Experimental Models Of Cerebral Ischemia Research Articles

Modeling of Cerebral Ischemic Stroke in Conscious Rats via Arterial Air Embolization.

The etiological factor of cerebral ischemia in the vast majority of cases is vascular embolism. In the present study we investigated embolism caused by atmospheric air bubbles injected into the internal carotid artery of conscious rats. Immediately after embolism modeling, behavioral abnormalities were observed in the animals, and after 24 h, foci of brain damage were detected. The death of animals was observed within 5 days after embolism. The proposed experimental model of cerebral ischemia in conscious rats is more relevant and better corresponds to real conditions than the model on narcotized animals and allows to perform physiological tests immediately after modeling.

Pretreatment with a dual antiplatelet and anticoagulant (APAC) reduces ischemia–reperfusion injury in a mouse model of temporary middle cerebral artery occlusion—implications for neurovascular procedures

BackgroundSeveral neurovascular procedures require temporary occlusion of cerebral arteries, leading to ischemia of unpredictable length, occasionally causing brain infarction. Experimental models of cerebral ischemia–reperfusion injury have established that platelet adhesion and coagulation play detrimental roles in reperfusion injury following transient cerebral ischemia. Therefore, in a model of cerebral ischemia–reperfusion injury (IRI), we investigated the therapeutic potential of a dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic which is able to bind to vascular injury sites.MethodsBrain ischemia was induced in mice by transient occlusion of the right middle cerebral artery for 60 min. APAC, unfractionated heparin (UFH) (both at heparin equivalent doses of 0.5 mg/kg), or vehicle was intravenously administered 10 min before or 60 min after the start of ischemia. At 24 h later, mice were scored for their neurological and motor behavior, and brain damage was quantified.ResultsBoth APAC and UFH administered before the onset of ischemia reduced brain injury. APAC and UFH pretreated mice had better neurological and motor functions (p < 0.05 and p < 0.01, respectively) and had significantly reduced cerebral infarct sizes (p < 0.01 and p < 0.001, respectively) at 24 h after transient occlusion compared with vehicle-treated mice. Importantly, no macroscopic bleeding complications were observed in either APAC- or UFH-treated animals. However, when APAC or UFH was administered 60 min after the start of ischemia, the therapeutic effect was lost, but without hemorrhaging either.ConclusionsPretreatment with APAC or UFH was safe and effective in reducing brain injury in a model of cerebral ischemia induced by transient middle cerebral artery occlusion. Further studies on the use of APAC to limit ischemic injury during temporary occlusion in neurovascular procedures are indicated.

Development of Pharmacological Strategies with Therapeutic Potential in Ischemic Stroke.

Acute ischemic stroke constitutes a health challenge with great social impact due to its high incidence, with the social dependency that it generates being an important source of inequality. The lack of treatments serving as effective neuroprotective therapies beyond thrombolysis and thrombectomy is presented as a need. With this goal in mind, our research group's collaborative studies into cerebral ischemia and subsequent reperfusion concluded that there is a need to develop compounds with antioxidant and radical scavenger features. In this review, we summarize the path taken toward the identification of lead compounds as potential candidates for the treatment of acute ischemic stroke. Evaluations of the antioxidant capacity, neuroprotection of primary neuronal cultures and in vivo experimental models of cerebral ischemia, including neurological deficit score assessments, are conducted to characterize the biological efficacy of the various neuroprotective compounds developed. Moreover, the initial results in preclinical development, including dose-response studies, the therapeutic window, the long-term neuroprotective effect and in vivo antioxidant evaluation, are reported. The results prompt these compounds for clinical trials and are encouraging regarding new drug developments aimed at a successful therapy for ischemic stroke.

