Mouse Model Of Cerebral Ischemia Research Articles

The HMGB1 Receptor RAGE Mediates Ischemic Brain Damage

In ischemic stroke, the necrotic core is surrounded by a zone of inflammation, in which delayed cell death aggravates the initial insult. Here, we provide evidence that the receptor for advanced glycation end products (RAGE) functions as a sensor of necrotic cell death and contributes to inflammation and ischemic brain damage. The RAGE ligand high mobility group box 1 (HMGB1) was elevated in serum of stroke patients and was released from ischemic brain tissue in a mouse model of cerebral ischemia. A neutralizing anti-HMGB1 antibody and HMGB1 box A, an antagonist of HMGB1 at the receptor RAGE, ameliorated ischemic brain damage. Interestingly, genetic RAGE deficiency and the decoy receptor soluble RAGE reduced the infarct size. In vitro, expression of RAGE in (micro)glial cells mediated the toxic effect of HMGB1. Addition of macrophages to neural cultures further enhanced the toxic effect of HMGB1. To test whether immigrant macrophages in the ischemic brain mediate the RAGE effect, we generated chimeric mice by transplanting RAGE(-/-) bone marrow to wild-type mice. RAGE deficiency in bone marrow-derived cells significantly reduced the infarct size. Thus, HMGB1-RAGE signaling links necrosis with macrophage activation and may provide a target for anti-inflammatory therapy in stroke.

Myocyte Enhancer Factor 2C as a Neurogenic and Antiapoptotic Transcription Factor in Murine Embryonic Stem Cells

Cell-based therapies require a reliable source of cells that can be easily grown, undergo directed differentiation, and remain viable after transplantation. Here, we generated stably transformed murine ES (embryonic stem) cells that express a constitutively active form of myocyte enhancer factor 2C (MEF2CA). MEF2C has been implicated as a calcium-dependent transcription factor that enhances survival and affects synapse formation of neurons as well as differentiation of cardiomyocytes. We now report that expression of MEF2CA, both in vitro and in vivo, under regulation of the nestin enhancer effectively produces "neuronal" progenitor cells that differentiate into a virtually pure population of neurons. Histological, electrophysiological, and behavioral analyses demonstrate that MEF2C-directed neuronal progenitor cells transplanted into a mouse model of cerebral ischemia can successfully differentiate into functioning neurons and ameliorate stroke-induced behavioral deficits.

Alpha-MSH promotes spontaneous post-ischemic pneumonia in mice via melanocortin-receptor-1

Pneumonia constitutes a serious medical complication and major cause of death in patients after cerebral stroke. In a mouse model of cerebral ischemia (MCAO), we have recently demonstrated that stroke animals spontaneously develop severe bacterial pneumonia which is preceded by a stress-mediated suppression of cellular immune responses in primary and secondary lymphoid organs. However, little is known about the mechanisms leading to impaired pulmonary antimicrobial immune response after cerebral ischemia. In this study, we demonstrate a rapid up-regulation of the immunomodulatory neuropeptide α-melanocyte-stimulating hormone (MSH) in the lung within 24 h after cerebral ischemia. Systemic administration of the naturally occurring α-MSH receptor-1 (MC-1R) antagonist agouti immediately after MCAO significantly reduced pulmonary bacterial burden at 72 h. In contrast, administration of recombinant α-MSH further increased bacterial load in lungs of MCAO animals. In addition, cerebral ischemia resulted in a strong modulation of local pulmonary immunity with increased production of IL-10 by lung macrophages, reduced pulmonary lymphocyte counts, as well as decreased lymphocytic IFN-γ but increased IL-4 production. However, α-MSH blockade by administration of agouti did not prevent changes in lung immune cell numbers or cytokine production suggesting that suppression of cellular immune responses is not the primary mechanism of α-MSH mediated inhibition of pulmonary antibacterial defenses. This study indicates an important role of α-MSH for the increased infectious susceptibility after cerebral ischemia and may provide new therapeutic strategies to prevent post-stroke infectious complications.

