibitors and iNOS null mice indicate that NO produced by iNOS contributes to ischemic brain injury. In the present study, we sought to determine whether iNOS is also expressed in the human brain after ischemic stroke. Studies were conducted using immunohistochemistry on autopsy brains with neuropathological evidence of acute cerebral infarction. iNOS immunoreactivity was observed in neutrophils infiltrating the ischemic brain and in blood vessels within the ischemic territory. iNOS-positive cells also were immunoreactive for nitrotyrosine, reflecting protein nitration by NO-derived peroxynitrite and nitrites. iNOS or nitrotyrosine immunoreactivity was not detected outside the region of the infarct. These observations provide evidence that iNOS is expressed in the human brain after ischemic infarction and support the hypothesis that iNOS inhibitors may be useful in the treatment of ischemic stroke in humans. Received: 8 June 1998 / Revised, accepted: 23 September 1998     

Pathophysiology and Neuroprotection of Global and Focal Perinatal Brain Injury: Lessons From Animal Models

BackgroundArterial ischemic stroke occurs more frequently in term newborns than in the elderly, and brain immaturity affects mechanisms of ischemic injury and recovery. The susceptibility to injury of the brain was assumed to be lower in the perinatal period as compared with childhood. This concept was recently challenged by clinical studies showing marked motor disabilities after stroke in neonates, with the severity of motor and cortical sensory deficits similar in both perinatal and childhood ischemic stroke. Our understanding of the triggers and the pathophysiological mechanisms of perinatal stroke has greatly improved in recent years, but many factors remain incompletely understood.MethodsIn this review, we focus on the pathophysiology of perinatal stroke and on therapeutic strategies that can protect the immature brain from the consequences of stroke by targeting inflammation and brain microenvironment.ResultsStudies in neonatal rodent models of cerebral ischemia have suggested a potential role for soluble inflammatory molecules as important modulators of injury and recovery. A great effort is underway to investigate neuroprotective molecules based on our increasing understanding of the pathophysiology.ConclusionIn this review, we provide a comprehensive summary of new insights concerning pathophysiology of focal and global perinatal brain injury and their implications for new therapeutic approaches.     

Inflammatory cell infiltration after endothelin-1-induced cerebral ischemia: histochemical and myeloperoxidase correlation with temporal changes in brain injury.

Accumulation of neutrophils in brain after transient focal stroke remains controversial with some studies showing neutrophils to be deleterious, whereas others suggest neutrophils do not contribute to ischemic injury. Myeloperoxidase (MPO) has been used extensively as a marker for quantifying neutrophil accumulation, but is an indirect method and does not detect neutrophils alone. To elucidate the interaction of macrophages in the neutrophil inflammatory response, we conducted double-label immunofluorescence in brain sections at 0, 1, 2, 3, 7, and 15 days after ischemia. Each of these results was obtained from the same animal to determine correlations between neutrophil infiltration and ischemic damage. It was found that MPO activity increased up to 3 days after cerebral ischemia. Dual-staining revealed that macrophages engulf neutrophils in the brain and that this engulfment of neutrophils increased with time, with 50% of neutrophils in the brain engulfed at 3 days and approximately 85% at 15 days (N=5, P<0.05). Interestingly, at 7 days the amount of dual-staining was decreased to 20% (N=5, P<0.05). Neutrophil infiltration was positively correlated with ischemic damage in both the cortex and striatum (r(2)=0.86 and 0.80, respectively, P<0.01). The results of this study indicate that the MPO from neutrophils phagocytized by macrophages may continue to contribute to the overall MPO activity, and that previous assessments that have utilized this marker to measure neutrophil accumulation may have mis-calculated the number of neutrophils within the ischemic territory and hence their contribution to the evolution of the infarct at later time points. Thus any biphasic infiltration of neutrophils may have been masked by the accumulation of macrophages.     

L-selectin inhibition does not reduce injury in a rabbit model of transient focal cerebral ischemia

