Experimental Cerebral Ischemia Research Articles

A Novel Biomarker in Experimental Cerebral Ischemia: Junctional Adhesion Molecule-A

Objectives: To investigate the role of blood brain barrier biomarkers for the detection of experimental cerebral ischemia in rats. Methods: Forty adult male Wistar albino rats with a mean age of 4–6 months and an average weight of 350–400 g were used in the study. The rats were divided into five ischemia groups (control, 1.5 h of ischemia, 4.5 h of ischemia, 6 h of ischemia, and 24 h of ischemia). Cerebral ischemia was achieved by unilateral ligating of CCA and ECA at the same time. After surgical preparation and awaiting for appropriate ischemia time we collected blood and brain tissue samples. Then we investigated serum occludin, claudin-5 and JAM-A levels from blood samples and the apoptotic index and percentages of pycnotic nucleus from brain tissues histologically. The obtained data were analyzed using IBM SPSS Statistics software package version 18 and the Jamovi software package. Results: Serum JAM-A level showed a statistically significant difference in all ischemia groups compared with the control group (p

Biological and Procedural Predictors of Outcome in the Stroke Preclinical Assessment Network (SPAN) Trial.

The SPAN trial (Stroke Preclinical Assessment Network) is the largest preclinical study testing acute stroke interventions in experimental focal cerebral ischemia using endovascular filament middle cerebral artery occlusion (MCAo). Besides testing interventions against controls, the prospective design captured numerous biological and procedural variables, highlighting the enormous heterogeneity introduced by the multicenter structure that might influence stroke outcomes. Here, we leveraged the unprecedented sample size achieved by the SPAN trial and the prospective design to identify the biological and procedural variables that affect experimental stroke outcomes in transient endovascular filament MCAo. The study cohort included all mice enrolled and randomized in the SPAN trial (N=1789). Mice were subjected to 60-minute MCAo and followed for a month. Thirteen biological and procedural independent variables and 4 functional (weight loss and 4-point neuroscore on days 1 and 2, corner test on days 7 and 28, and mortality) and 3 tissue (day 2, magnetic resonance imaging infarct volumes and swelling; day 30, magnetic resonance imaging tissue loss) outcome variables were prospectively captured. Multivariable regression with stepwise elimination was used to identify the predictors and their effect sizes. Older age, active circadian stage at MCAo, and thinner and longer filament silicone tips predicted higher mortality. Older age, larger body weight, longer anesthesia duration, and longer filament tips predicted worse neuroscores, while high-fat diet and blood flow monitoring predicted milder neuroscores. Older age and a high-fat diet predicted worse corner test performance. While shorter filament tips predicted more ipsiversive turning, longer filament tips appeared to predict contraversive turning. Age, sex, and weight interacted when predicting the infarct volume. Older age was associated with smaller infarcts on day 2 magnetic resonance imaging, especially in animals with larger body weights; this association was most conspicuous in females. High-fat diet also predicted smaller infarcts. In contrast, the use of cerebral blood flow monitoring and more severe cerebral blood flow drop during MCAo, longer anesthesia, and longer filament tips all predicted larger infarcts. Bivariate analyses among the dependent variables highlighted a disconnect between tissue and functional outcomes. Our analyses identified variables affecting endovascular filament MCAo outcome, an experimental stroke model used worldwide. Multiple regression refuted some commonly reported predictors and revealed previously unrecognized associations. Given the multicenter prospective design that represents a sampling of real-world conditions, the degree of heterogeneity mimicking clinical trials, the large number of predictors adjusted for in the multivariable model, and the large sample size, we think this is the most definitive analysis of the predictors of preclinical stroke outcome to date. Future multicenter experimental stroke trials should standardize or at least ensure a balanced representation of the biological and procedural variables identified herein as potential confounders.

