Ignacio Lizasoain

Toll-Like Receptor 4 Is Involved in Brain Damage and Inflammation After Experimental Stroke

Background— Stroke is the second to third leading cause of death. Toll-like receptor 4 (TLR4) is a signaling receptor in innate immunity that is a specific immunologic response to systemic bacterial infection and cerebral injury. The role of TLR4 in brain ischemia has not been examined yet. We have therefore investigated whether cerebral ischemia and inflammation produced by permanent occlusion of the middle cerebral artery differ in mice that lack a functional TLR4 signaling pathway. Methods and Results— Permanent occlusion of the middle cerebral artery was performed on 2 strains of TLR4-deficient mice (C3H/HeJ and C57BL/10ScNJ) and respective controls (C3H/HeN and C57BL/10ScSn). Stroke outcome was evaluated by determination of infarct volume and assessment of neurological scores. Brains were collected 24 hours and 7 days after stroke. When compared with control mice, TLR4-deficient mice had lower infarct volumes and better outcomes in neurological and behavioral tests. Mice that lacked TLR4 had minor expression of stroke-induced interferon regulatory factor-1, inducible nitric oxide synthase, and cyclooxygenase-2, mediators implicated in brain damage. The levels of interferon-β and of the lipid peroxidation marker malondialdehyde were also lower in brains from TLR4-deficient mice than in those from control mice. In addition, the expression of matrix metalloproteinase-9, which is induced and mediates brain damage, was also reduced in TLR4-deficient mice after experimental stroke. Conclusions— TLR4-deficient mice have minor infarctions and less inflammatory response after an ischemic insult. These data demonstrate that TLR4 signaling and innate immunity are involved in brain damage and in inflammation triggered by ischemic injury.

Plasma Metalloproteinase-9 Concentration Predicts Hemorrhagic Transformation in Acute Ischemic Stroke

Background and Purpose— Matrix metalloproteinase-9 (MMP-9) activity has been associated with hemorrhagic transformation (HT) in experimental models of cerebral ischemia. Our aim was to investigate the relationship between MMP-9 concentrations in blood within 24 hours of stroke onset and subsequent HT of cerebral infarction. Methods— We studied 250 patients with a hemispheric ischemic stroke of 7.8±4.5 hours’ duration. Early CT signs of cerebral infarction were evaluated on admission. The HT and infarct volume were analyzed from the CT performed on days 4 through 7. MMP-9 levels were determined by enzyme-linked immunosorbent assay in blood samples obtained on admission. Results— HT was observed in 38 patients (15.2%): 24 (63.2%) had a hemorrhagic infarction, and 14 (36.8%) had a parenchymal hematoma. A total of 108 patients (43%) received anticoagulants before the second CT scan. Systolic and diastolic blood pressures, body temperature, frequency of early CT signs of ischemia (92% versus 22%), and treatment with anticoagulants (79% versus 37%) were significantly higher in the group with HT (P <0.001). Mean infarct volume was 126±60 cm3 in the HT group and 90±68 cm3 in the group without HT (P =0.003). Median (quartiles) plasma MMP-9 concentrations were higher in the HT group (193 [163, 213] versus 62 [40, 93] ng/mL, P <0.001), even in the 24 patients seen within 3 hours of symptom onset (P =0.014). MMP-9 levels ≥140 ng/mL had a positive and negative predictive value of HT of 61% and 97%, respectively. MMP-9 ≥140 ng/mL was associated with HT (odds ratio, 12; 95% confidence interval, 3 to 51;P <0.001) after adjustment for potential confounders and final infarct volume. Conclusions— High plasma MMP-9 concentration in the acute phase of a cerebral infarct is an independent biochemical predictor of HT in all stroke subtypes.

Glutathione depletion, lipid peroxidation and mitochondrial dysfunction are induced by chronic stress in rat brain.

