Ischemic Rat Brain Research Articles

Carotid artery transplantation of brain endothelial cells enhances neuroprotection and neurorepair in ischaemic stroke rats.

Brain microvascular endothelial cells (BMECs), an important component of the neurovascular unit, can promote angiogenesis and synaptic formation in ischaemic mice after brain parenchyma transplantation. Since the therapeutic efficacy of cell-based therapies depends on the extent of transplanted cell residence in the target tissue and cell migration ability, the delivery route has become a hot research topic. In this study, we investigated the effects of carotid artery transplantation of BMECs on neuronal injury, neurorepair, and neurological dysfunction in rats after cerebral ischaemic attack. Purified passage 1 endothelial cells (P1-BMECs) were prepared from mouse brain tissue. Adult rats were subjected to transient middle cerebral artery occlusion (MCAO) for 30 min. Then, the rats were treated with 5 × 105 P1-BMECs through carotid artery infusion or tail vein injection. We observed that carotid artery transplantation of BMECs produced more potent neuroprotective effects than caudal injection in MCAO rats, including reducing infarct size and alleviating neurological deficits in behavioural tests. Carotid artery-transplanted BMECs displayed a wider distribution in the ischaemic rat brain. Immunostaining for endothelial progenitor cells and the mature endothelial cell markers CD34 and RECA-1 showed that carotid artery transplantation of BMECs significantly increased angiogenesis. Carotid artery transplantation of BMECs significantly increased the number of surviving neurons, decreased the cerebral infarction volume, and alleviated neurological deficits. In addition, we found that carotid artery transplantation of BMECs significantly enhanced ischaemia-induced hippocampal neurogenesis, as measured by doublecortin (DCX) and Ki67 double staining within 2 weeks after ischaemic injury. We conclude that carotid artery transplantation of BMECs can promote cerebral angiogenesis, neurogenesis, and neurological function recovery in adult rats after ischaemic stroke. Our results suggest that carotid injection of BMECs may be a promising new approach for treating acute brain injuries.

Effects of intranasal drug delivery on the autonomic system in rehabilitation neurology

Autonomic disorders predominate or are present in most patients with pathological conditions, especially traumatic and ischemic brain injuries. The use of physical factors of therapy, especially electric influence, easily changes the activity of the autonomic system, which adapts the body to external conditions. Of particular interest is the intranasal drug delivery, including neuropeptides, in combination with electric influence while using olfactory autonomic pathways in the central nervous system, a number of visceral reflexes, neurohumoral effects, active moments of galvanization and others. The conducted clinical and experimental studies confirm the pathogenetic mechanisms of intranasal use of peptide compounds and their effectiveness. In experimental brain trauma or ischemia in rats, intranasal administration of neuropeptides normalized bioelectrogenesis, behavioral, and cognitive activity, neurological deficit, and morphological damage. The antioxidant effect was separately confirmed. The work presents several authors’ techniques for intranasal electric influence with drugs for correcting the state of the autonomic system, cerebral hemodynamics in case of ischemic or traumatic damage to the central nervous system. The positive effects of therapy for cognitive deficits, vascular and autonomic disorders are described. Intranasal delivery of neuropeptides to the central nervous system makes it possible to decrease a medication load by reducing their therapeutic doses. Thus, a number of positive effects of the presented technique are described, which should be used in rehabilitation neurology and can be a “trigger” of neurological processes in the central nervous system.

Synergistic effect of chrysin and kaempferol in ameliorating Cerebral Ischemic Reperfusion injury in rat by controlling expression of proinflammatory mediators NF-κB and STAT3

ABSTRACT Objectives The two flavonoids kaempferol and chrysin are known to possess anti-inflammatory and antioxidant activities. In addition, these two flavonoids were reported to display synergistic effects against inflammation. The present study aims to provide an analysis of the combined effects of kaempferol and chrysin on ischemic rat brain induced by endothelin-1. Methods The neurological deficit score and infarct area of the brain were determined post drug treatment. Histopathological sections displayed the morphological changes in the brain tissue. The brain tissues were processed for assessing the antioxidant and anti-inflammatory activity by measuring superoxide dismutase activity, catalase activity, level of reduced glutathione, brain malondialdehyde, and amount of calcium. The expression level of inflammatory molecules was analyzed by western blotting and immunohistochemistry. Results The infarct area, neurological score and NF-κB and STAT3 expression levels were significantly reduced. Discussion The analysis of neuroprotective synergistic activity of kaempferol and chrysin indicated the therapeutic potential of the combination in alleviating cerebral ischemia by controlling expression of proinflammatory mediators.