Diet Supplementation with Polyphenol-Rich Salicornia ramosissima Extracts Protects against Tissue Damage in Experimental Models of Cerebral Ischemia

Strokes are the second most common cause of death worldwide and a leading cause of disability. Regular consumption of polyphenols has been shown to reduce the risk of suffering a cardiovascular event. For this reason, we have investigated the protective effect of Salicornia ramosissima, a seasonal halophyte that synthetizes high amounts of bioactive compounds, including polyphenols, in response to environmental stress. Aqueous, hydroalcoholic, and ethanolic extracts were prepared to investigate if dietary supplementation prior to ischemic challenge can prevent subsequent damage using two animal models. First, we screened the protective effect against hypoxia-reoxygenation in Drosophila melanogaster and observed that both ethanolic and hydroalcoholic extracts protected flies from the deleterious effects of hypoxia. Second, we confirmed the protective effect of S. ramosissima ethanolic extract against brain ischemia using the transient middle cerebral artery occlusion mice model. Four weeks of oral supplementation with the ethanolic extract before artery occlusion reduced infarct volume and lowered the plasma levels of the DNA peroxidant product 8-hydroxydeoxyguanosine. Phytochemical profiling of S. ramosissima ethanolic extract revealed 50 compounds. Thus, it represents a valuable source of bioactive compounds that show promising disease-modifying activities and could be further developed as an effective food supplement for the prevention or treatment of neurovascular disorders.

Pharmacological Effect of Panax notoginseng Saponins on Cerebral Ischemia in Animal Models.

Panax notoginseng saponins (PNS), bioactive compounds, are commonly used to treat ischemic heart and cerebral diseases in China and other Asian countries. Most previous studies of PNS have focused on the mechanisms underlying their treatment of ischemic cardiovascular diseases but not cerebral ischemic diseases. This study sought to explore the pharmacological mechanisms underlying the effectiveness of PNS in treating cerebral ischemic diseases. Different experimental cerebral ischemia models (including middle cerebral artery occlusion (MCAO) and the blockade of four arteries in rats, collagen-adrenaline-induced systemic intravascular thrombosis in mice, thrombosis of carotid artery-jugular vein blood flow in the bypass of rats, and hypoxia tolerance in mice) were used to investigate the mechanisms underlying the actions of PNS on cerebral ischemia. The results indicated that (1) PNS improved neurological function and reduced the cerebral ischemia infraction area in MCAO rats; (2) PNS improved motor coordination function in rats with complete cerebral ischemia (blockade of four arteries), decreased Ca2+ levels, and ameliorated energy metabolism in the brains of ischemia rats; (3) PNS reduced thrombosis in common carotid artery-jugular vein blood flow in the bypass of rats; (4) PNS provided significant promise in antistroke hemiplegia and hypoxia tolerance in mice. In conclusion, PNS showed antagonistic effects on ischemic stroke, and pharmacological mechanisms are likely to be associated with the reduction of cerebral pathological damage, thrombolysis, antihypoxia, and improvement in the intracellular Ca2+ overload and cerebral energy metabolism.

Preclinical Characterization of Antioxidant Quinolyl Nitrone QN23 as a New Candidate for the Treatment of Ischemic Stroke.

Nitrones are encouraging drug candidates for the treatment of oxidative stress-driven diseases such as acute ischemic stroke (AIS). In a previous study, we found a promising quinolylnitrone, QN23, which exerted a neuroprotective effect in neuronal cell cultures subjected to oxygen–glucose deprivation and in experimental models of cerebral ischemia. In this paper, we update the biological and pharmacological characterization of QN23. We describe the suitability of intravenous administration of QN23 to induce neuroprotection in transitory four-vessel occlusion (4VO) and middle cerebral artery occlusion (tMCAO) experimental models of brain ischemia by assessing neuronal death, apoptosis induction, and infarct area, as well as neurofunctional outcomes. QN23 significantly decreased the neuronal death and apoptosis induced by the ischemic episode in a dose-dependent manner and showed a therapeutic effect when administered up to 3 h after post-ischemic reperfusion onset, effects that remained 11 weeks after the ischemic episode. In addition, QN23 significantly reduced infarct volume, thus recovering the motor function in a tMCAO model. Remarkably, we assessed the antioxidant activity of QN23 in vivo using dihydroethidium as a molecular probe for radical species. Finally, we describe QN23 pharmacokinetic parameters. All these results pointing to QN23 as an interesting and promising preclinical candidate for the treatment of AIS.