Damage to the mitochondrial electron transport chain has been suggested to be an important factor in the pathogenesis of a range of neurodegenerative disorders. We have previously demonstrated that chronic stress induced an increase in nitric oxide (NO) production via an expression of inducible NO synthase (iNOS) in brain. Since it has been demonstrated that NO regulates mitochondrial function, we sought to study the susceptibility of the mitochondrial respiratory chain complexes to chronic restrain stress exposure in brain cortex. In adult male rats, stress (immobilization for six hours during 21 days) inhibits the activities of the first complexes of the mitochondrial respiratory chain (inhibition of 69% in complex I-III and of 67% in complex II-III), without affecting complex IV activity, ATP production and oxygen consumption. The mitochondrial marker citrate synthase is not significantly affected by stress after 21 days, indicating that at this time the mitochondrial structure is still intact. Moreover, the administration of the preferred inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) protects against the inhibition of the activity of complexes of the mitochondrial respiratory chain as well as prevents NOx− accumulation, lipid peroxidation and glutathione depletion induced by stress. These results suggest that a sustained overproduction of NO via iNOS is responsible, at least in part, of the inhibition of mitochondrial respiratory chain caused by stress and that this pathway also accounts for the oxidative stress found in this situation.

Statin treatment withdrawal in ischemic stroke A controlled randomized study

Background: Pretreatment with statins has been shown to reduce brain injury in cerebral ischemia. In this controlled randomized study, we investigated the influence of statin pretreatment and its withdrawal on the outcome of acute ischemic stroke patients. Methods: From 215 patients admitted within 24 hours of a hemispheric ischemic stroke, 89 patients on chronic statin treatment were randomly assigned either to statin withdrawal for the first 3 days after admission (n = 46) or to immediately receive atorvastatin 20 mg/day (n = 43). The primary outcome event was death or dependency (modified Rankin Scale [mRS] score > 2) at 3 months. Early neurologic deterioration (END) and infarct volume at days 4 to 7 were secondary outcome variables. In a secondary analysis, outcome variables were compared with the nonrandomized patients without previous statin therapy (n = 126). Results: Patients with statin withdrawal showed a higher frequency of mRS score > 2 at the end of follow-up (60.0% vs 39.0%; p = 0.043), END (65.2% vs 20.9%; p < 0.0001), and greater infarct volume (74 [45, 126] vs 26 [12, 70] mL; p = 0.002) compared with the non–statin-withdrawal group. Statin withdrawal was associated with a 4.66 (1.46 to 14.91)–fold increase in the risk of death or dependency, a 8.67 (3.05 to 24.63)–fold increase in the risk of END, and an increase in mean infarct volume of 37.63 mL (SE 10.01; p < 0.001) after adjusting for age and baseline stroke severity. Compared with patients without previous treatment with statins, statin withdrawal was associated with a 19.01 (1.96 to 184.09)–fold increase in the risk of END and an increase in mean infarct volume of 43.51 mL (SE 21.91; p = 0.048). Conclusion: Statin withdrawal is associated with increased risk of death or dependency at 90 days. Hence, this treatment should be continued in the acute phase of ischemic stroke.

Chronic Stress Induces the Expression of Inducible Nitric Oxide Synthase in Rat Brain Cortex

Abstract: Long‐term exposure to stress has detrimental effects on several brain functions in many species, including humans, and leads to neurodegenerative changes. However, the underlying neural mechanisms by which stress causes neurodegeneration are still unknown. We have investigated the role of endogenously released nitric oxide (NO) in this phenomenon and the possible induction of the inducible NO synthase (iNOS) isoform. In adult male rats, stress (immobilization for 6 h during 21 days) increases the activity of a calcium‐independent NO synthase and induces the expression of iNOS in cortical neurons as seen by immunohistochemical and western blot analysis. Three weeks of repeated immobilization increases immunoreactivity for nitrotyrosine, a nitration product of peroxynitrite. Repeated stress causes accumulation of the NO metabolites NO2‐ + NO3‐ (NOx‐) accumulation in cortex, and these changes occur in parallel with lactate dehydrogenase (LDH) release and impairment of glutamate uptake in synaptosomes. Administration of the selective iNOS inhibitor aminoguanidine (400 mg/kg i.p. daily from days 7 to 21 of stress) prevents NOx‐ accumulation in cortex, LDH release, and impairment of glutamate uptake in synaptosomes. Taken together, these findings indicate that a sustained overproduction of NO via iNOS expression may be responsible, at least in part, for some of the neurodegenerative changes caused by stress and support a possible neuroprotective role for specific iNOS inhibitors in this situation.