Peptidoglycan recognition protein-S (PGRP-S) is upregulated by NF-κB

Cerebral ischemia triggers inflammation and apoptosis, and the transcription factor NF-kappaB is a key regulator of both events. Here, we report on the induction of the peptidoglycan recognition protein-S (PGRP-S) in a mouse model of cerebral ischemia. Upregulation was reduced if the NF-kappaB subunit RelA was conditionally deleted in the brain. Regulation of PGRP-S transcription by RelA was confirmed in vitro. Cotransfection of a RelA expression plasmid stimulated the expression of a PGRP-S luciferase fusion gene. Mutation of two NF-kappaB response elements in the PGRP-S promoter disrupted stimulation by RelA. To investigate the function of PGRP-S in cerebral ischemia, we subjected PGRP-S(-/-) mice to cerebral ischemia. However, there was no difference in the infarct size in PGRP-S-deficient mice compared to controls. In summary, the data show that PGRP-S is induced in cerebral ischemia by RelA, but its role in ischemia is unclear.

Enantio-selective effects of clenbuterol in cultured neurons and astrocytes, and in a mouse model of cerebral ischemia

Neuroprotective effects of the lipophilic β 2-adrenoceptor agonist clenbuterol have been established in neuronal cultures and in various rodent models of stroke. In previous studies, however, clenbuterol was always applied as a racemate, while it has not been established whether the enantiomers differ in their neuroprotective activities. Here, we demonstrate that R,S-clenbuterol and S(+)-clenbuterol, but not the R(−)-enantiomer protect cultured neurons against glutamate-mediated excitotoxicity and staurosporine-induced apoptosis. Similar to previous findings with clenbuterol racemate, the neuroprotective effect of S(+) -clenbuterol correlated well with morphological changes of astrocytes which transformed into dense stellate cells with dendritic processes indicating β 2-adrenoceptor-mediated activation. Most importantly, the S(+)-enantiomer but not R(−)-clenbuterol reduced ischemic brain damage similar to the effect of the racemate. The selective β 2-adrenoceptor antagonist butoxamine blocked this neuroprotective effect of S(+)-clenbuterol. In addition, S(+)-clenbuterol significantly reduced blood pressure, enhanced blood glucose levels and increased glucocorticoid levels compared to vehicle-or R(−)-clenbuterol-treated controls. These results clearly demonstrate that S(+)-clenbuterol is the eutomer that mediates neuroprotective effects of the β 2-adrenoceptor agonist but also according changes of physiological parameters.