Abstract

Neutrophils are known to mediate injury in acute ischemic stroke especially during reperfusion. Migration of neutrophils into regions of ischemic injury involves binding to the endothelial cells via interactions with various adhesion molecules. One adhesion molecule, L-selectin, is found on the surface of leukocytes, and is shed prior to leukocyte infiltration. We studied whether a humanized antibody to L-selectin (HuDREG200) might limit ischemic injury in an experimental stroke model. New Zealand White rabbits underwent transorbital occlusion of the left middle cerebral, anterior cerebral and internal carotid arteries using aneurysm clips for 2 h followed by 6 h of reperfusion. Treatment with a saturating dose (4 mg kg^-1) of HuDREG200 (n = 8) or vehicle (n = 8) was administered 20 min after occlusion and given as a single i.v. bolus. Hemispheric ischemic neuronal damage (IND) as seen on hematoxylin and eosin stained sections was no different between groups (HuDREG200, 23.3% ± 6%; vehicle, 19.6% ± 6%; mean ± SEM, n.s., t-test). Immunohistochemical staining with neutrophil elastase confirmed the presence of neutrophils within regions of IND in control brains, but treatment did not alter their numbers within ischemic tissue. We conclude that antagonism of neutrophil adhesion at the level of L-selectin does not alter ischemic injury in experimental stroke. [Neurol Res 2001; 23: 72-78]     

Treatment with Evasin-3 reduces atherosclerotic vulnerability for ischemic stroke,but not brain injury in mice

Neutrophilic inflammation might have a pathophysiological role in both carotid plaque rupture and ischemic stroke injury. Here, we investigated the potential benefits of the CXC chemokine-binding protein Evasin-3, which potently inhibits chemokine bioactivity and related neutrophilic inflammation in two mouse models of carotid atherosclerosis and ischemic stroke, respectively. In the first model, the chronic treatment with Evasin-3 as compared with Vehicle (phosphate-buffered saline (PBS)) was investigated in apolipoprotein E-deficient mice implanted of a ‘cast'' carotid device. In the second model, acute Evasin-3 treatment (5 minutes after cerebral ischemia onset) was assessed in mice subjected to transient left middle cerebral artery occlusion. Although CXCL1 and CXCL2 were upregulated in both atherosclerotic plaques and infarcted brain, only CXCL1 was detectable in serum. In carotid atherosclerosis, treatment with Evasin-3 was associated with reduction in intraplaque neutrophil and matrix metalloproteinase-9 content and weak increase in collagen as compared with Vehicle. In ischemic stroke, treatment with Evasin-3 was associated with reduction in ischemic brain neutrophil infiltration and protective oxidants. No other effects in clinical and histological outcomes were observed. We concluded that Evasin-3 treatment was associated with reduction in neutrophilic inflammation in both mouse models. However, Evasin-3 administration after cerebral ischemia onset failed to improve poststroke outcomes.     

Stroke and T-cells

The microvasculature of the brain region affected by a stroke assumes an inflammatory phenotype that is characterized by endothelial cell activation and barrier dysfunction and the recruitment of adherent leukocytes. Although most attention has been devoted to the possible role of neutrophils in the tissue responses to ischemic stroke there is evidence that T-lymphocytes also accumulate in the postischemic brain. Although comparable detailed analyses of lymphocyte involvement in ischemic brain injury have not been performed, emerging findings suggest a role for T-cells in the pathogenesis of ischemic stroke. The recruitment of T-cells to the site of brain injury is critically dependent on the coordinated expression of adhesion molecules on the activated capillary endothelium. Whether the recruited lymphocytes are acting directly on brain tissue or indirectly through activation of other circulating blood cells and/or extravascular cells remain unclear. Cytotoxic CD8+ T-cells may induce brain injury through molecules released from their cytotoxic granules. CD4+ T-helper 1 (TH1) cells, which secrete proinflammatory cytokines, including interleukin-2 (IL-2), IL-12, interferon-gamma, and tumor necrosis factor-alpha, may play a key role in the pathogenesis of stroke, whereas CD4+TH2 cells may play a protective role through anti-inflammatory cytokines such as IL-4, IL-5, IL-10, and IL-13. T-cells should be considered as therapeutic targets for ischemic stroke. However, because infection is a leading cause of mortality in the postacute phase of ischemic stroke, and considering anti-inflammatory role of CD4+TH2, treatment targeting T-cells should be carefully designed to reduce deleterious and enhance protective actions of T-cells.     

Cerebrovascular complications in patients with cancer

Stroke in the cancer patient is most often caused by disorders of coagulation that are induced by the cancer, by cancer metastatic to the central nervous system, or by coagulation disorders or vascular injury induced by cancer therapy. Nonbacterial thrombotic endocarditis with diffuse thrombosis of cerebral vessels is often the cause of cerebral infarction. Venous occlusion is most common in leukemic patients but can also result from growth of solid tumor in the adjacent skull or dura. Chemotherapy administration is associated with a small risk of cerebral arterial or venous thrombosis. Radiation that is administered to the neck can result in delayed carotid atherosclerosis. Tumor embolization to the brain is a rare cause of stroke. Fungal septic cerebral emboli occur most commonly in leukemic patients who have undergone bone marrow transplant. Hemorrhages occur in the brain parenchyma or subdural space and are most commonly caused by acute disseminated intravascular coagulation or metastatic tumor. Hemolysis from chemotherapy administration is a rare cause of brain hemorrhage. Neuroimaging studies, measurement of coagulation function, and echocardiography are the must useful modalities to identify the cause of stroke.     