Human-Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Showed Neuronal Differentiation, Neurite Extension, and Formation of Synaptic Structures in Rodent Ischemic Stroke Brains.

Ischemic stroke is a major cerebrovascular disease with high morbidity and mortality rates; however, effective treatments for ischemic stroke-related neurological dysfunction have yet to be developed. In this study, we generated neural progenitor cells from human leukocyte antigen major loci gene-homozygous-induced pluripotent stem cells (hiPSC-NPCs) and evaluated their therapeutic effects against ischemic stroke. hiPSC-NPCs were intracerebrally transplanted into rat ischemic brains produced by transient middle cerebral artery occlusion at either the subacute or acute stage, and their in vivo survival, differentiation, and efficacy for functional improvement in neurological dysfunction were evaluated. hiPSC-NPCs were histologically identified in host brain tissues and showed neuronal differentiation into vGLUT-positive glutamatergic neurons, extended neurites into both the ipsilateral infarct and contralateral healthy hemispheres, and synaptic structures formed 12 weeks after both acute and subacute stage transplantation. They also improved neurological function when transplanted at the subacute stage with γ-secretase inhibitor pretreatment. However, their effects were modest and not significant and showed a possible risk of cells remaining in their undifferentiated and immature status in acute-stage transplantation. These results suggest that hiPSC-NPCs show cell replacement effects in ischemic stroke-damaged neural tissues, but their efficacy is insufficient for neurological functional improvement after acute or subacute transplantation. Further optimization of cell preparation methods and the timing of transplantation is required to balance the efficacy and safety of hiPSC-NPC transplantation.

Early exercise intervention promotes myelin repair in the brains of ischemic rats by inhibiting the MEK/ERK pathway.

Our previous studies have shown that early exercise intervention after stroke increases neural activity and synaptic plasticity and promotes the recovery of nerve fiber bundle integrity in the brain. However, the effect of exercise on the repair of myelin in the brain and the related mechanism are still unclear. In this study, we randomly divided the rats into three groups. Before and after 28 days of intervention, body weight, nerve function, the infarct size, white matter fiber bundle integrity, and nerve myelin structure and function were observed by measuring body weight, analysis of modified neurological severity score, CatWalk gait analysis, MRI, luxol fast blue staining, immunofluorescence, and transmission electron microscopy. Changes in the expression of proteins in the MEK/ERK pathway were assessed. The results showed that early exercise intervention resulted in neurological recovery, decreased the infarct volume and increased nerve fiber integrity, the myelin coverage area, myelin basic protein (MBP) fluorescence intensity expression, and myelin thickness. Furthermore, the expression level of MBP was significantly increased after early exercise intervention, while the expression levels of p-MEK1/2 and p-ERK1/2 were significantly reduced. In the cell study, MBP expression levels were significantly higher in the oxygen and glucose deprivation and administration group.In summary, early exercise intervention after stroke can promote myelin repair by inhibiting the MEK/ERK signaling pathway.

Hydrogen Sulfide Protects against Rat Ischemic Brain Injury by Promoting RhoA Phosphorylation at Serine 188.