Inhibition of neutrophil rolling and migration by caADAMTS13 in vitro and in mouse models of thrombosis and inflammation

Recent investigation of a constitutively active ADAMTS13 variant (caADAMTS13) in murine models of acute ischaemic stroke (AIS) have revealed a potential anti-inflammatory mechanism of action contributing to its protective effect. However, it remains unclear whether these observations are a direct result of VWF proteolysis by caADAMTS13. We have implemented state of the art in vitro assays of neutrophil rolling and transmigration to quantify the impact of caADAMTS13 on these processes. Moreover, we have tested caADAMTS13 in two in vivo assays of neutrophil migration to confirm the impact of the treatment on the neutrophil response to sterile inflammation. Neutrophil rolling, over an interleukin-1β stimulated hCMEC/D3 monolayer, is directly inhibited by caADAMTS13, reducing the proportion of neutrophils rolling to 9.5 ± 3.8 % compared to 18.0 ± 4.5 % in untreated controls. Similarly, neutrophil transmigration recorded in real-time, was significantly suppressed in the presence of caADAMTS13 which reduced the number of migration events to a level like that in unstimulated controls (18.0 ± 4.5 and 15.8 ± 7.5 cells/mm2/h, respectively). Brain tissue from mice undergoing experimental focal cerebral ischaemia has indicated the inhibition of this process by caADAMTS13. This is supported by caADAMTS13’s ability to reduce neutrophil migration into the peritoneal cavity in an ischaemia-independent model of sterile inflammation, with the VWF-dependent mechanism by which this occurs being confirmed using a second experimental stroke model. These findings will be an important consideration in the further development of caADAMTS13 as a potential therapy for AIS and other thromboinflammatory pathologies, including cardiovascular disease.

Ischemic Neuroprotection by Insulin with Down-Regulation of Divalent Metal Transporter 1 (DMT1) Expression and Ferrous Iron-Dependent Cell Death.

Background: The regulation of divalent metal transporter-1 (DMT1) by insulin has been previously described in Langerhans cells and significant neuroprotection was found by insulin and insulin-like growth factor 1 treatment during experimental cerebral ischemia in acute ischemic stroke patients and in a rat 6-OHDA model of Parkinson's disease, where DMT1 involvement is described. According to the regulation of DMT1, previously described as a target gene of NF-kB in the early phase of post-ischemic neurodegeneration, both in vitro and in vivo, and because insulin controls the NFkB signaling with protection from ischemic cell death in rat cardiomyocytes, we evaluated the role of insulin in relation to DMT1 expression and function during ischemic neurodegeneration. Methods: Insulin neuroprotection is evaluated in differentiated human neuroblastoma cells, SK-N-SH, and in primary mouse cortical neurons exposed to oxygen glucose deprivation (OGD) for 8 h or 3 h, respectively, with or without 300 nM insulin. The insulin neuroprotection during OGD was evaluated in both cellular models in terms of cell death, and in SK-N-SH for DMT1 protein expression and acute ferrous iron treatment, performed in acidic conditions, known to promote the maximum DMT1 uptake as a proton co-transporter; and the transactivation of 1B/DMT1 mouse promoter, already known to be responsive to NF-kB, was analyzed in primary mouse cortical neurons. Results: Insulin neuroprotection during OGD was concomitant to the down-regulation of both DMT1 protein expression and 1B/DMT1 mouse promoter transactivation. We also showed the insulin-dependent protection from cell death after acute ferrous iron treatment. In conclusion, although preliminary, this evaluation highlights the peculiar role of DMT1 as a possible pharmacological target, involved in neuroprotection by insulin during in vitro neuronal ischemia and acute ferrous iron uptake.

Duration of spreading depression is the electrophysiological correlate of infarct growth in malignant hemispheric stroke.