The role of physical exercise in apoptosis in an experimental cerebral ischemia model

IntroductionStroke is a major public health problem, with high mortality rates and high levels of disability. Between 1990 and 2010 for example, stroke increased from the fifth to the third leading cause of disability. In addition, its incidence has increased among younger people, a situation in which there are significant health consequences and elevated social costs. There is evidence that physical exercise promotes neuroprotective effects when used as a therapeutic treatment in an experimental ischemia model. However, the mechanisms of neuroprotection are still not well understood.ObjectiveThe objective of this study was to evaluate the expression profile of the microRNAs miR‐21, miR‐16, miR‐155 and caspase‐3 and Bcl‐2 genes by real‐time PCR in brain tissue (ischemic focus) of the animals after the performance of physical training before focal cerebral ischemia.Materials and Methods48 Wistar rats were used. The animals were divided into four experimental groups: control group, ischemia group, exercise group and exercise + ischemia group. The real‐time PCR methodology was used to analyze the expression of miRNAs; miR16, miR‐21, miR‐155, caspase‐3 and Bcl‐2.Results and conclusionsIn our study, we did not observe statistically significant differences in the expression of miRNAs: miR‐16, miR‐21, miR‐155 or in the Bcl‐2 and caspase‐3 genes in brain tissue in the groups undergoing cerebral ischemia and exercise when compared to the control group.

Effect of Adiponectin on Acute Experimental Cerebral Ischemia/Reperfusion Injury.

To examine the effect of adiponectin administration on acute brain injury in an experimental model of cerebral ischemia/ reperfusion (I/R) in rats. The study animals were divided into the following four groups: group I, sham (did not undergo surgical intervention and did not receive drugs); group II, the I/R model (received the intervention, but did not receive drugs); group III (I/Radiponectin) (the I/R model was used, and the animals were treated with 5 mg/kg adiponectin peritoneally 30 minutes after the ischemia); and group IV (I/R-tirofiban)( the I/R model was used, and the animals were treated with 0.5 mg/kg tirofiban peritoneally 30 minutes after the ischemia). Tumor necrosis factor-? (TNF-?) and interleukin (IL)-1? levels were statistically higher in the I/R group (group II) than in other groups. In the post-hoc (Tukey) test analysis, groups I, III, and IV had significantly lower TNF-? and IL-1? levels after treatment with both tirofiban and adiponectin than group II. No statistically significant difference was found between groups III and IV in terms of TNF-? levels. However, the decreased IL-1? level was more pronounced in group IV (tirofiban) than in other groups. The mean neurologic deficit scores were statistically significantly different among the groups. In the post-hoc (Tukey) test analysis, neurologic deficit scores were statistically significantly lower in groups III and IV than in group II. Adiponectin has anti-inflammatory and cerebral protective effects in experimental cerebral I/R injury.

Neuroprotection against cerebral ischemia/reperfusion by dietary phytochemical extracts from Tibetan turnip (Brassica rapa L.)

Ethnopharmacological relevanceThe Tibetan turnip (Brassica rapa L.) has a wide array of medicine properties including heat-clearing, detoxifying and anti-hypoxia as listed in the famous centuries-old Tibetan medicine classic “The Four Medical Tantras”. Evidence-based medicine also indicated the anti-hypoxic effect of turnips, suggesting a potential link to neuroprotective effect on ischemic stroke. This thereby enables turnips to serve as a novel nontoxic agent in related treatment. Aim of the studyThis study aimed to investigate the neuroprotective effect and elucidate the mechanism of aqueous extract of turnip (AET) on cerebral ischemia/reperfusion. Materials and methodsThe experimental models of cerebral ischemia included transient middle cerebral artery occlusion/reperfusion (MCAO) in C57BL/6J mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in HT-22 cells. Long-term effect of AET on infarct volume was evaluated by microtubule-associated protein 2 (MAP2) immunofluorescence 28 days after MCAO, and on neurofunctional outcomes determined by rotarod, grid walking, and cylinder tests in the meantime. Efficacy of AET was determined by the cell viability, the release of lactate dehydrogenase (LDH) and reactive oxygen species (ROS) in neurons. The underlying mechanism of AET rescued OGD/R cells were characterized by PI3K, Akt and mTOR expressions, which were further used to validate AET's role in the pathway. ResultsAET can reduce cerebral infarct volume and ameliorate behavioral deficits of MCAO/R mice dose-dependently. In vitro experiment further demonstrated that suitable concentrations of AET inhibited ROS, LDH production and restored mitochondrial expression induced by OGD/R. AET pretreatment can reverse the OGD/R-induced decreased level of phosphorylation of PI3K, Akt, mTOR, whereas this effect was blocked in the LY294002 (PI3K inhibitor) treatment group. ConclusionsAET improved the survival of OGD/R-injured HT-22 cells by activating the PI3K/Akt/mTOR pathway. Based on the results above, aqueous extract of turnip has a protective effect on focal cerebral ischemic injury.

