Model Of Focal Ischemia Research Articles

A molecular brain atlas reveals cellular shifts during the repair phase of stroke.

Ischemic stroke triggers a cascade of pathological events that affect multiple cell types and often lead to incomplete functional recovery. Despite advances in single-cell technologies, the molecular and cellular responses that contribute to long-term post-stroke impairment remain poorly understood. To gain better insight into the underlying mechanisms, we generated a single-cell transcriptomic atlas from distinct brain regions using a mouse model of permanent focal ischemia at one month post-injury. Our findings reveal cell- and region-specific changes within the stroke-injured and peri-infarct brain tissue. For instance, GABAergic and glutamatergic neurons exhibited upregulated genes in signaling pathways involved in axon guidance and synaptic plasticity, and downregulated pathways associated with aerobic metabolism. Using cell-cell communication analysis, we identified increased strength in predicted interactions within stroke tissue among both neural and non-neural cells via signaling pathways such as those involving collagen, protein tyrosine phosphatase receptor, neuronal growth regulator, laminin, and several cell adhesion molecules. Furthermore, we found a strong correlation between mouse transcriptome responses after stroke and those observed in human nonfatal brain stroke lesions. Common molecular features were linked to inflammatory responses, extracellular matrix organization, and angiogenesis. Our findings provide a detailed resource for advancing our molecular understanding of stroke pathology and for discovering therapeutic targets in the repair phase of stroke recovery.

Impact of inflammatory preconditioning on murine microglial proteome response induced by focal ischemic brain injury.

Preconditioning with lipopolysaccharide (LPS) induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are resident immune cells of the central nervous system (CNS) that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, - using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins signal transducer and activator of transcription1/2 (STAT1/2).

Disrupting PIAS3-mediated SUMOylation of MLK3 ameliorates poststroke neuronal damage and deficits in cognitive and sensorimotor behaviors

Activated small ubiquitin-like modifiers (SUMOs) have been implicated in neuropathological processes following ischemic stroke. However, the target proteins of SUMOylation and their contribution to neuronal injury remain to be elucidated. MLK3 (mixed-lineage kinase 3), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, is a critical regulator of neuronal lesions following cerebral ischemia. Here, we found that SUMOylation of MLK3 increases in both global and focal ischemic rodent models and primary neuronal models of oxygen and glucose deprivation (OGD). SUMO1 conjugation at the Lys401 site of MLK3 promoted its activation, stimulated its downstream p38/c-Jun N-terminal kinase (JNK) cascades, and led to cell apoptosis. The interaction of MLK3 with PIAS3, a SUMO ligase, was elevated following ischemia and reperfusion. The PINIT domain of PIAS3 was involved in direct interactions with MLK3. Overexpression of the PINIT domain of PIAS3 disrupted the MLK3-PIAS3 interaction, inhibited SUMOylation of MLK3, suppressed downstream signaling, and reduced cell apoptosis and neurite damage. In rodent ischemic models, the overexpression of the PINIT domain reduced brain lesions and alleviated deficits in learning, memory, and sensorimotor functions. Our findings demonstrate that brain ischemia-induced MLK3 SUMOylation by PIAS3 is a potential target against poststroke neuronal lesions and behavioral impairments.

Mitochondrial transplantation confers protection against the effects of ischemic stroke by repressing microglial pyroptosis and promoting neurogenesis.