Spreading depolarizations (SD) contribute to lesion progression after experimental focal cerebral ischemia while such correlation has never been shown in stroke patients. In this prospective, diagnostic study, we investigate the association of SDs and secondary infarct progression after malignant hemispheric stroke. SDs were continuously monitored for 3-9 days with electrocorticography after decompressive hemicraniectomy for malignant hemispheric stroke. To ensure valid detection and analysis of SDs, a threshold based on the electrocorticographic baseline activity was calculated to identify valid electrocorticographic recordings. Subsequently SD characteristics were analyzed in association to infarct progression based on serial MRI. Overall, 62 patients with a mean stroke volume of 289.6 ± 68 cm3 were included. Valid electrocorticographic recordings were found in 44/62 patients with a mean recording duration of 139.6 ± 26.5 hours and 52.5 ± 39.5 SDs per patient. Infarct progression of more than 5% was found in 21/44 patients. While the number of SDs was similar between patients with and without infarct progression, the SD-induced depression duration per day was significantly longer in patients with infarct progression (593.8 vs. 314.1 minutes; *p = 0.046). Therefore, infarct progression is associated with a prolonged SD-induced depression duration. Real-time analysis of electrocorticographic recordings may identify secondary stroke progression and help implementing targeted management strategies.

Insulin resistance and stroke: mechanisms and therapeutic approaches

The review analyzed literature data on the epidemio­logy, risk factors, and mechanisms of acute cerebrovascular accident (ACVA) in patients with diabetes mellitus. The role of insulin resistance and the effectiveness of therapeutic approaches to its correction in cerebral stroke are considered. Diabetes mellitus is recognized as an independent modifiable risk factor for ACVA. In people with diabetes of different age, the risk of stroke is increased by 2–6 times, and the indicators are especially high in patients of young working age. The presence of diabetes mellitus is associated with more severe symptoms, increased risk of complications, longer hospitalization, and higher mortality. Research results show that insulin resistance is one of the main triggers for the development of ischemic stroke due to embolism caused by oxidative stress, endothelial dysfunction and platelet hyperactivation, as well as due to atherosclerotic changes caused by inflammation, proliferation of smooth muscle cells of the vascular wall, dyslipidemia and hypertension on the background of hyperglycemia and hyperinsulinemia. It has been proven that insulin resistance not only provokes ACVA, but also negatively affects their prognosis. Metformin is a key drug for improving insulin sensitivity and is recognized as one of the most important first-line therapeutic agents to achieve and maintain treatment goals in patients with type 2 diabetes. The results of expe­rimental and clinical studies proved that this agent has a whole range of neuroprotective properties, which generally prevent the development of cerebral ischemia and reduce the negative consequences in case of its occurrence. Animals with experimental acute cerebral ischemia who have been treated with metformin had a better overall neurological score, significantly smaller infarct size, better coordination scores, and higher numbers of neurons and microglia. The neuroprotective effect of metformin in stroke is realized through the AMPK (5’AMP-activated protein kinase) signaling pathway with reduction of oxidative stress, neuroinflammation, stimulation of angiogenesis and neurogenesis, autophagy, and inhibition of apoptosis. According to data from cohort and randomized clinical trials, the use of metformin is associated with a significantly lower risk of developing ACVA. Long-term use of this drug in type 2 diabetes contributes to a milder course of stroke, is associated with better functional recovery, and a decrease in disability and mortality rates.

Mitochondria-associated endoplasmic reticulum membranes tethering protein VAPB-PTPIP51 protects against ischemic stroke through inhibiting the activation of autophagy.

Mitochondria-associated endoplasmic reticulum membranes (MAMs) serve as a crucial bridge connecting the endoplasmic reticulum (ER) and mitochondria within cells. Vesicle-associated membrane protein-associated protein B (VAPB) and protein tyrosine phosphatase interacting protein 51 (PTPIP51) are responsible for the formation and stability of MAMs, which have been implicated in the pathogenesis of various diseases. However, the role of MAMs in ischemic stroke (IS) remains unclear. We aimed to investigate the role of MAMs tethering protein VAPB-PTPIP51 in experimental cerebral ischemia. We simulated cerebral ischemia-reperfusion injury (CIRI) by using a mouse middle cerebral artery occlusion (MCAO) model. We observed a decrease in VAPB-PTPIP51 expression in the brain tissue. Our findings suggested compromised MAMs after MCAO, as a decreased mitochondria-ER contact (MERC) coverage and an increased distance were observed through the transmission electron microscope (TEM). Upon VAPB or PTPIP51 knockdown, the damage to MAMs was exacerbated, accompanied by excessive autophagy activation and increased reactive oxygen species (ROS) production, resulting in an enlarged infarct area and exacerbated neurological deficits. Notably, we observed that this damage was concomitant with the inhibition of the PI3K/AKT/mTOR pathway and was successfully mitigated by the treatment with the PI3K activator. Our findings suggest that the downregulation of VAPB-PTPIP51 expression after IS mediates structural damage to MAMs. This may exacerbate CIRI by inhibiting the PI3K pathway and activating autophagy, thus providing new therapeutic targets for IS.