The protective role of hydrogen sulfide against cerebral ischemia-reperfusion injury involves the inhibition of the RhoA-/Rho-associated coiled-coil kinase (ROCK) pathway. However, the specific mechanism remains elusive. This study investigates the impact of hydrogen sulfide on RhoA phosphorylation at serine 188 (Ser188) in vivo, aiming to test the hypothesis that hydrogen sulfide exerts neuroprotection by enhancing RhoA phosphorylation at Ser188, subsequently inhibiting the RhoA/ROCK pathway. Recombinant RhoAwild-pEGFP-N1 and RhoAS188A-pEGFP-N1 plasmids were constructed and administered via stereotaxic injection into the rat hippocampus. A rat global cerebral ischemia-reperfusion model was induced by bilateral carotid artery ligation to elucidate the neuroprotective mechanisms of hydrogen sulfide. Both RhoAwild-pEGFP-N1 and RhoAS188A-pEGFP-N1 plasmids expressed RhoAwild and RhoAS188A proteins, respectively, in rat hippocampal tissues, alongside the intrinsic RhoA protein. Systemic administration of the exogenous hydrogen sulfide donor sodium hydrosulfide led to an increase in Ser188 phosphorylation of transfected RhoAwild and intrinsic RhoA protein within the hippocampus. However, this effect was not observed in tissues transfected with RhoAS188A. Sodium hydrosulfide-mediated RhoA phosphorylation correlated with decreased RhoA and ROCK2 activity in rat hippocampal tissues. Furthermore, sodium hydrosulfide administration reduced cerebral ischemia-reperfusion-induced neuronal damage and apoptosis in rat hippocampal tissues transfected with RhoAwild. However, this neuroprotective effect was attenuated in rats transfected with RhoAS188A. These findings suggest that the neuroprotective mechanism of hydrogen sulfide against cerebral ischemia/reperfusion injury involves increased RhoA phosphorylation at Ser188. Promoting this phosphorylation may represent a potential intrinsic therapeutic target for ischemic stroke.

Effect of 2-Week Naringin Supplementation on Neurogenesis and BDNF Levels in Ischemia–Reperfusion Model of Rats

Background: Ischemic stroke is the leading cause of mortality and disability worldwide with more than half of survivors living with serious neurological sequelae; thus, it has recently attracted a lot of attention in the field of medical study. Purpose: The aim of this study was to determine the effect of naringin supplementation on neurogenesis and brain-derived neurotrophic factor (BDNF) levels in the brain in experimental brain ischemia–reperfusion. Study design: The research was carried out on 40 male Wistar-type rats (10–12 weeks old) obtained from the Experimental Animals Research and Application Center of Selçuk University. Experimental groups were as follows: (1) Control group, (2) Sham group, (3) Brain ischemia–reperfusion group, (4) Brain ischemia–reperfusion + vehicle group (administered for 14 days), and (5) Brain ischemia–reperfusion + Naringin group (100 mg/kg/day administered for 14 days). Methods: In the ischemia–reperfusion groups, global ischemia was performed in the brain by ligation of the right and left carotid arteries for 30 min. Naringin was administered to experimental animals by intragastric route for 14 days following reperfusion. The training phase of the rotarod test was started 4 days before ischemia–reperfusion, and the test phase together with neurological scoring was performed the day before and 1, 7, and 14 days after the operation. At the end of the experiment, animals were sacrificed, and then hippocampus and frontal cortex tissues were taken from the brain. Double cortin marker (DCX), neuronal nuclear antigen marker (NeuN), and BDNF were evaluated in hippocampus and frontal cortex tissues by Real-Time qPCR analysis and immunohistochemistry methods. Results: While ischemia–reperfusion increased the neurological score values, DCX, NeuN, and BDNF levels decreased significantly after ischemia in the hippocampus and frontal cortex tissues. However, naringin supplementation restored the deterioration to a certain extent. Conclusion: The results of the study show that 2 weeks of naringin supplementation may have protective effects on impaired neurogenesis and BDNF levels after brain ischemia and reperfusion in rats.

YTHDF2-regulated matrilin-3 mitigates post-reperfusion hemorrhagic transformation in ischemic stroke via the PI3K/AKT pathway.