An experimental model of acute cerebral ischemia with transplantation of mesenchymal stem cells into the carotid artery

Transplantation of mesenchymal cells is a perspective paradigm for treatment of stroke. However, after intravenous injection most infusion cells get in filter organs, such as lungs. Experimental model of cerebral ischemia with transplantation of mesenchymal cells in a carotid was developed. Experiments on 15 animals conducted on by the females of crawls in age 12-36 weeks weighing 4-6 kg. The general and internal carotid arteries were projected under local and intravenous anesthesia. General carotid artery was ligated and mesenchymal stem cells were injected. The population of the cages abstracted from a brain was folded on 96% from cages positive for by the markers of mesenchymal cells, and less than on 2% from hematopoietic cells and cells of endothelia, positive after corresponding markers. In 6 hour after introduction to the right internal carotid of mesenchymal cells, the histological encephaloscopy of crawls was executed for visualization of GFP-positive cages mark also the marker of lipophil of PKH26. In 6 hour after transplantation of cage distributed at a right hemisphere in area of bark and basale kernels (in the zone of blood supply of right internal carotid) and visualized along the midwall of cerebral vessels both in the zone of heart attack of brain and for peripheries. Experimental confinnation of that therapeutic activity of mesenchymal cells arises up during their system transplantation and delivery on an artery that supplies with blood the zone of ischemic damage of brain is first got. Keywords: carotid arteries, mesenchymal stem cells, ischemic stroke.

Sumoylation of sodium/calcium exchanger in brain ischemia and ischemic preconditioning

The small ubiquitin-like modifier (SUMO) conjugation (or SUMOylation) is a post-translational protein modification mechanism activated by different stress conditions that has been recently investigated in experimental models of cerebral ischemia. The expression of SUMOylation enzymes and substrates is not restricted to the nucleus, since they are present also in the cytoplasm and on plasma membrane and are involved in several physiological and pathological conditions.In the last decades, convincing evidence have supported the idea that the increased levels of SUMOylated proteins may induce tolerance to ischemic stress. In particular, it has been established that protein SUMOylation may confer neuroprotection during ischemic preconditioning.Considering the increasing evidence that SUMO can modify stability and expression of ion channels and transporters and the relevance of controlling ionic homeostasis in ischemic conditions, the present review will resume the main aspects of SUMO pathways related to the key molecules involved in maintenance of ionic homeostasis during cerebral ischemia and ischemic preconditioning, with a particular focus on the on Na+/Ca2+ exchangers.

Abstract WP318: Prediction of Leptomeningeal Collateral Recruitment in Experimental Ischemic Stroke by Cerebrovascular Reactivity to Carbon Dioxide

Purpose: Immediate pial collateral recruitment during acute ischemic stroke is an endogenous response limiting permanent tissue damage due to proximal cerebral artery occlusion. There is presently no known way to predict the degree of pial collateral recruitment. This work sought to determine if cerebrovascular reactivity (CVR) to CO 2 predicts pial collateral recruitment in an experimental model of cerebral ischemia. Methods: Eight mongrel dogs underwent cerebral blood flow (CBF) measurements using stable isotope neutron activated microspheres during normocapnia (PaCO 2 30-35mmHg) using medical air, and subsequently during hypercapnia (PaCO 2 60-70mmHg) using 7% CO 2 . Anesthesia (propofol, remifentanil, rocuronium and 1% isofluorane) and normal range physiologic parameters were maintained to avoid undue influence on CO 2 reactivity. Animals underwent middle cerebral artery occlusion using a previously described endovascular method. Pial collateral recruitment was angiographically assessed using a previously published 11-point scale. Animals subsequently underwent MRI to derive 4-hour infarct volumes calculated using an automated threshold technique using mean diffusivity maps. Bivariate linear fit analyses were used to assess correlations between CVR versus pial collateral score and infarct volume. Results: CBF values derived from cortical gray matter during simultaneous CO 2 challenge demonstrate a statistically significant correlation to infarct volumes (p=0.0041, r 2 adj= 0.734) and pial collateral recruitment (p=0.0006, r 2 adj= 0.856) (figure 1). CBF at PaCO 2 of 65mmHg was secondarily found to correlate with infarct volume (p=0.0013, r 2 adj= 0.817) and pial collateral recruitment (p=0.0005, r 2 adj= 0.862). Conclusion: Preliminary data presented suggest that cerebrovascular reactivity to CO 2 may be predictive of pial collateral recruitment and infarct volumes during acute ischemic stroke