P-Glycoprotein Exacerbates Brain Injury Following Experimental Cerebral Ischemia by Promoting Proinflammatory Microglia Activation.

Microglia are activated following cerebral ischemic insult. P-glycoprotein (P-gp) is an efflux transporter on microvascular endothelial cells and upregulated after cerebral ischemia. This study evaluated the effects and possible mechanisms of P-gp on microglial polarization/activation in mice after ischemic stroke. P-gp-specific siRNA and adeno-associated virus (p-AAV) were used to silence and overexpress P-gp, respectively. Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) were performed in mice and cerebral microvascular endothelial cells (bEnd.3) in vitro, respectively. OGD/R-injured bEnd.3 cells were cocultured with mouse microglial cells (BV2) in Transwell. Influences on acute ischemic stroke outcome, the expression of inflammatory cytokines, and chemokines and chemokines receptors, microglial polarization, glucocorticoid receptor (GR) nuclear translocation, and GR-mediated mRNA decay (GMD) activation were evaluated via reverse transcription real-time polymerase chain reaction, western blot, or immunofluorescence. Silencing P-gp markedly alleviated experimental ischemia injury as indicated by reduced cerebral infarct size, improved neurological deficits, and reduced the expression of interleukin-6 (IL-6) and IL-12 expression. Silencing P-gp also mitigated proinflammatory microglial polarization and the expression of C-C motif chemokine ligand 2 (CCL2) and its receptor CCR2 expression, whereas promoted anti-inflammatory microglia polarization. Additionally, P-gp silencing promoted GR nuclear translocation and the expression of GMD relative proteins in endothelial cells. Conversely, overexpressing P-gp via p-AAV transfection offset all these effects. Furthermore, silencing endothelial GR counteracted all effects mediated by silencing or overexpressing P-gp. Elevated P-gp expression aggravated inflammatory response and brain damage after ischemic stroke by augmenting proinflammatory microglial polarization in association with increased endothelial CCL2 release due to GMD inhibition by P-gp.

Downregulation of Nrp1 transcription promotes blood–brain barrier disruption following experimental cerebral ischemia–reperfusion

Disruption of the blood–brain barrier (BBB) following cerebral ischemia–reperfusion injury (CIRI) is a major factor in the pathophysiology of stroke. Endothelial cell–cell communication is essential for maintaining BBB integrity. By analyzing GSE227651 data, we found that a decrease in endothelial cell–cell communication mediated by Sema3/Nrp1 may be due to the downregulation of Nrp1 transcription, which could contribute to BBB breakdown after CIRI. We confirmed this hypothesis by using western blot analysis to show a reduction in Nrp1 protein levels in penumbra endothelial cells after CIRI in mice. We then overexpressed Nrp1 specifically in brain endothelial cells using adeno-associated virus in mice. Furthermore, Nrp1 overexpression had a protective effect on BBB integrity, as evidenced by a decrease in IgG and albumin leakage caused by CIRI in mice. Finally, we found that Nrp1 overexpression also reduced brain cell death and neurological deficits induced by cerebral ischemia–reperfusion in mice. Our findings suggest that Nrp1 downregulation may be a key factor in the breakdown of endothelial cell–cell communication and subsequent BBB disruption following CIRI. Targeting Nrp1-mediated pathways may be a promising approach for mitigating BBB damage and alleviating neurological consequences in stroke patients.

Effects of transcranial direct current stimulation on the glutamatergic pathway in the male rat hippocampus after experimental focal cerebral ischemia.

