Simvastatin blocks soluble SSAO/VAP-1 release in experimental models of cerebral ischemia: Possible benefits for stroke-induced inflammation control.

Beyond cholesterol reduction, statins mediate their beneficial effects on stroke patients through pleiotropic actions. They have shown anti-inflammatory properties by a number of different mechanisms, including the inhibition of NF-κB transcriptional activity and the consequent increase and release of adhesion molecules. We have studied simvastatin's effects on the vascular enzyme semicarbazide-sensitive amine oxidase/vascular adhesion protein 1 (SSAO/VAP-1), which is involved in stroke-mediated brain injury. SSAO/VAP-1 has leukocyte-binding capacity and mediates the expression of other adhesion proteins through signaling molecules generated by its catalytic activity. Our results indicate that soluble SSAO/VAP-1 is released into the bloodstream after an ischemic stimulus, in parallel with an increase in E-selectin and VCAM-1 and correlating with infarct volume. Simvastatin blocks soluble SSAO/VAP-1 release and prevents E-selectin and VCAM-1 overexpression as well. Simvastatin also effectively blocks SSAO/VAP-1-mediated leukocyte adhesion, although it is not an enzymatic inhibitor of SSAO in vitro. In addition, simvastatin-induced changes in adhesion molecules are greater in human brain endothelial cell cultures expressing SSAO/VAP-1, compared to those not expressing it, indicating some synergic effect with SSAO/VAP-1. We think that part of the beneficial effect of simvastatin in stroke is mediated by the attenuation of the SSAO/VAP-1-dependent inflammatory response.

Brain proteomics identifies potential simvastatin targets in acute phase of stroke in a rat embolic model.

Finding an efficient neuroprotectant is of urgent need in the field of stroke research. The goal of this study was to test the effect of acute simvastatin administration after stroke in a rat embolic model and to explore its mechanism of action through brain proteomics. To that end, male Wistar rats were subjected to a Middle Cerebral Arteria Occlusion and simvastatin (20 mg/kg s.c) (n = 11) or vehicle (n = 9) were administered 15 min after. To evaluate the neuroprotective mechanisms of simvastatin, brain homogenates after 48 h were analyzed by two-dimensional fluorescence Difference in Gel Electrophoresis (DIGE) technology. We confirmed that simvastatin reduced the infarct volume and improved neurological impairment at 48 h after the stroke in this model. Considering our proteomics analysis, 66 spots, which revealed significant differences between groups, were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry allowing the identification of 27 proteins. From these results, we suggest that simvastatin protective effect can be partly explained by the attenuation of the oxidative and stress response at blood-brain barrier level after cerebral ischemia. Interestingly, analyzing one of the proteins (HSP75) in plasma from stroke patients who had received simvastatin during the acute phase, we confirmed the results found in the pre-clinical model. Our aim was to study statins benefits when administered during the acute phase of stroke and to explore its mechanisms of action through brain proteomics assay. Using an embolic model, simvastatin-treated rats showed significant infarct volume reduction and neurological improvement compared to vehicle-treated group. Analyzing their homogenated brains by two-dimensional fluorescence Difference in Gel Electrophoresis (DIGE) technology, we concluded that the protective effect of simvastatin can be attributable to oxidative stress response attenuation and blood-brain barrier protection after cerebral ischemia.

Mild hypothermia protects against oxygen glucose deprivation (OGD)-induced cell death in brain slices from adult mice.

Most of neuroprotective strategies in stroke have failed to move from bench to bedside. One explanation might be the use of excessive uniform and smooth experimental models. Therefore, we have employed a more stringent in vitro model based on cultured brain slices from adult mice submitted to OGD. Using this acute model, we have confirmed that mild hypothermia protects against OGD-induced cell death when cooling the tissue during and after OGD, but not when hypothermia is induced only during reoxygenation.

Immunological biomarkers improve the accuracy of clinical risk models of infection in the acute phase of ischemic stroke.