Pathogenic mechanisms following ischemic stroke

Stroke is the second most common cause of death and the leading cause of disability worldwide. Brain injury following stroke results from a complex series of pathophysiological events including excitotoxicity, oxidative and nitrative stress, inflammation, and apoptosis. Moreover, there is a mechanistic link between brain ischemia, innate and adaptive immune cells, intracranial atherosclerosis, and also the gut microbiota in modifying the cerebral responses to ischemic insult. There are very few treatments for stroke injuries, partly owing to an incomplete understanding of the diverse cellular and molecular changes that occur following ischemic stroke and that are responsible for neuronal death. Experimental discoveries have begun to define the cellular and molecular mechanisms involved in stroke injury, leading to the development of numerous agents that target various injury pathways. In the present article, we review the underlying pathophysiology of ischemic stroke and reveal the intertwined pathways that are promising therapeutic targets.     

Neuroprotective properties of statins in cerebral ischemia and stroke.

BACKGROUND: The atheroma-retarding properties of beta-hydroxy-beta-methylglutaryl coenzyme A reductase (HMG-CoA) inhibitors, or "statins," in both the coronary and carotid arterial beds are well established. However, a growing body of recent data suggests that statins possess important adjunctive properties that may confer additional benefit beyond the retardation of atherosclerosis. In this article, we review the emerging evidence that statins have beneficial effects within the cerebral circulation and brain parenchyma during ischemic stroke and reperfusion. SUMMARY OF REVIEW: Clinical studies show that statins reduce the incidence of ischemic stroke through probable effects on precerebral atherosclerotic plaque and through antithrombotic mechanisms. Additionally, statins have been shown to reduce infarct size in experimental animal models of stroke. Statins both upregulate endothelial nitric oxide synthase (eNOS) and inhibit inducible nitric oxide synthase (iNOS), effects that are potentially neuroprotective. The preservation of eNOS activity in cerebral vasculature, particularly in the ischemic penumbra, may be especially important in preserving blood flow and limiting neurological loss. Statins may also attenuate the inflammatory cytokine responses that accompany cerebral ischemia, and they possess antioxidant properties that likely ameliorate ischemic oxidative stress in the brain. CONCLUSIONS: In addition to reducing stroke, the statin class of drugs exhibits a number of important neuroprotective properties that likely attenuate the effects of ischemia on the brain vasculature and parenchyma. Further investigation of the role of statins in human neuroprotection by use of neuroimaging and cognitive studies is warranted to explore these preliminary observations. In addition to reducing ischemic stroke, early evidence indicates that statins may also be neuroprotective.     

MicroRNA‐365 modulates astrocyte conversion into neuron in adult rat brain after stroke by targeting Pax6

Reactive astrocytes induced by ischemia can transdifferentiate into mature neurons. This neurogenic potential of astrocytes may have therapeutic value for brain injury. Epigenetic modifications are widely known to involve in developmental and adult neurogenesis. PAX6, a neurogenic fate determinant, contributes to the astrocyte‐to‐neuron conversion. However, it is unclear whether microRNAs (miRs) modulate PAX6‐mediated astrocyte‐to‐neuron conversion. In the present study we used bioinformatic approaches to predict miRs potentially targeting Pax6, and transient middle cerebral artery occlusion (MCAO) to model cerebral ischemic injury in adult rats. These rats were given striatal injection of glial fibrillary acidic protein targeted enhanced green fluorescence protein lentiviral vectors (Lv‐GFAP‐EGFP) to permit cell fate mapping for tracing astrocytes‐derived neurons. We verified that miR‐365 directly targets to the 3′‐UTR of Pax6 by luciferase assay. We found that miR‐365 expression was significantly increased in the ischemic brain. Intraventricular injection of miR‐365 antagomir effectively increased astrocytic PAX6 expression and the number of new mature neurons derived from astrocytes in the ischemic striatum, and reduced neurological deficits as well as cerebral infarct volume. Conversely, miR‐365 agomir reduced PAX6 expression and neurogenesis, and worsened brain injury. Moreover, exogenous overexpression of PAX6 enhanced the astrocyte‐to‐neuron conversion and abolished the effects of miR‐365. Our results demonstrate that increase of miR‐365 in the ischemic brain inhibits astrocyte‐to‐neuron conversion by targeting Pax6, whereas knockdown of miR‐365 enhances PAX6‐mediated neurogenesis from astrocytes and attenuates neuronal injury in the brain after ischemic stroke. Our findings provide a foundation for developing novel therapeutic strategies for brain injury.     