This study aimed to assess the focal cerebral ischemia-induced changes in learning and memory together with glutamatergic pathway in rats and the effects of treatment of the animals with transcranial Direct Current Stimulation (tDCS). One hundred male rats were divided into five groups as sham, tDCS, Ischemia/Reperfusion (IR), IR + tDCS, and IR + E-tDCS groups. Learning, memory, and locomotor activity functions were evaluated by behavioral experiments in rats. Glutamate and glutamine levels, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR1), N-Methyl-D-Aspartate receptors (NMDAR1 and NMDAR2A), vesicular glutamate transporter-1 (VGLUT-1), and excitatory amino acid transporters (EAAT1-3) mRNA expressions in hippocampus tissues were measured. Ischemic areas were analyzed by TTC staining. The increase was observed in IR + tDCS, and IR + E-tDCS groups compared to the IR group while a significant decrease was observed in IR group compared to the sham in the locomotor activity, learning, and memory tests. While glutamate and glutamine levels, AMPAR1, NMDAR1, NMDAR2A, VGLUT1, and EAAT1 mRNA expressions were significantly higher in IR group compared to the sham group, it was found to be significantly lower in IR + tDCS and IR + E-tDCS groups compared to the IR group. EAAT2 and EAAT3 mRNA expressions were significantly higher in IR + tDCS and IR + E-tDCS groups compared to the IR group. Ischemic areas were significantly decreased in IR + tDCS and IR + E-tDCS groups compared to the IR group. Current results suggest that tDCS application after ischemia improves learning and memory disorders and these effects of tDCS may be provided through transporters that regulate glutamate levels.

Neuroimmune Support of Neuronal Regeneration and Neuroplasticity following Cerebral Ischemia in Juvenile Mice.

Ischemic damage to the brain and loss of neurons contribute to functional disabilities in many stroke survivors. Recovery of neuroplasticity is critical to restoration of function and improved quality of life. Stroke and neurological deficits occur in both adults and children, and yet it is well documented that the developing brain has remarkable plasticity which promotes increased post-ischemic functional recovery compared with adults. However, the mechanisms underlying post-stroke recovery in the young brain have not been fully explored. We observed opposing responses to experimental cerebral ischemia in juvenile and adult mice, with substantial neural regeneration and enhanced neuroplasticity detected in the juvenile brain that was not found in adults. We demonstrate strikingly different stroke-induced neuroimmune responses that are deleterious in adults and protective in juveniles, supporting neural regeneration and plasticity. Understanding age-related differences in neuronal repair and regeneration, restoration of neural network function, and neuroimmune signaling in the stroke-injured brain may offer new insights for the development of novel therapeutic strategies for stroke rehabilitation.

Preconditioning with interleukin-1 alpha is required for the neuroprotective properties of mesenchymal stem cells after ischemic stroke in mice

Mesenchymal stem cell (MSC) pre-conditioning with interleukin-1 alpha (IL-1ɑ) drives MSCs toward a potent anti-inflammatory phenotype. The aim of this study was to assess the therapeutic potential of intra-arterially administered IL-1ɑ preconditioned MSCs, after experimental cerebral ischaemia in mice. After 3 h from the start of middle cerebral artery occlusion, animals were treated with vehicle, 9.1 × 104 non-conditioned or IL-1ɑ preconditioned MSCs by intra-arterial administration. Animals were allowed to recover for 1.5 h after treatment to measure cerebral blood flow (CBF), and 3 days or 14 days post-stroke to evaluate lesion volume and functional outcomes. At 3-days post-stroke preconditioned MSCs reduced (by 67%) lesion volume and increased CBF (by 32%) compared to vehicle, while non-conditioned MSCs had no effect. A separate cohort of animals recovered to 14 days post-stroke also showed reduced infarct volume (by 51%) at 48 h (assessed by MRI) and better functional recovery at 14 days when treated with preconditioned MSCs when compared to vehicle. Preconditioning MSCs with IL-1α increases their neuroprotective capability and improves functional recovery after delayed intra-arterial administration. With increasing use of thrombectomy, the adjunct use of preconditioned MSCs therefore represents a highly relevant therapy to improve outcomes in ischemic stroke.