UNLABELLED: Infection is an independent risk factor for adverse outcome in stroke patients. The risk of developing an infection in this setting is partly related to a stroke-induced immunodepression, in which a shift to a predominant Th2 (immunosuppressive) phenotype has been postulated to play a major role. Our aim was to study whether clinical variables or changes in plasma cytokine expression can predict poststroke infections. PATIENTS AND METHODS: Medical records of 92 stroke patients were reviewed, and the baseline concentration of cytokines from the Th1/Th2 system was determined. Clinical and serological predictors of incident infections and their prognostic significance were sought by means of univariate and multivariate analysis, and two predictive models for developing an infection were constructed by combining independent predictors (strictly clinical in one, and both clinical and serological in the other) for this outcome. The improvement conferred by the addition of immunological markers to the clinical model was assessed by comparing their respective ROC curves and by improvement (Net Reclassification Index and Integrated Discriminator Improvement) analysis. RESULTS: Nineteen patients (20.7% of the study sample) developed an infection. Ongoing antiplatelet therapy at symptom onset (OR 0.02, 95% CI 0.001-0.23, p = 0.001), diabetes mellitus (OR 9.96, 95% CI 1.32-75.29, p = 0.03), IL-13 level <33 pg/ml (OR 84.16, 95% CI 2.53-2795.18, p = 0.01) and interferon-γ level >8.4 pg/ml (OR 60.17, 95% CI 1.78-2037.23, p = 0.02) were independently associated with the development of infections during hospital admission. The combined regression model predicted infection with an accuracy of 93.4%, an improvement in the predictive capacity of 17% (p < 0.001). Infection was associated with a worse neurological status at hospital discharge (median NIHSS score 11 (6-18) vs. 4 (1-11.5), p = 0.014). CONCLUSIONS: This study shows that bloodstream biomarkers are useful to improve the accuracy of clinical prognostic models for infection in the acute phase of stroke. The clinical predictors of infection in the acute phase of stroke are relatively well established in the medical literature, but further research to identify the optimal combination of biomarkers (possibly inflammatory and stress markers) to be included in a clinically useful model is needed. Such a model could be subsequently used in clinical trials to assess the effect of prophylactic and/or early antibiotic therapy in this setting, a currently controversial issue in this field.

A new method for focal transient cerebral ischaemia by distal compression of the middle cerebral artery.

AIMS: Rodent experimental models are essential for in vivo study of stroke. Our aim was to develop a reproducible method of mouse transient focal cerebral ischaemia by distal artery compression. METHODS: The distal middle cerebral artery (dMCA) was occluded by compression with a blunted needle, and cerebral blood flow was monitored by laser Doppler flowmetry to ensure appropriate occlusion and reperfusion in Balb/c mice. The ischaemic lesion was evaluated 24 h after occlusion by TTC staining and immunolabelling (NeuN, CD31, GFAP and Iba-1) while the established permanent dMCA occlusion (dMCAO) model was used as a control. The corner test was performed to evaluate neurological behaviour. RESULTS: Laser Doppler flowmetry register showed a homogenous arterial occlusion among animals. Forty-five minutes of arterial occlusion did not lead brain infarction when evaluated by TTC staining 24 h after occlusion. Extending the cerebral ischaemia period to 60 min induced a cortically localized homogeneous brain infarct. No differences in infarct volume were detected between animals submitted to permanent or 60-min transient dMCAO (42.33 ± 9.88 mm³ and 37.63 ± 12.09 mm³ respectively). The ischaemic injury was confirmed by immunohistochemistry in the 60-min transient dMCAO model but not in the 45-min model. Neurological deficits assessed with the corner test were significant only during the first 48 h but not at long term. CONCLUSIONS: This work shows an easy-to-perform method for the induction of brain ischaemia and reperfusion to assess stroke repair and treatment screening, with cortically localized ischaemic cell damage, low mortality and neurological impairment in the acute phase.

Charge effect of a liposomal delivery system encapsulating simvastatin to treat experimental ischemic stroke in rats.