Neonatal arterial ischemic stroke and cerebral sinovenous thrombosis are more commonly diagnosed in boys

The risk factors for arterial ischemic stroke and cerebral sinovenous thrombosis in neonates are not well understood. We looked at gender, birthweight, and gestational age in neonates with arterial ischemic stroke and cerebral sinovenous thrombosis to see if there were trends suggesting that these were risk factors. We identified neonates with a gestational age at birth > or = 36 weeks and a diagnosis of arterial ischemic stroke or cerebral sinovenous thrombosis made by computed tomography or magnetic resonance imaging during the neonatal period from a consecutive cohort study of children with arterial ischemic stroke and cerebral sinovenous thrombosis in Ontario. Data on gender, birthweight, and gestational age were obtained by health record review. Sixty-six children with neonatal arterial ischemic stroke were identified. Forty-one (62.1%; 95% CI 49.3-73.8%) were male. Thirty-two children with neonatal cerebral sinovenous thrombosis were identified. Twenty-five (78.1%; 95% CI 60.0-90.7%) were male. One male child was identified with both arterial ischemic stroke and cerebral sinovenous thrombosis. There was a trend toward higher than average birthweights among neonates with arterial ischemic stroke and a trend toward older gestational age in female neonates with arterial ischemic stroke. Our data suggest that neonatal arterial ischemic stroke and cerebral sinovenous thrombosis are more commonly diagnosed in boys. The slightly larger size of male neonates may be contributory in arterial ischemic stroke. It is not known whether boys are at higher risk of developing arterial ischemic stroke and cerebral sinovenous thrombosis or are simply more likely to present with symptoms resulting in diagnosis. These issues need further study.     

Modification of neutrophil adhesion to human endothelial cell line in acute ischemic stroke by dipyridamole and candesartan

Ischemic stroke is a leading cause of disability. Inflammation of the vessel wall following neutrophil adhesion to vascular endothelium may contribute to ischemic damage. We studied the effect of a platelet inhibitor and an angiotensin II receptor antagonist: alone or in combination, on the adhesion of neutrophils to endothelial cell line in stroke patients. Neutrophils were collected from 12 patients with ischemic stroke within 48 h. Six patients with previous stroke and six healthy volunteers served as control. Neutrophils were incubated with dipyridamole, candesartan or both and allowed to adhere to human endothelial cell line (ECV-304). Adhesion and expression of adhesion molecules (AM) were determined using fluorescence-activated cell-sorting (FACS). Dipyridamole and the combination of dipyridamole and candesartan inhibited significantly the adhesion of neutrophils from ischemic stroke patients as compared to controls with a prominent additive effect. No inhibition was seen in the control groups. These drugs also reduced significantly the expression of the AM Mac-1. Both candesartan and dipyridamole inhibited the adhesion of neutrophils to vascular endothelium in ischemic stroke patients but not in chronic stroke patients or healthy persons. This effect may be related to specific downregulation of Mac-1 by these drugs or other intracellular events.     

Cryptogenic embolic stroke in a girl with a trisomy 21 mosaic

Stroke in trisomy 21 may be due to cardioembolism, atherosclerosis, vasculitis, moyamoya disease, sinus venous thrombosis, internal carotid hypoplasia or infections like endocarditis with septic emboli, meningitis or brain abscess. In rare cases, however, stroke etiology remains unexplained. We present a 19 year old Caucasian girl with trisomy 21 with a 47XX+21 karyotype who suffered at age 11?years from a transient ischemic attack with left hemiparesis, and at age 17?years from an ischemic stroke in the territory of the right cerebral medial artery. She suffered from arterial hypertension, obesity and hypercholesterolemia. Since blood coagulation studies, immunologic parameters, blood cultures, 24-h Holter monitoring, transthoracic and transesophageal echocardiography, magnetic resonance angiography of the extra- and intracranial vessels, thoracic and abdominal aorta and renal arteries did not provide any explanation for the stroke, implantation of a loop recorder is considered in order to detect episodes of clinically silent atrial fibrillation.     