Tricellulin, α-Catenin and Microfibrillar-Associated Protein 5 Exhibit Concomitantly Altered Immunosignals along with Vascular, Extracellular and Cytoskeletal Elements after Experimental Focal Cerebral Ischemia.

Along with initiatives to understand the pathophysiology of stroke in detail and to identify neuroprotective targets, cell-stabilizing elements have gained increasing attention. Although cell culture experiments have indicated that tricellulin, α-catenin and microfibrillar-associated protein 5 (MFAP5) contribute to cellular integrity, these elements have not yet been investigated in the ischemic brain. Applying immunofluorescence labeling, this study explored tricellulin, MFAP5 and α-catenin in non-ischemic and ischemic brain areas of mice (24, 4 h of ischemia) and rats (4 h of ischemia), along with collagen IV and fibronectin as vascular and extracellular matrix constituents and microtubule-associated protein 2 (MAP2) and neurofilament light chain (NF-L) as cytoskeletal elements. Immunosignals of tricellulin and notably MFAP5 partially appeared in a fiber-like pattern, and α-catenin appeared more in a dotted pattern. Regional associations with vascular and extracellular constituents were found for tricellulin and α-catenin, particularly in ischemic areas. Due to ischemia, signals of tricellulin, MFAP5 and α-catenin decreased concomitantly with MAP2 and NF-L, whereby MFAP5 provided the most sensitive reaction. For the first time, this study demonstrated ischemia-related alterations in tricellulin, MFAP5 and α-catenin along with the vasculature, extracellular matrix and cytoskeleton. Confirmatory studies are needed, also exploring their role in cellular integrity and the potential for neuroprotective approaches in stroke.

Distinct Alterations in Oxygenation, Ion Composition and Acid-Base Balance in Cerebral Collaterals During Large-Vessel Occlusion Stroke

PurposeDisturbances of blood gas and ion homeostasis including regional hypoxia and massive sodium (Na+)/potassium (K+) shifts are a hallmark of experimental cerebral ischemia but have not been sufficiently investigated for their relevance in stroke patients.MethodsWe report a prospective observational study on 366 stroke patients who underwent endovascular thrombectomy (EVT) for large-vessel occlusion (LVO) of the anterior circulation (18 December 2018–31 August 2020). Intraprocedural blood gas samples (1 ml) from within cerebral collateral arteries (ischemic) and matched systemic control samples were obtained according to a prespecified protocol in 51 patients.ResultsWe observed a significant reduction in cerebral oxygen partial pressure (−4.29%, paO2ischemic = 185.3 mm Hg vs. paO2systemic = 193.6 mm Hg; p = 0.035) and K+ concentrations (−5.49%, K+ischemic = 3.44 mmol/L vs. K+systemic = 3.64 mmol/L; p = 0.0083). The cerebral Na+:K+ ratio was significantly increased and negatively correlated with baseline tissue integrity (r = −0.32, p = 0.031). Correspondingly, cerebral Na+ concentrations were most strongly correlated with infarct progression after recanalization (r = 0.42, p = 0.0033). We found more alkaline cerebral pH values (+0.14%, pHischemic = 7.38 vs. pHsystemic = 7.37; p = 0.0019), with a time-dependent shift towards more acidotic conditions (r = −0.36, p = 0.055).ConclusionThese findings suggest that stroke-induced changes in oxygen supply, ion composition and acid-base balance occur and dynamically progress within penumbral areas during human cerebral ischemia and are related to acute tissue damage.