BACKGROUND AND AIMS: Although the beneficial effects of statins on stroke have been widely demonstrated both in experimental studies and in clinical trials, the aim of this study is to prepare and characterize a new liposomal delivery system that encapsulates simvastatin to improve its delivery into the brain. MATERIALS AND METHODS: In order to select the optimal liposome lipid composition with the highest capacity to reach the brain, male Wistar rats were submitted to sham or transitory middle cerebral arterial occlusion (MCAOt) surgery and treated (intravenous [IV]) with fluorescent-labeled liposomes with different net surface charges. Ninety minutes after the administration of liposomes, the brain, blood, liver, lungs, spleen, and kidneys were evaluated ex vivo using the Xenogen IVIS(®) Spectrum imaging system to detect the load of fluorescent liposomes. In a second substudy, simvastatin was assessed upon reaching the brain, comparing free and encapsulated simvastatin (IV) administration. For this purpose, simvastatin levels in brain homogenates from sham or MCAOt rats at 2 hours or 4 hours after receiving the treatment were detected through ultra-high-protein liquid chromatography. RESULTS: Whereas positively charged liposomes were not detected in brain or plasma 90 minutes after their administration, neutral and negatively charged liposomes were able to reach the brain and accumulate specifically in the infarcted area. Moreover, neutral liposomes exhibited higher bioavailability in plasma 4 hours after being administered. The detection of simvastatin by ultra-high-protein liquid chromatography confirmed its ability to cross the blood-brain barrier, when administered either as a free drug or encapsulated into liposomes. CONCLUSION: This study confirms that liposome charge is critical to promote its accumulation in the brain infarct after MCAOt. Furthermore, simvastatin can be delivered after being encapsulated. Thus, simvastatin encapsulation might be a promising strategy to ensure that the drug reaches the brain, while increasing its bioavailability and reducing possible side effects.

Rat middle cerebral artery occlusion is not a suitable model for the study of stroke-induced spontaneous infections.

BACKGROUND: Infections related to stroke-induced immunodepression are an important complication causing a high rate of death in patients. Several experimental studies in mouse stroke models have described this process but it has never been tested in other species such as rats. METHODS: Our study focused on the appearance of secondary systemic and pulmonary infections in ischemic rats, comparing with sham and naive animals. For that purpose, male Wistar rats were subjected to embolic middle cerebral artery occlusion (eMCAO) or to transient MCAO (tMCAO) inserting a nylon filament. Forty-eight hours after ischemia, blood and lung samples were evaluated. RESULTS: In eMCAO set, ischemic rats showed a significant decrease in blood-peripheral lymphocytes (naive = 58.8±18.1%, ischemic = 22.9±16.4%) together with an increase in polymorphonuclears (PMNs) (naive = 29.2±14.7%, ischemic = 71.7±19.5%), while no change in monocytes was observed. The increase in PMNs counts was positively correlated with worse neurological outcome 48 hours after eMCAO (r = 0.55, p = 0.043). However, sham animals showed similar changes in peripheral leukocytes as those seen in ischemic rats (lymphocytes: 40.1±19.7%; PMNs: 51.7±19.2%). Analysis of bacteriological lung growth showed clear differences between naive (0±0 CFU/mL; log10) and both sham (3.9±2.5 CFU/mL; log10) and ischemic (4.3±2.9 CFU/mL; log10) groups. Additionally, naive animals presented non-pathological lung histology, while both sham and ischemic showed congestion, edema or hemorrhage. Concordant results were found in the second set of animals submitted to a tMCAO. CONCLUSIONS: Inflammatory and infection changes in Wistar rats subjected to MCAO models may be attributed not only to the brain ischemic injury but to the surgical aggression and/or anaesthetic stress. Consequently, we suggest that stroke-induced immunodepression in ischemic experimental models should be interpreted with caution in further experimental and translational studies, at least in rat stroke models that entail cervicotomy and cranial trepanation.