Neutrophils scan for activated platelets to initiate inflammation

Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with an asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream. The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in the redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thromboinflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and they suggest that the neutrophils’ bipolarity allows the integration of signals present at both the endothelium and the circulation before inflammation proceeds. Blood neutrophil cells sample platelets in the bloodstream before infiltrating surrounding tissue. A two-cell collaboration for inflammation Immune cells called neutrophils are first responders to infection. Neutrophils move within and through blood vessels to get to sites of infection quickly. Sreeramkumar et al. found that mouse neutrophils rely on platelets to help find such sites. Neutrophils extended protrusions into blood vessels. When these protrusions came into contact with platelets, the neutrophils migrated into the surrounding tissue to carry out their inflammatory functions. Preventing these neutrophilplatelet interactions alleviated collateral inflammatory damage to tissues in several injury models in mice. Science, this issue p. 1234

The Increase in TNF-α Levels Is Implicated in NF-κB Activation and Inducible Nitric Oxide Synthase Expression in Brain Cortex after Immobilization Stress ☆

The underlying mechanisms by which physical or psychological stress causes neurodegeneration are still unknown. We have demonstrated that the high-output and long-lasting synthesizing source of nitric oxide (NO), inducible NO synthase (iNOS), is expressed in brain cortex after three weeks of repeated stress and that its overexpression accounts for the neurodegenerative changes found in this situation. Now we have found that a short duration of stress (immobilization for 6 h) also induces the expression of iNOS in brain cortex in adult male rats. In order to elucidate the possible mechanisms involved in iNOS expression, we have studied the role of the cytokine tumor necrosis factor-α (TNF-α) released in brain during stress. We have shown that there is an increase in soluble TNF-α levels after 1 h of stress in cortex and that this is preceded by an increase in TNF-α-convertase (TACE) activity in brain cortex as soon as 30 min after immobilization. Stress-induced increase in both TACE activity and TNF-α levels seems to be mediated by excitatory amino acids since they can be blocked by MK-801 (dizocilpine) (0.2 mg/kg i.p.), an antagonist of the N-methyl-D-aspartate subtype of glutamate receptor. In order to study the role of TACE and TNF-α in iNOS induction, a group of animals were i.p. injected with the preferred TACE inhibitor BB1101 (2 and 10 mg/kg). Indeed, BB1101 inhibited iNOS expression induced by six hours of stress. In addition, we studied the role of the transcription factor nuclear factor κB (NF-κB), which is required for iNOS expression. We have found that the administration of the TACE inhibitor BB1101 inhibited the stress-stimulated translocation of NF-κB to the nucleus. Taken together, these findings indicate that glutamate receptor activation induces TACE up-regulation and subsequent increase in TNF-α levels, and this account for stress-induced iNOS expression via NF-κB activation, supporting a possible neuroprotective role for specific TACE inhibitors in this situation.

The Increase of Circulating Endothelial Progenitor Cells After Acute Ischemic Stroke Is Associated With Good Outcome

Background and Purpose— Increased circulating endothelial progenitor cells (EPC) have been associated with a low cardiovascular risk and may be involved in endothelial cell regeneration. The present study was designed to evaluate the prognostic value of EPC in acute ischemic stroke. Methods— Forty-eight patients with a first-ever nonlacunar ischemic stroke were prospectively included in the study within 12 hours of symptoms onset. Stroke severity was evaluated by the National Institutes of Health Stroke Scale, and functional outcome was assessed at 3 months by the modified Rankin Scale (mRS). Infarct volume growth between admission and days 4 to 7 was measured on multiparametric MRI. EPC colonies were defined as early outgrowth colony-forming unit-endothelial cell (CFU-EC). The increment of CFU-EC was quantified during the first week and defined as the absolute difference between the number of CFU-EC at day 7 and admission. The influence of CFU-EC increase on good functional outcome (mRS ≤2) and infarct growth was analyzed by logistic regression and linear models. Results— Patients with good outcome (n=25) showed a higher CFU-EC increment during the first week (median [quartiles], 23 [11, 36] versus −3 [−7, 1], P<0.0001) compared with patients with poor outcome. CFU-EC increment ≥4 during the first week was associated with good functional outcome at 3 months (odds ratio, 30.7; 95% CI, 2.4 to 375.7; P=0.004) after adjustment for baseline stroke severity, ischemic volume and thrombolytic treatment. For each unit increase in the CFU-EC the mean reduction in the growth of infarct volume was 0.39 (0.03 to 0.76) mL (P=0.033). Conclusions— The increase of circulating EPC after acute ischemic stroke is associated with good functional outcome and reduced infarct growth. These findings suggest that EPC might participate in neurorepair after ischemic stroke.