Взаимосвязь амилоидогенеза и таупатии с клиническими исходами ишемии головного мозга. Экспериментальное исследование

Currently, it has been established that increased formation of β-amyloid and tau protein play a significant role in the pathogenesis of cerebral ischemia. At the same time, these molecules can act as prognostic markers of the severity of cerebral ischemia. The aim of the study. To evaluate the peculiarities of changes in the concentration of β-amyloid and tau protein in blood serum and cerebrospinal fluid in rats with experimental subtotal cerebral ischemia, as well as to establish their relationship with the severity of post-ischemic clinical changes. Materials and methods. Subtotal cerebral ischemia was modeled by simultaneous occlusion of common carotid arteries in Wistar rats. The study assessed changes in neurological, sensorimotor and cognitive deficits. The content of β-amyloid and tau protein was assessed in blood serum and cerebrospinal fluid on the 1st, 3rd, 5th and 7th days of ischemia. During the analysis, species-specific kits for solid-phase enzyme immunoassay were used. Results. It was found that in animals with subtotal cerebral ischemia, as the duration of the ischemic period increases from day 1 to day 7, the concentration of β-amyloid and tau protein in both blood serum and cerebrospinal fluid progressively increases. It is worth noting that these changes strongly correlated with changes in the clinical manifestations of cerebral ischemia — neurological, sensorimotor and cognitive deficits.

Изменения содержания АТФ-синтазы в нейронах мозга при экспериментальной церебральной ишемии

Introduction. ATP synthase is a key component of ATP synthesis. The study of its content in brain neurons in experimental cerebral ischemia may reflect changes in the functional state of different neurons and their sensitivity to the pathological effect. The study aimed to reveal patterns in ATP synthase immunoreactivity in neurons of various parts of the rat brain during subtotal ischemia of various durations. Materials and methods. Modeling of subtotal cerebral ischemia (SCI) was carried out by ligation of both common carotid arteries (n=12: control group – n=4, 30-minute SCI – n=4, 3-hour SCI – n=4); the control animals underwent a sham surgery. Frontal paraffin sections were stained according to the Nissl method to identify brain structures and immunohistochemically for ATP synthase. ATP synthase immunoreactivity was expressed in units of optical density ×103. Results. Changes in the immunoreactivity of ATP synthase in brain structures occur to varying degrees and not in all studied structures. The most pronounced and rapid decrease in the content of ATP synthase was observed in telencephalon, namely in the temporal and retrosplenial agranular cortex. The least pronounced changes in the cortex were in the structures of the hippocampus, in the neurons of layer II of the CA2 field. In the structures of the thalamus, hypothalamus, and midbrain the dynamic varied. In the structures of the medulla oblongata, the decrease in immunoreactivity occurs more slowly and less pronounced. Conclusion. During experimental cerebral ischemia the degree and rate of change in the content of ATP synthase in rat brain structures vary greatly and depend both on the brain region and the neurotransmitter nature of neurons. Keywords: ATP synthase, ischemia, immunohistochemistry, brain, rat

Transgenic rat with overproduction of ubiquitous angiotensin-(1-7) presents neuroprotection in a model of ischemia and reperfusion

Recent studies showed that angiotensin-(1-7) has cerebroprotective actions in stroke. In the present study, we aim to test whether tissue overexpression of Angiotensin-(1-7), mainly in the brain provides neuroprotection in a model of ischemia/reperfusion by bilateral common carotid arteries occlusion/reperfusion (BCCAo/R). Evaluation of neurological deficit scores and bilateral asymmetry test (BAT) were performed seven days after transient BCCAo/R in transgenic rats (TG-7371) overexpressing Angiotensin-(1-7) and Sprague-Dawley (SD) rats. To assess blood-brain barrier (BBB) permeability Evans blue dye (EB) was intravenously injected. Cytokine levels were quantified in the whole brain through Elisa assay and oxidative stress was measured 7 days after ischemia. The expression of AT1 and Mas receptors and inducible nitric oxide synthase (iNOS) was evaluated by RT-PCR. Neurological deficits were observed in both SD-BCCAo/R and TG-BCCAo/R, contrasting to sham-operated groups. However, TG-BCCAo/R showed a significant lower neurological score and latency in BAT when compared with SD-BCCAo/R. BBB integrity in TG-BCCAo/R was improved, since these animals showed lower extravasation of EB than SD-BCCAo/R. Interestingly, TG-BCCAo/R presented lower levels of pro-inflammatory cytokines when compared to SD-BCCAo/R. Levels of IL-10 were higher in SD-BCCAo/R than in SD control and even higher in TG-BCCAo/R. TG-BCCAo/R animals presented decreased levels of TBARS and increase in SOD activity and GSH levels when compared to SD sham rats. RT-PCR results showed higher levels of AT1 receptor and iNOS in SD-BCCAo/R compared to TG-BCCAo/R, but no difference was observed for Mas receptor. The present study shows that lifetime increase in cerebral expression of an Ang-(1-7)-producing fusion protein induces neuroprotection in experimental global cerebral ischemia and reperfusion, reassuring that, pharmacological strategies leading to increase in Ang-(1-7) can be an additional tool for stroke therapy.