Hemorrhagic transformation can complicate ischemic strokes after recanalization treatment within a time window that requires early intervention. To determine potential therapeutic effects of matrilin-3, rat cerebral ischemia-reperfusion was produced using transient middle cerebral artery occlusion (tMCAO); intracranial hemorrhage and infarct volumes were assayed through hemoglobin determination and 2,3,5-triphenyltetrazoliumchloride (TTC) staining, respectively. Oxygen-glucose deprivation (OGD) modeling of ischemia was performed on C8-D1A cells. Interactions between matrilin-3 and YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) were determined using RNA immunoprecipitation assay and actinomycin D treatment. Reperfusion after tMCAO modeling increased hemorrhage, hemoglobin content, and infarct volumes; these were alleviated by matrilin treatment. Matrilin-3 was expressed at low levels and YTHDF2 was expressed at high levels in ischemic brains. In OGD-induced cells, matrilin-3 was negatively regulated by YTHDF2. Matrilin-3 overexpression downregulated p-PI3K/PI3K, p-AKT/AKT, ZO-1, VE-cadherin and occludin, and upregulated p-JNK/JNK in ischemic rat brains; these effects were reversed by LY294002 (a PI3K inhibitor). YTHDF2 knockdown inactivated the PI3K/AKT pathway, inhibited inflammation and decreased blood-brain barrier-related protein levels in cells; these effects were reversed by matrilin-3 deficiency. These results indicate that YTHDF2-regulated matrilin-3 protected ischemic rats against post-reperfusion hemorrhagic transformation via the PI3K/AKT pathway and that matrilin may have therapeutic potential in ischemic stroke.

Preparation of novel S-allyl cysteine chitosan based nanoparticles for use in ischemic brain treatment.

Objective: To enhance the brain bioavailability of S-allyl-l-cysteine (SC) by developing novel S-allyl-l-cysteine chitosan nanoparticles (SC CS NPs) and examining the quantity of SC by developing a novel method of ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in ischemic rat brain treatment. Methods: The ionotropic gelation method was used to develop S-allyl cysteine-loaded CS NPs. The 4-factor, 5-level central composite design was optimized to determine the effect of independent variables, i.e., particle size, polydispersity index (PDI), zeta potential, EE, and loading capacity, together with their characterization, followed by drug release and intranasal permeation to enhance the brain bioavailability and examination of their neurobehavioral and biochemical parameters with their histopathological examination. Results: SC CS NPs were optimized at the particle size of 93.21 ± 3.31 nm (PDI: 0.317 ± 0.003), zeta potential of 44.4 ± 2.93, and drug loading of 41.23 ± 1.97% with an entrapment efficiency of 82.61 ± 4.93% having sustain and controlled release (79.92 ± 3.86%) with great permeation (>80.0%) of SC. SC showed the retention time of 1.021 min and 162.50/73.05 m/z. SC showed good linearity in the range of 5.0-1300.0 ng mL-1, % inter-and-intraday accuracy of 96.00-99.06% and CV of 4.38-4.38%. We observed significant results, i.e., p < 0.001 for improved (AUC)0-24 and Cmax delivered via i.v. and i.n. dose. We also observed the highly significantly observations of SC CS NPs (i.n.) based on their treatment results for the biochemical, neurobehavioral, and histopathological examination in the developed ischemic MCAO brain rat model. Conclusion: The excellent significant role of mucoadhesive CS NPs of SC was proven based on the enhancement in the brain bioavailability of SC via i.n. delivery in rats and easy targeting of the brain for ischemic brain treatment followed by an improvement in neuroprotection based on a very small dose of SC.

The potential effect of salvianolic acid B against rat ischemic brain injury in combination with mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) and Salvianolic acid B (SAB) are known to exert potent anti-inflammatory and anti-oxidative properties. But the effect of SAB and MSCs combination treatment on the cerebral ischemia/reperfusion injury (CI/RI) is not clear. MethodsAfter the CI/RI animal model established, rats were administered with MSCs and SAB individually or combination treatment. To evaluate the therapeutic potential, behavioral tests, TTC staining, Hematoxylin-eosin (HE) staining, and immunofluorescence assays were performed to evaluate the neuroprotection and endogenous neurogenesis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and enzyme linked immunosorbent assay (ELISA) were performed to evaluate the anti-apoptosis and anti-inflammatory effect. Meanwhile, the expression of the TLR4/NF-ĸB/MYD88 signal pathway-related proteins was evaluated by Western blot. ResultsMSCs and SAB individually or combination treatment have protective effect in CI/RI rats. More importantly, the rats with the combination treatment showed a better behavioral recovery, neurogenesis and smaller infarct size compared with the rats administered with MSCs or SAB individually. Further research showed that the combination treatment decreased CI/RI induced inflammatory cytokines and oxidative stress, including inhibiting the production of IL-1β, IL-6, TNF-α, decreasing the levels of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). In addition, the neuroprotection effect of SAB and MSCs combination was achieved through the regulation of TLR4/NF-κB/MyD88 signaling pathway related proteins, including inhibition the protein levels of TLR4, MYD88, p-NF-κB p65, TRAF6-and action of SIRT1 in brain tissues. ConclusionThe present study indicated that the MSCs and SAB combination treatment had better protective effect against rat ischemic brain injury. The combination of SAB and MSCs may provide a potent and promising strategy for the treatment of ischemic stroke and is worthy for further development.