Inducible nitric oxide synthase expression in brain cortex after acute restraint stress is regulated by nuclear factor κB-mediated mechanisms

The underlying mechanisms by which physical or psychological stress causes neurodegeneration are still unknown. We have demonstrated that the high‐output and long‐lasting synthesizing source of nitric oxide (NO), inducible NO synthase (iNOS), is expressed in brain cortex during stress and that its overexpression accounts for the neurodegenerative changes seen after 3 weeks of repeated stress. Now we have found that acute stress (restraint for 6 h) increases the activity of a calcium‐independent NOS and induces the expression of iNOS in brain cortex in adult male rats. In order to elucidate the possible mechanisms involved in this induction, we studied the role of transcription nuclear factor κB (NF‐κB), which is required for iNOS synthesis. We have observed that an acute restraint stress session stimulates the translocation of the NF‐κB to the nucleus after 4 h and that the administration of the NF‐κB inhibitor pyrrolidine dithiocarbamate [PDTC, 75 and 150 mg/kg intraperitoneally (i.p.)] at the onset of stress inhibits the stress‐induced increase in iNOS expression. Since glutamate release and subsequent NMDA (N‐methyl‐d‐aspartate) receptor activation has been recognized as an early change after exposure to stressful stimuli, and glutamate has been shown to induce iNOS in brain via a NF‐κB‐dependent mechanism, we studied the possible role of excitatory amino acids in the induction of iNOS in our model. Pretreatment with the NMDA receptor antagonist dizocilpine (MK‐801, 0.1 and 0.3 mg/kg i.p.) inhibits the stress‐induced NF‐κB activation as well as the stress‐induced increase in iNOS expression. Taken together, these findings indicate that excitatory amino acids and subsequent activation of NF‐κB account for stress‐induced iNOS expression in cerebral cortex, and support a possible neuroprotective role for specific inhibitors in this situation.

Toll-Like Receptor 4 Is Involved in Subacute Stress–Induced Neuroinflammation and in the Worsening of Experimental Stroke

Background and Purpose— Psychological stress causes an inflammatory response in the brain and is able to exacerbate brain damage caused by experimental stroke. We previously reported that subacute immobilization stress in mice worsens stroke outcome through mechanisms that involve inflammatory mechanisms, such as accumulation of oxidative/nitrosative mediators and expression of inducible nitric oxide synthase and cyclooxygenase-2 in the brain. Some of these inflammatory mediators could be regulated by innate immunity, the activation of which takes place in the brain and produces an inflammatory response mediated by toll-like receptors (TLRs). Recently, we described the implications of TLR4 in ischemic injury, but the role of TLR4 in stress has not yet been examined. We therefore investigated whether inflammation produced by immobilization stress differs in mice that lack a functional TLR4 signaling pathway. Methods— We used an experimental paradigm consisting of the exposure of mice to repeated immobilization sessions (1 hour daily for 7 days) before permanent middle cerebral artery occlusion. Results— We found that TLR4-deficient mice subjected to subacute stress had a better behavioral condition compared with normal mice (C3H/HeN) and that this effect was associated with a minor inflammatory response (cyclooxygenase-2 and inducible nitric oxide synthase expression) and lipid peroxidation (malondialdehyde levels) in brain tissue. Furthermore, previous exposure to stress was followed by a smaller infarct volume after permanent middle cerebral artery occlusion in TLR4-deficient mice than in mice that express TLR4 normally. Conclusions— Our results indicate that TLR4 is involved in the inflammatory response after subacute stress and its exacerbating effect on stroke. These data implicate the effects of innate immunity on inflammation and damage in the brain after stroke.