Cannabidiol Exerts a Neuroprotective and Glia-Balancing Effect in the Subacute Phase of Stroke.

Pharmacological agents limiting secondary tissue loss and improving functional outcomes after stroke are still limited. Cannabidiol (CBD), the major non-psychoactive component of Cannabis sativa, has been proposed as a neuroprotective agent against experimental cerebral ischemia. The effects of CBD mostly relate to the modulation of neuroinflammation, including glial activation. To investigate the effects of CBD on glial cells after focal ischemia in vivo, we performed time-lapse imaging of microglia and astroglial Ca2+ signaling in the somatosensory cortex in the subacute phase of stroke by in vivo two-photon laser-scanning microscopy using transgenic mice with microglial EGFP expression and astrocyte-specific expression of the genetically encoded Ca2+ sensor GCaMP3. CBD (10 mg/kg, intraperitoneally) prevented ischemia-induced neurological impairment, reducing the neurological deficit score from 2.0 ± 1.2 to 0.8 ± 0.8, and protected against neurodegeneration, as shown by the reduction (more than 70%) in Fluoro-Jade C staining (18.8 ± 7.5 to 5.3 ± 0.3). CBD reduced ischemia-induced microglial activation assessed by changes in soma area and total branch length, and exerted a balancing effect on astroglial Ca2+ signals. Our findings indicate that the neuroprotective effects of CBD may occur in the subacute phase of ischemia, and reinforce its strong anti-inflammatory property. Nevertheless, its mechanism of action on glial cells still requires further studies.

Transcranial-Direct-Current-Stimulation Accelerates Motor Recovery After Cortical Infarction in Mice: The Interplay of Structural Cellular Responses and Functional Recovery

Background Transcranial direct current stimulation (tDCS) promotes recovery after stroke in humans. The underlying mechanisms, however, remain to be elucidated. Animal models suggest tDCS effects on neuroinflammation, stem cell proliferation, neurogenesis, and neural plasticity. Objective In a longitudinal study, we employed tDCS in the subacute and chronic phase after experimental focal cerebral ischemia in mice to explore the relationship between functional recovery and cellular processes. Methods Mice received photothrombosis in the right motor cortex, verified by Magnetic Resonance Imaging. A composite neuroscore quantified subsequent functional deficits. Mice received tDCS daily: either 5 sessions from day 5 to 9, or 10 sessions with days 12 to 16 in addition. TDCS with anodal or cathodal polarity was compared to sham stimulation. Further imaging to assess proliferation and neuroinflammation was performed by immunohistochemistry at different time points and Positron Emission Tomography at the end of the observation time of 3 weeks. Results Cathodal tDCS at 198 kC/m2 (220 A/m2) between days 5 and 9 accelerated functional recovery, increased neurogenesis, decreased microglial activation, and mitigated CD16/32-expression associated with M1-phenotype. Anodal tDCS exerted similar effects on neurogenesis and microglial polarization but not on recovery of function or microglial activation. TDCS on days 12 to 16 after stroke did not induce any further effects, suggesting that the therapeutic time window was closed by then. Conclusion Overall, data suggest that non-invasive neuromodulation by tDCS impacts neurogenesis and microglial activation as critical cellular processes influencing functional recovery during the early phase of regeneration from focal cerebral ischemia.

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.