Illustrate the distribution and metabolic regulatory effects of pterostilbene in cerebral ischemia-reperfusion rat brain by mass spectrometry imaging and spatial metabolomics

Pterostilbene is a promising molecule with superior pharmacological activities and pharmacokinetic characteristics compared to its structural analogue resveratrol, which could be used to treat ischemic stroke. However, its mechanism is still unclear. The cutting-edge air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) and spatial metabolomics analysis were applied to investigate the distribution of pterostilbene in ischemic rat brain and the changes of related small molecule metabolic pathways to further explore the potential mechanisms of pterostilbene against cerebral ischemia-reperfusion injury. This research found that pterostilbene could significantly restore cerebral microcirculation blood flow, reduce infarct volume, improve neurological function and ameliorate neuronal damage in ischemic rats. Moreover, pterostilbene was widely and abundantly distributed in ischemic brain tissue, laying a solid foundation for the rescue of ischemic penumbra. Further study revealed that pterostilbene played a therapeutic role in restoring energy supply, rebalancing neurotransmitters, reducing abnormal polyamine accumulation and phospholipid metabolism. These findings offer an opportunity to illustrate novel mechanisms of pterostilbene in the treatment of cerebral ischemia/reperfusion injury resulting from ischemic stroke.

Isoflurane Anesthesia's Impact on Gene Expression Patterns of Rat Brains in an Ischemic Stroke Model.

Ischemic stroke (IS) is one of the most severe brain diseases. Animal models with anesthesia are actively used to study stroke genomics and pathogenesis. However, the anesthesia-related gene expression patterns of ischemic rat brains remain poorly understood. In this study, we sought to elucidate the impact of isoflurane (ISO) anesthesia on the extent of ischemic brain damage and gene expression changes associated with stroke. We used the transient middle cerebral artery occlusion (tMCAO) model under long-term and short-term ISO anesthesia, magnetic resonance imaging (MRI), RNA sequencing, and bioinformatics. We revealed that the volume of cerebral damage at 24 h after tMCAO was inversely proportional to the duration of ISO anesthesia. Then, we revealed hundreds of overlapping ischemia-related differentially expressed genes (DEGs) with a cutoff of >1.5; Padj < 0.05, and 694 and 1557 DEGs only under long-term and short-term anesthesia, respectively, using sham-operated controls. Concomitantly, unique DEGs identified under short-term anesthesia were mainly associated with neurosignaling systems, whereas unique DEGs identified under long-term anesthesia were predominantly related to the inflammatory response. We were able to determine the effects of the duration of anesthesia using isoflurane on the transcriptomes in the brains of rats at 24 h after tMCAO. Thus, specific genome responses may be useful in developing potential approaches to reduce damaged areas after cerebral ischemia and neuroprotection.

Chronic Kombucha Beverage Consumption Attenuates Inflammatory Markers and Histopathology of Brain Tissue in Transnet Global Brain Ischemia in Rats.

There is evidence that kombucha beverage (KB), a traditional fermented beverage, has a preventive effect on experimental brain ischemia. According to our previous studies, pre-treatment of KB attenuates brain edema and improves motor skills and oxidative stress in a rat model of global brain ischemia. This study was designed to evaluate the effects of the pre-treatment of KB, as a novel agent, on pro-inflammatory parameters and brain histopathology changes following global brain ischemia. Adult male Wistar rats were randomly divided into the sham, the control, and the groups treated with kombucha (KB1 and KB2 groups). KB at doses 1 and 2 mL/kg was prescribed two-week consecutive days before induction of global brain ischemia. Global brain ischemia was induced by blocking common carotid arteries for 60min and the following reperfusion by 24h. The serum and brain levels of tumor necrosis factor-α(TNF-α), IL-1β, histopathological change, and infarct volume are determined using the ELISA, hematoxylin and eosin (H&E), and 2,3,5-triphenyl tetrazolium chloride (TTC) staining, respectively. This study indicated that pre-treatment of KB significantly reduced infarct volume, the serum, and brain levels of TNF-α and IL-1β. The histopathological finding of the brain tissue confirmed a protective role for pre-treatment KB in the ischemic rats. Thus, the present study showed that the beneficial effects of KB pre-treatment on brain ischemic may be mediated by decreasing pro-inflammatory parameters.