Issue Highlights

HomeCirculationVol. 116, No. 4Issue Highlights Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIssue Highlights Originally published24 Jul 2007https://doi.org/10.1161/CIRCULATIONAHA.107.183537Circulation. 2007;116:359BURDEN AND PROGNOSTIC IMPORTANCE OF SUBCLINICAL CARDIOVASCULAR DISEASE IN OVERWEIGHT AND OBESE INDIVIDUALS, by Ingelsson et al.Subclinical cardiovascular disease can be assessed using several routinely available tests that can measure atherosclerotic burden and target organ damage. The extent of subclinical disease in obesity and its prognostic importance has not been investigated systematically. In this issue of Circulation, Ingelsson and colleagues used data from 5 tests (electrocardiography, echocardiography, carotid ultrasound, ankle-brachial pressure, urinary albumin excretion) performed in the community-based Framingham Heart Study sample. The authors report a higher cross-sectional prevalence of subclinical disease in overweight and obese individuals (compared with those with a normal body mass index), and in those with increased waist circumference compared with those with a normal waist circumference. On prospective follow-up, the risk of overt cardiovascular disease was higher in overweight and obese individuals with evidence of subclinical disease, as compared with individuals without subclinical disease. These data suggest that overweight and obesity are associated with a high prevalence of subclinical disease, which in part contributes to the increased risk of overt cardiovascular disease in individuals with excess adiposity. See p 375.PREDICTION OF MODE OF DEATH IN HEART FAILURE: THE SEATTLE HEART FAILURE MODEL, by Mozaffarian et al.Therapeutic strategies to treat symptomatic heart failure are directed toward improving prognosis. Clinicians are very aware that the risk of death and other complications of heart failure vary considerably The Seattle Heart Failure Model incorporates commonly available clinical features, laboratory measurements, and medications used to provide some quantization of this nonhomogeneous risk. In this issue of Circulation, Mozaffarian and colleagues use information from several clinical trials and registries to evaluate the Seattle Heart Failure Model score to assess mode of death in over 10 000 ambulatory patients with symptomatic heart failure. With over 2000 deaths at an annualized rate of approximately 12%, there was a relatively greater increase in the proportion of deaths attributed to progressive pump failure versus sudden, unanticipated heart failure in those with higher scores. The authors appropriately conclude that such information warrants further study and may facilitate decision making regarding improvements in recommendations for implantable cardioverter-defibrillators. See p 392 (editorial p 360).MAST CELL STABILIZATION REDUCES HEMORRHAGE FORMATION AND MORTALITY AFTER ADMINISTRATION OF THROMBOLYTICS IN EXPERIMENTAL ISCHEMIC STROKE, by Strbian et al.Although thrombolysis is an established effective treatment of ischemic stroke, hemorrhagic complications and reperfusion injury limit the outcome of patients after this intervention. Mast cells are known to increase ischemic and hemorrhagic brain edema, possibly because of the release of vasoactive, proteolytic, and fibrinolytic mediators upon degranulation. In a mouse model of cerebral ischemia and reperfusion, the authors investigated this question and found that application of a tissue plasminogen activator, a commonly used treatment strategy in these patients, induces strong mast cell degranulation. Similarly, postischemic administration of a tissue plasminogen activator markedly increased hemorrhagic edema formation, an effect that was significantly reduced by pretreatment of the animals with the mast cell stabilizor, cromoglycate. These results were confirmed in genetically modified mast cell–deficient mice, suggesting that degranulation of these cells is involved in this major complication, as well as in stroke. Further, the potential clinical relevance of these results is interesting, as neurological outcome was improved and mortality reduced in this experimental animal model. Overall, these results point to new treatment modalities in acute stroke using mast cell stabilizers, such as cromoglycate or other molecules, in a relevant clinical setting. See p 411 (editorial p 363).Visit http://circ.ahajournals.org:Images in Cardiovascular MedicineApicoaortic Valve–Containing Conduit in a Patient With Relapsing Prosthetic Endocarditis. See p e88. Download figureDownload PowerPointIsolated Left Ventricular Noncompaction Enhanced by Echocontrast Agent. See p e90.CorrespondenceSee p e92. Previous Back to top Next FiguresReferencesRelatedDetails July 24, 2007Vol 116, Issue 4 Advertisement Article InformationMetrics https://doi.org/10.1161/CIRCULATIONAHA.107.183537 Originally publishedJuly 24, 2007 PDF download Advertisement

Calpain-Cleavage of α-Synuclein: Connecting Proteolytic Processing to Disease-Linked Aggregation

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are both characterized pathologically by the presence of neuronal inclusions termed Lewy bodies (LBs). A common feature found in LBs are aggregates of alpha-synuclein (alpha-Syn), and although it is now recognized that alpha-Syn is the major building block for these toxic filaments, the mechanism of how this occurs remains unknown. In the present study, we demonstrate that proteolytic processing of alpha-Syn by the protease calpain I leads to the formation of aggregated high-molecular weight species and adoption of a beta-sheet structure. To determine whether calpain-cleavage of alpha-Syn occurs in PD and DLB, we designed site-directed calpain-cleavage antibodies to alpha-Syn and tested their utility in several animal model systems. Detection of calpain-cleaved alpha-Syn was evident in mouse models of cerebral ischemia and PD and in a Drosophila model of PD. In the human PD and DLB brain, calpain-cleaved alpha-Syn antibodies immunolabeled LBs and neurites in the substantia nigra. Moreover, calpain-cleaved alpha-Syn fragments identified within LBs colocalized with activated calpain in neurons of the PD and DLB brains. These findings suggest that calpain I may participate in the disease-linked aggregation of alpha-Syn in various alpha-synucleinopathies.