Comparative evaluation of pre- and postoperative administration of citicoline: clinical outcomes and the volume of ischemic brain damage after meningioma removal

In the experimental model of cerebral ischemia, citicoline has shown greater effectiveness with its preventive use compared with therapeutic one.Aim. To study the main clinical outcomes and the dynamics of morphometric indicators of ischemic brain damage according to computed tomography (CT) data in patients with meningiomas of the skull base in the postoperative period against the background of prophylactic and therapeutic administration of citicoline.Materials and methods. The study included 53 patients aged 40 to 69 years with the skull base meningiomas. The first (control) group (n=17) and the third group, in which citicoline was administered in the postoperative period for 7–21 days at a dose of 1000 mg 2 times a day intravenously (n=25), were formed retrospectively. The second group (n=11), in which citicoline was administered prophylactically 1.5–2.5 hours before surgery at a dose of 2000 mg intravenously, was formed prospectively. We evaluated the main clinical outcomes and CT morphometry data.Results. The average time spent in the intensive care unit was minimal in the second group: 9.6±3.2 days vs 17.6±3.7 days in the control (p=0.049). Postoperative mortality was 24% in the control group, 9% in the second group, and 20% in the third group. The survival time in the first group was less than 21 days, over 21 days in the second and third groups. The groups did not differ in neurological outcomes and overall in-hospital stay. The average volume of ischemic brain damage on the first day after surgery in the second group was less than in the control group, and amounted to 111.7±15.2 cm3 against 151.3±17.1 cm3, respectively (p=0.044).Conclusion. The prophylactic administration of citicoline before a tumor removal operation may have effective potential for reducing the severity and prevalence of ischemic cerebral edema. Further randomized clinical trials are needed.

Neuroprotective potential of adenosine A1 receptor partial agonists in experimental models of cerebral ischemia

Cerebral ischemia is the second most common cause of death and a major cause of disability worldwide. Available therapies are based only on anticoagulants or recombinant tissue plasminogen activator. Extracellular adenosine increases during ischemia and acts as a neuroprotective endogenous agent mainly by activating adenosine A1 receptors (A1 Rs) which control calcium influx, glutamate release, membrane potential, and metabolism. Accordingly, in many experimental paradigms it has been already demonstrated that the stimulation of A1 R with full agonists is able to reduce ischemia-related structural and functional brain damage; unfortunately, cardiovascular side effects and desensitization of A1 R induced by these compounds have strongly limited their exploitation in stroke therapy so far. Among the newly emerging compounds, A1 R partial agonists could be almost free of side effects and equally effective. Therefore, we decided to evaluate the neuroprotective potential of two A1 R partial agonists, namely 2'-dCCPA and 3'-dCCPA, in invitro and exvivo experimental models of cerebral ischemia. Within the experimental paradigm of oxygen-glucose deprivation invitro in human neuroblastoma (SH-SY5Y) cells both A1 R partial agonists increased cell viability. Considering the high level of expression of A1 Rs in the hippocampus and the susceptibility of CA1 region to hypoxia, we performed electrophysiological experiments in this subfield. The application of 7min of oxygen-glucose deprivation constantly produces an irreversible synaptic failure in all the C57Bl/6 mice hippocampal slices evaluated; both tested compounds allowed a significant recovery of synaptic transmission. These findings demonstrate that A1 R and its partial agonists are still of interest for cerebral ischemia therapy. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Brain Peptides for the Treatment of Neuropsychiatric Disorders.