Hierarchical K-means clustering method for accelerated Lorentzian estimation (KALE) in chemical exchange saturation transfer-magnetic resonance imaging quantification.

Quantification of in vivo chemical exchange saturation transfer (CEST) magnetic resonance signals is challenging due to contamination from coexisting effects, including the direct water effect and asymmetric magnetization transfer. Fitting-based analysis allows the calculation of multiple types of signals from the line shape of Z-spectra. However, the conventional voxelwise method has several drawbacks, including its long computation time and its susceptibility to image noise and Z-spectra oscillations, and it is difficult to determine the initial fitting parameters. Herein, we propose a K-means clustering method for accelerated Lorentzian estimation (KALE) in CEST quantification. Briefly, voxels in CEST images are clustered into K groups according to their Z-spectra characteristics. A 'groupwise' fitting process is then performed with preset initial values, yielding a set of fitted spectra and fitted parameters for each group. With the updated initial values, each group is further clustered into subgroups, and groupwise fitting is performed again. This hierarchical K-means clustering and parameter updating process continues until the pixel number or intensity error meets the termination criteria. Voxelwise fitting could be further conducted to improve the quantification images (termed voxel-K) by utilizing the previous groupwise KALE results as the initial values (termed group-K). Incorporated with Lorentzian difference (LD) quantification, KALE was first optimized and evaluated on 5 healthy human brain datasets at 3 Tesla. Compared with traditional voxel-by-voxel LD quantification, the computation times of group-K and voxel-K were significantly reduced by ~85% and ~70%, respectively (P<0.001). Furthermore, the group-K images exhibited better denoising performance than traditional LD and voxel-K. KALE was further validated on six ischemic rat brains acquired at 7 Tesla, with both LD_group-K and LD_voxel-K displaying almost identical contrast maps with traditional voxelwise maps. When incorporated with the five-pool Lorentzian fitting (LF), KALE exhibited an improved contrast-to-noise ratio (CNR) for amplitude maps of each pool [P=0.003, 0.015, 0.047, and 0.047 for amide, nuclear Overhauser effect (NOE), magnetic transfer (MT) and guanidine amine, respectively] and improved fitting goodness (P=0.033). KALE quantification provides comparable or even superior contrast maps to traditional voxelwise fitting, with significantly reduced computation time. The 'smart' and hierarchical voxel-clustering and parameter updating process of KALE may facilitate more preclinical and clinical CEST applications.

Abstract TP221: Protective Effects Of Galectin-3 On Endothelial Progenitor Cells Under Ischemic Conditions

Stroke remains the major cause of long term functional deficits and death with limited treatment options. Ischemic stroke leads to endothelial dysfunction, blood brain barrier disruption, poor angiogenesis, inflammation, and neuronal cell death. Stem/Progenitor cells including mesenchymal, neural, and endothelial progenitor cells (EPC) can enhance tissue regeneration and repair. However, the beneficial effects of progenitor cells have been modest presumably due to their poor recruitment and survival in an ischemic microenvironment. Galectin-3 (Gal3), a β-galactoside-binding protein controls cell proliferation, survival, angiogenesis, and migration/recruitment. We have shown that the expression of Gal3 is significantly up-regulated in ischemic rat brain, and that blocking of Gal3 decreases the cerebral blood vessel density. We recently demonstrated that Gal3 reduces cerebral infarction and enhances recovery of motor/sensory functions in an animal stroke model. In this study we examined the effects of Gal3 on bone marrow derived EPC. EPCs were characterized by the uptake of acetylated LDL and expression of CD34 and VEGFR2/KDR. We further demonstrate that Gal3 enhances the proliferation, angiogenesis, migration, and survival of bone marrow derived EPCs in an in vitro ischemic conditions following oxygen glucose deprivation. Gal3 effects were associated with up-regulation of chemoattractant SDF1 and VEGF, further emphasizing the role of Gal3 in stem/progenitor function. Overall, these results indicate a key role for Gal3 in neuro-vascular protection and functional recovery following ischemic stroke through modulation of angiogenic and apoptotic pathways in progenitor cells.