NLRP3 deficiency ameliorates neurovascular damage in experimental ischemic stroke

Although the innate immune response to induce postischemic inflammation is considered as an essential step in the progression of cerebral ischemia injury, the role of innate immunity mediator NLRP3 in the pathogenesis of ischemic stroke is unknown. In this study, focal ischemia was induced by middle cerebral artery occlusion in NLRP3^−/−, NOX2^−/−, or wild-type (WT) mice. By magnetic resonance imaging (MRI), Evans blue permeability, and electron microscopic analyses, we found that NLRP3 deficiency ameliorated cerebral injury in mice after ischemic stroke by reducing infarcts and blood–brain barrier (BBB) damage. We further showed that the contribution of NLRP3 to neurovascular damage was associated with an autocrine/paracrine pattern of NLRP3-mediated interleukin-1β (IL-1β) release as evidenced by increased brain microvessel endothelial cell permeability and microglia-mediated neurotoxicity. Finally, we found that NOX2 deficiency improved outcomes after ischemic stroke by mediating NLRP3 signaling. This study for the first time shows the contribution of NLRP3 to neurovascular damage and provides direct evidence that NLRP3 as an important target molecule links NOX2-mediated oxidative stress to neurovascular damage in ischemic stroke. Pharmacological targeting of NLRP3-mediated inflammatory response at multiple levels may help design a new approach to develop therapeutic strategies for prevention of deterioration of cerebral function and for the treatment of stroke.     

Polymorphonuclear leukocytes and monocytes/macrophages in the pathogenesis of cerebral ischemia and stroke.

BACKGROUND: The extent to which polymorphonuclear leukocytes and monocytes/macrophages contribute to the pathobiology of cerebral ischemia and stroke is an issue of long-standing contradiction and controversy. Recent developments in the ability to selectively modify leukocyte adhesion with antiadhesion antibodies and the potential clinical application of this therapeutic approach have spurred a resurgence of experimental studies examining the role of leukocytes in cerebral ischemia and stroke. SUMMARY OF REVIEW: We review studies examining leukocyte accumulation, initiation of thrombosis, and exacerbation of ischemic brain injury in stroke, and we examine other proposed contributions of leukocytes to cerebrovascular pathophysiology. CONCLUSIONS: The importance of specific characteristics of a given ischemia model and of underlying stroke risk factors in determining the degree of leukocyte involvement and effectiveness of therapies directed against these cells is discussed.     

Targeting neutrophils as a novel therapeutic strategy after stroke

Stroke is followed by an intricate immune interaction involving the engagement of multiple immune cells, including neutrophils. As one of the first responders recruited to the brain, the crucial roles of neutrophils in the ischemic brain damage are receiving increasing attention in recent years. Notably, neutrophils are not homogenous, and yet there is still a lack of full knowledge about the extent and impact of neutrophil heterogeneity. The biological understanding of the neutrophil response to both innate and pathological conditions is rapidly evolving as single-cell-RNA sequencing uncovers overall neutrophil profiling across maturation and differentiation contexts. In this review, we scrutinize the latest research that points to the multifaceted role of neutrophils in different conditions and summarize the regulatory signals that may determine neutrophil diversity. In addition, we list several potential targets or therapeutic strategies targeting neutrophils to limit brain damage following ischemic stroke.     

Lack of Evidence for Neutrophil Participation during Infarct Formation Following Focal Cerebral Ischemia in the Rat

The involvement of neutrophils in the pathogenesis of cerebral ischemic injury in two rat models of focal ischemia was investigated. In Experiment I, a model of focal ischemia with partial reperfusion was used. Although significant and discrete ischemic damage within the neocortex was nearly maximal at 12 h postocclusion, no elevation in neutrophils was seen at this time point. Even after 21 h postocclusion, only a subtle increase in neutrophils within the ischemic tissue was observed. To further investigate the possible role of neutrophils in cerebral ischemia, the effect of cyclophosphamide-induced neutropenia was investigated (Experiment II). While a marked reduction (>98%) in systemic neutrophils was achieved in advance of and during the ischemic challenge, no reduction in the volume of ischemic damage was observed. In Experiment III, variations in the rat model of focal ischemia were made to produce a larger area of ischemic damage, as well as to permit complete reperfusion of blood to the affected cortex. While more neutrophils were seen in this variation of the model, very few were observed (<1 per field) prior to the time that maximal ischemic damage had already occurred. Together, these experiments revealed that substantial brain necrosis occurred prior to the appearance of neutrophils, under conditions of partial, as well as complete, reperfusion. Moreover, at the time points when elevations in neutrophils were observed, no further increase in volume of ischemic damage was noted. Finally, pharmacologic removal of neutrophils prior to ischemia did not alter the size of the ischemic region. These data therefore fail to support the hypothesis that neutrophils play a general and essential role in infarct formation following focal brain ischemia and argue that further studies are required to more clearly elucidate the conditions under which neutrophils might participate in ischemic pathogenesis.