The realization of the importance of growth factors in adult CNS led to several studies investigating their roles in neuropsychiatric disorders. Based on the observations that chronic stress decreases brain-derived neurotrophic factor (BDNF) and antidepressant treatments reverse BDNF to normal levels, "neurotrophic hypothesis of depression" was proposed. Subsequent studies found that several other growth factors, including fibroblast growth factor (FGF), vascular endothelial growth factor, nerve growth factor were also decreased by chronic stress. Growth factors promote stem cell survival, angiogenesis and neurogenesis in addition to having anti-apoptotic and anti-inflammatory effects, all of which make them potential drug candidates as neuroprotective or neurorestorative agents. Indeed, certain peptides have consistently been shown to improve stroke outcome in experimental models of cerebral ischemia. Recent developments in nanotechnology appear promising in overcoming the blood-brain barrier and in delivering sufficient amounts of these large peptides to the brain after systemic administration. In addition to the translational potential resulting from application of nanotechnical approaches for delivering these large peptide growth factors, recent success obtained with small molecule and peptide antagonists of calcitonin gene-related peptide has created renewed enthusiasm to elucidate the role of neuropeptides in migraine headache, one of the most common health problems in the world. In this review, we will first focus on the role of FGF2 in mood disorders as well as in ischemic stroke. We will also introduce the nanomedicines developed to efficiently deliver FGF2 to the brain. In the last section, we will explore roles of the neuropeptides in migraine and its acute and prophylactic treatment.

Low Levels of Caveolin-1 Predict Symptomatic Bleeding After Thrombolytic Therapy in Patients With Acute Ischemic Stroke.

Experimental models of cerebral ischemia demonstrate that the decrease in the caveolin-1 membrane protein results in an increase in endothelial permeability. Because this phenomenon is responsible for hemorrhagic transformation (HT) after cerebral ischemia, we aimed to determine whether caveolin-1 levels may predict bleeding after recombinant tissue-type plasminogen activator (r-tPA) administration in patients with acute stroke. We studied 133 patients with a first hemispheric stroke treated with r-tPA within 4.5 hours of symptom onset. HT was evaluated and classified on cranial computed tomography at 24 hours and was considered as symptomatic HT (sHT) if associated with neurological deterioration. Serum caveolin-1 levels were analyzed before and at 2, 24, and 72 hours post-r-tPA administration in patients and in 40 healthy controls. Baseline caveolin-1 levels were higher in patients than controls (0.24 [0.17-0.40] versus 0.07 [0.0-0.20] ng/mL; P<0.000). Twenty six (19.5%) patients had HT, which was symptomatic in 7 (5.3%). Patients with parenchymal hemorrhage-2 and sHT had lower baseline caveolin-1 levels than the rest of patients (0.08 [0.04-0.19] versus 0.26 [0.14-0.40]; P=0.019 and 0.08 [0.02-0.17] versus 0.26 [0.13-0.41]; P=0.019, respectively). The levels remained stable in the first 72 hours in patients with parenchymal hemorrhage-2 and sHT, whereas in the rest of patients levels decreased in this time. Caveolin-1 levels ≤0.17 ng/mL had the highest predictive capacity of sHT (86% sensitivity, 65% specificity, 99% negative predictive value, 12% positive predictive value). After adjustment for confounders, caveolin-1 levels ≤0.17 ng/mL independently predicted sHT (odds ratio, 11.6; 95% confidence interval, 11.3-102.8; P=0.027). Low serum levels of caveolin-1 are an independent predictor of sHT after r-tPA administration. Because of the small sample size, further research is needed to validate these data.