Dynamic MRI of the Mesenchymal Stem Cells Distribution during Intravenous Transplantation in a Rat Model of Ischemic Stroke.

Systemic transplantation of mesenchymal stem cells (MSCs) is a promising approach for the treatment of ischemia-associated disorders, including stroke. However, exact mechanisms underlying its beneficial effects are still debated. In this respect, studies of the transplanted cells distribution and homing are indispensable. We proposed an MRI protocol which allowed us to estimate the dynamic distribution of single superparamagnetic iron oxide labeled MSCs in live ischemic rat brain during intravenous transplantation after the transient middle cerebral artery occlusion. Additionally, we evaluated therapeutic efficacy of cell therapy in this rat stroke model. According to the dynamic MRI data, limited numbers of MSCs accumulated diffusely in the brain vessels starting at the 7th minute from the onset of infusion, reached its maximum by 29 min, and gradually eliminated from cerebral circulation during 24 h. Despite low numbers of cells entering brain blood flow and their short-term engraftment, MSCs transplantation induced long lasting improvement of the neurological deficit, but without acceleration of the stroke volume reduction compared to the control animals during 14 post-transplantation days. Taken together, these findings indicate that MSCs convey their positive action by triggering certain paracrine mechanisms or cell-cell interactions or invoking direct long-lasting effects on brain vessels.

Neuroprotective effects of meloxicam on transient brain ischemia in rats: the two faces of anti-inflammatory treatments.

The inflammatory response plays an important role in neuroprotection and regeneration after ischemic insult. The use of non-steroidal anti-inflammatory drugs has been a matter of debate as to whether they have beneficial or detrimental effects. In this context, the effects of the anti-inflammatory agent meloxicam have been scarcely documented after stroke, but its ability to inhibit both cyclooxygenase isoforms (1 and 2) could be a promising strategy to modulate post-ischemic inflammation. This study analyzed the effect of meloxicam in a transient focal cerebral ischemia model in rats, measuring its neuroprotective effect after 48 hours and 7 days of reperfusion and the effects of the treatment on the glial scar and regenerative events such as the generation of new progenitors in the subventricular zone and axonal sprouting at the edge of the damaged area. We show that meloxicam's neuroprotective effects remained after 7 days of reperfusion even if its administration was restricted to the two first days after ischemia. Moreover, meloxicam treatment modulated glial scar reactivity, which matched with an increase in axonal sprouting. However, this treatment decreased the formation of neuronal progenitor cells. This study discusses the dual role of anti-inflammatory treatments after stroke and encourages the careful analysis of both the neuroprotective and the regenerative effects in preclinical studies.

The immunolocalization of cluster of differentiation 31, phalloidin and alpha smooth muscle actin on vascular network of normal and ischemic rat brain

Cluster of differentiation 31 (CD31), phalloidin and alpha smooth muscle actin (α-SMA) have been widely applied to label the cerebral blood vessels in the past years. Although CD31 is mainly used as endothelial marker in determining the cerebral capillaries, it seems likely that its labeling efficiency is closely correlated with the antibodies from the polyclonal or monoclonal one, as well as the conditions of blood vessels. In order to test this phenomenon, we compared the labeling characteristics of goat polyclonal anti-CD31 (gP-CD31) and mouse monoclonal anti-CD31 (mM-CD31) with those of phalloidin and α-SMA on the rat brain in health and ischemia/reperfusion (I/R) with the middle cerebral artery occlusion. By multiple immunofluorescence staining, it was found that gP-CD31 labeling expressed extensively on the cerebral capillaries forming the vascular networks on the normal and ischemic regions, but mM-CD31 labeling mainly presented on the capillaries in the ischemic region. In contrast to the vascular labeling with gP-CD31, phalloidin and α-SMA were mainly expressed on the wall of cortical penetrating arteries, and less on that of capillaries. By three-dimensional reconstruction analysis, it was clearly shown that gP-CD31 labeling was mainly located on the lumen side of vascular wall and was surrounded by phalloidin labeling and α-SMA labeling. These results indicate that gP-CD31 is more sensitive than mM-CD31 for labeling the cerebral vasculature, and is highly compatible with phalloidin and α-SMA for evaluating the cerebral vascular networks under the physiological and pathological conditions.