Stroke Propagates Bacterial Aspiration to Pneumonia in a Model of Cerebral Ischemia

Bacterial pneumonia is the most common cause of death in patients sustaining acute stroke and is believed to result from an increased aspiration. Recently, stroke-induced immunodeficiency was described in a mouse model of cerebral ischemia, which is primarily caused by overactivation of sympathetic nervous system. We tested if stroke-induced immunodeficiency increases the risk of pneumonia after aspiration in a newly developed model of poststroke pneumonia. Experimental stroke in mice was induced by occlusion of the middle cerebral artery (MCAO) for 60 minutes. Aspiration pneumonia was induced by intranasal application of 20 microL of a defined suspension of Streptococcus pneumoniae in phosphate-buffered saline 4 or 14 days after MCAO. Treatment comprised moxifloxacin (100 mg/kg body weight, six times every 2 hours after operation) or propranolol (30 mg/kg body weight, immediately before as well as 4 and 8 hours after MCAO). Readout was lung histology and bacterial counts in lung and blood. Nasal inoculation of only 200 colony-forming units of S. pneumoniae caused severe pneumonia and bacteremia after experimental stroke, whereas 200,000 colony-forming units are needed to induce comparable disease in sham animals. Aspiration pneumonia in stroke animals outlasted acute stroke state but was preventable by beta-adrenoreceptor blockade. Experimental stroke propagates bacterial aspiration from harmless intranasal colonization to harmful pneumonia. Prevention of infections by beta-adrenoreceptor blockade suggests that immunodepression by sympathetic hyperactivity is essential for progression of bacterial aspiration to pneumonia.

Withdrawal of statin treatment abrogates stroke protection in mice.

Statins (3-hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase inhibitors) reduce stroke damage independent of lipid lowering by upregulation of endothelial nitric oxide synthase (eNOS). Acute withdrawal of statin treatment may suppress endothelial NO production and impair vascular function. To test this hypothesis, we treated 129/SV mice with atorvastatin (10 mg/kg) for 14 days and then withdrew treatment. Treatment with atorvastatin conferred stroke protection by 40% after filamentous occlusion of the middle cerebral artery followed by reperfusion. Withdrawal of statin treatment, however, resulted in the loss of stroke protection after 2 and 4 days. In mouse aortas and brain vasculature, statins upregulated eNOS message 2.3- and 1.7-fold, respectively, as measured by reverse transcription-polymerase chain reaction. Withdrawal of statins resulted in 5- and 2.7-fold downregulation of eNOS in aorta and brain, respectively, after 2 days. Statin treatment decreased RhoA GTPase membrane expression to 48%, while withdrawal of statins resulted in 4-fold increase of RhoA in the membrane. Moreover, platelet factor 4 and beta-thromboglobulin in plasma were significantly downregulated by statin treatment, but withdrawal of statins resulted in a 2.9- and 3.1-fold upregulation after 2 days, respectively. Thrombus formation induced by ligature of the inferior vena cava was significantly reduced by statin treatment. When statin treatment was withdrawn, however, protection was lost between 2 and 4 days. Acute termination of statin treatment results in a rapid loss of protection in mouse models of cerebral ischemia and thrombus formation independent of lipid lowering. In patients with acute or impending stroke, withdrawal of statins may impair outcome.

CD44 deficiency in mice protects brain from cerebral ischemia injury.

CD44 is a transmembrane glycoprotein known to be involved in endothelial cell recognition, lymphocyte trafficking, and regulation of cytokine gene expression in inflammatory diseases. In the present study, we demonstrated that the expression of CD44 mRNA was induced in a mouse model of cerebral ischemia. A potential role of CD44 in ischemic brain injury was investigated using CD44-deficient (CD44-/-) mice. Over 50% (p < 0.05, n = 14) and 78% (p < 0.05, n = 10) reduction in ischemic infarct was observed in CD44-/- mice compared with that of wild-type mice following transient (30 min ischemia) and permanent (24 h) occlusion of the middle cerebral artery (MCAO), respectively. Similarly, significant improvement was observed in neurological function in CD44-/- mice as evidenced by spontaneous and forced motor task scores. The marked protection from ischemic brain injury in CD44-/- mice was associated with normal physiological parameters, cytokine gene expression, astrocyte and microglia activation as compared with wild-type mice. However, in CD44-/- mice, significantly lower expression of soluble interleukin-1beta protein was noted after brain ischemia. Our data provide new evidence on the potential role of CD44 in brain tissue in response to ischemia and may suggest that this effect might be associated with selective reduction in inflammatory cytokines such as interleukin-1beta.