Environmental enrichment cognitive neuroprotection in an experimental model of cerebral ischemia: biochemical and molecular aspects

Stroke is considered a major cause of global morbidity. Currently, there are no effective treatments for post-stroke cognitive impairment. Enriched environment (EE) has been brought forward as a preconditioning method to induce cerebral tolerance in an ischemic event. However, the subjacent mechanisms involved in this tolerance are not yet clear. Herein we aimed to identify the mechanisms of neuroprotection triggered by EE preconditioning in a murine model of ischemic stroke. In order to do so, C57Bl/6 mice were kept for five weeks either in EE or in standard environment (SC) prior to ischemic injury through bilateral carotid occlusion (BCCAo) or sham surgery. To evaluate cognitive deficits resulting from ischemia, animals were subjected to a set of behavioral test to assess short-term (STM), long-term (LTM) and working memory (WM) performance. Despite no effect of EE having been observed in LTM and WM, EE preconditioning was able to prevent short-term deficits in response to ischemia. This improvement was accompanied by a reduction in the infarct volume in animals following EE pre-exposure. Next, we aimed to analyze the expression of genes involved in cholinergic (M1 and alpha 7 receptors) and glutamatergic (NMDA subunits GluN1, GluN2A, GluN2B and GluN2C) neurotransmission, inflammatory mediators (GFAP and IL-1β) and of the neurotrophin BDNF. Animals tested for STM did not present alterations in the expression of glutamatergic or cholinergic receptors; however, EE was shown to prevent increased expression of IL1-β. On the other hand, in animals submitted to LTM task, EE exposure lead to increased GFAP expression in EE animals that underwent ischemic injury, affecting also the expression of NMDA subunits. In spite of that, no alterations in glutamate content were observed in either group. Altogether, this study suggests that the changes observed in the expression of glutamatergic receptors, the reduction of the inflammatory cytokine IL1-β expression and the increased expression of GFAP in ischemic animals might contribute to the cognitive improvement induced by the EE paradigm.

Multi-targeting effects of a new synthetic molecule (JM-20) in experimental models of cerebral ischemia.

Ischemic stroke is a major cause of death and disability worldwide. Thrombolysis by tissue plasminogen activator is the only pharmacological treatment approved for clinical practice, but has a narrow therapeutic window and poor efficacy when the cell death cascade is activated. Numerous drugs that are thought to protect neurons against injury have previously failed in human trials despite showing efficacy in experimental models of stroke. Herein, we reviewed the main pre-clinical results of the neuroprotective effects of JM-20, a new hybrid molecule, against brain ischemia. JM-20 appears to protect the brain from ischemic damage by interfering with several elements of the ischemic cascade: antiexcitotoxic, anticalcic, antioxidant, antiapoptotic, and anti-inflammatory. Its ability to protect not only neurons but also glial cells together with its ability to target and preserve mitochondrial function makes JM-20 a promising molecule that may be able to shield the whole neurovascular unit. The multimodal and multi-cell action of JM-20 may explain the high degree of protection observed in a rat model of brain ischemia, as assayed through histological (hematoxylin-eosin, and luxol fast blue staining), neurochemical (glutamate and aspartate levels in cerebrospinal fluid), mitochondrial functionality and behavioural (neurological scale) analysis at doses of 4 and 8mg/kg. Furthermore, the wide therapeutic window of JM-20 of 8h also suggests that this molecule could be of potential interest in situations where brain perfusion is compromised.

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.

Cerebrospinal Fluid Metabolomics After Natural Product Treatment in an Experimental Model of Cerebral Ischemia.

Cerebrospinal fluid (CSF) is an important biofluid for diagnosis of and research on neurological diseases. However, in-depth metabolomic profiling of CSF remains an analytical challenge due to the small volume of samples, particularly in small animal models. In this work, we report the application of a high-performance chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) workflow for CSF metabolomics in Gastrodia elata and Uncaria rhynchophylla water extract (GUW)-treated experimental cerebral ischemia model of rat. The GUW is a commonly used Traditional Chinese Medicine (TCM) for hypertension and brain disease. This study investigated the amine- and phenol-containing biomarkers in the CSF metabolome. After GUW treatment for 7 days, the neurological deficit score was significantly improved with infarct volume reduction, while the integrity of brain histological structure was preserved. Over 1957 metabolites were quantified in CSF by dansylation LC-MS. The analysis of this comprehensive list of metabolites suggests that metabolites associated with oxidative stress, inflammatory response, and excitotoxicity change during GUW-induced alleviation of ischemic injury. This work is significant in that (1) it shows CIL LC-MS can be used for in-depth profiling of the CSF metabolome in experimental ischemic stroke and (2) identifies several potential molecular targets (that might mediate the central nervous system) and associate with pharmacodynamic effects of some frequently used TCMs.