The Weakened Interaction Between HECTD4 and GluN2B in Ischemic Stroke Promotes Calcium Overload and Brain Injury Through a Mechanism Involving the Decrease of GluN2B and MALT1 Ubiquitination.

Glutamate receptor ionotropic NMDA 2B (GluN2B) plays an essential role in calcium overload during excitotoxicity. Reverse-phase nano-liquid chromatography-tandem mass spectrometry has revealed an interaction between GluN2B and HECT domain E3 ubiquitin protein ligase 4 (HECTD4), an E3 ubiquitin ligase highly expressed in the brain. As a potential substrate for HECTD4, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) acts as a scaffold with hydrolysis activity. This study explores the relationship between HECTD4, GluN2B, and MALT1, focusing on their role in brain injury in ischemic stroke. Rats were subjected to 2h-ischemia followed by 24-h reperfusion to establish an ischemic stroke model. We observed the downregulation of HECTD4 and the upregulation of MALT1. Additionally, an increased GluN2B phosphorylation was concomitant with weakened interactions between HECTD4 and GluN2B, followed by decreased striatal-enriched protein phosphatase (STEP61). Knockdown of HECTD4 exacerbated hypoxia- or NMDA-induced injury in nerve cells coincident with a decrease in GluN2B and MALT1 ubiquitination, and an increase in GluN2B phosphorylation as well as an increase in intracellular calcium level, which were counteracted by MALT1 siRNA. Blockage of MALT1 with its inhibitor or siRNA reduced STEP61 degradation, accompanied by a decrease in GluN2B phosphorylation, intracellular calcium concentration, and brain cell injury, which were reversed by overexpression of MALT1. Based on these observations, we conclude that the downregulation of HECTD4 in ischemic stroke rat brain accounts for calcium overload and brain injury due to activating GluN2B directly and indirectly through a mechanism involving the reduced ubiquitination of GluN2B and MALT1, respectively.

Protective Effect of Beta-Carotene against Myeloperoxidase- Mediated Oxidative Stress and Inflammation in Rat Ischemic Brain Injury.

Oxidative stress and inflammatory reaction play critical roles in ischemia/reperfusion (I/R) injury in the brain. β-carotene (βCAR) is a naturally occurring pigment present in fruits and vegetables that expresses antioxidant and anti-inflammatory activities. This study was conducted to investigate the involvement of Bcl2/Bax and NF-κB signaling pathways in the potential protective role of βCAR against brain injury in a middle cerebral artery occlusion (MCAO) rat model. A focal brain ischemia model was created for 2 h, followed by reperfusion. Rats were given 10 and 20 mg/kg of βCAR for 7 days orally before induction of ischemia, at the start of reperfusion, and 3 days after ischemia. Scores of neurological deficit were rated 24 h after induction of ischemia. Motor coordination and spontaneous coordinate activities were assessed using rotarod and activity cage, respectively. After 2 h of the last dose, the animals were killed and their brains were extracted for further examinations. The results of the study show that βCAR diminished the score of neurological deficits and ameliorated motor coordination, balance, and locomotor activity in the I/R control group. Further, βCAR resulted in diminution of malondialdehyde (MDA) and augmentation of reduced glutathione (GSH) contents, as well as the elevation of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) enzyme activities in the brain homogenates of I/R rats. βCAR treatment significantly reduced nuclear factor kappa B (NF-κB) brain content and myeloperoxidase (MPO) activity and ameliorated the histological alterations in the brain tissues. βCAR significantly suppressed Bcl-2-associated X protein (Bax) and caspase-3 expression, as well as upregulated B-cell lymphoma-2 (Bcl-2) expression, suggesting a neuroprotective potential via downregulating NF-kB and protecting the rat brain against the I/R-associated apoptotic injury.