Abstract JOURNAL/nrgr/04.03/01300535-202406000-00037/inline-graphic1/v/2023-10-30T152229Z/r/image-tiff Transferring healthy and functional mitochondria to the lateral ventricles confers neuroprotection in a rat model of ischemia-reperfusion injury. Autologous mitochondrial transplantation is also beneficial in pediatric patients with cardiac ischemia-reperfusion injury. Thus, transplantation of functional exogenous mitochondria may be a promising therapeutic approach for ischemic disease. To explore the neuroprotective effect of mitochondria transplantation and determine the underlying mechanism in ischemic stroke, in this study we established a photo-thrombosis-induced mouse model of focal ischemia and administered freshly isolated mitochondria via the tail vein or to the injury site (in situ). Animal behavior tests, immunofluorescence staining, 2,3,5-triphenyltetrazolium chloride (TTC) staining, mRNA-seq, and western blotting were used to assess mouse anxiety and memory, cortical infarct area, pyroptosis, and neurogenesis, respectively. Using bioinformatics analysis, western blotting, co-immunoprecipitation, and mass spectroscopy, we identified S100 calcium binding protein A9 (S100A9) as a potential regulator of mitochondrial function and determined its possible interacting proteins. Interactions between exogenous and endogenous mitochondria, as well as the effect of exogenous mitochondria on recipient microglia, were assessed in vitro. Our data showed that: (1) mitochondrial transplantation markedly reduced mortality and improved emotional and cognitive function, as well as reducing infarct area, inhibiting pyroptosis, and promoting cortical neurogenesis; (2) microglial expression of S100A9 was markedly increased by ischemic injury and regulated mitochondrial function; (3) in vitro, exogenous mitochondria enhanced mitochondrial function, reduced redox stress, and regulated microglial polarization and pyroptosis by fusing with endogenous mitochondria; and (4) S100A9 promoted internalization of exogenous mitochondria by the microglia, thereby amplifying their pro-proliferation and anti-inflammatory effects. Taken together, our findings show that mitochondrial transplantation protects against the deleterious effects of ischemic stroke by suppressing pyroptosis and promoting neurogenesis, and that S100A9 plays a vital role in promoting internalization of exogenous mitochondria.

Inhibiting metabotropic glutamate receptor 5 after stroke restores brain function and connectivity.

Stroke results in local neural disconnection and brain-wide neuronal network dysfunction leading to neurological deficits. Beyond the hyper-acute phase of ischaemic stroke, there is no clinically-approved pharmacological treatment that alleviates sensorimotor impairments. Functional recovery after stroke involves the formation of new or alternative neuronal circuits including existing neural connections. The type-5 metabotropic glutamate receptor (mGluR5) has been shown to modulate brain plasticity and function and is a therapeutic target in neurological diseases outside of stroke. We investigated whether mGluR5 influences functional recovery and network reorganization rodent models of focal ischaemia. Using multiple behavioural tests, we observed that treatment with negative allosteric modulators (NAMs) of mGluR5 (MTEP, fenobam and AFQ056) for 12 days, starting 2 or 10 days after stroke, restored lost sensorimotor functions, without diminishing infarct size. Recovery was evident within hours after initiation of treatment and progressed over the subsequent 12 days. Recovery was prevented by activation of mGluR5 with the positive allosteric modulator VU0360172 and accelerated in mGluR5 knock-out mice compared with wild-type mice. After stroke, multisensory stimulation by enriched environments enhanced recovery, a result prevented by VU0360172, implying a role of mGluR5 in enriched environment-mediated recovery. Additionally, MTEP treatment in conjunction with enriched environment housing provided an additive recovery enhancement compared to either MTEP or enriched environment alone. Using optical intrinsic signal imaging, we observed brain-wide disruptions in resting-state functional connectivity after stroke that were prevented by mGluR5 inhibition in distinct areas of contralesional sensorimotor and bilateral visual cortices. The levels of mGluR5 protein in mice and in tissue samples of stroke patients were unchanged after stroke. We conclude that neuronal circuitry subserving sensorimotor function after stroke is depressed by a mGluR5-dependent maladaptive plasticity mechanism that can be restored by mGluR5 inhibition. Post-acute stroke treatment with mGluR5 NAMs combined with rehabilitative training may represent a novel post-acute stroke therapy.

TRPC Channels Activated by G Protein-Coupled Receptors Drive Ca2+ Dysregulation Leading to Secondary Brain Injury in the Mouse Model

Canonical transient receptor potential (TRPC) non-selective cation channels, particularly those assembled with TRPC3, TRPC6, and TRPC7 subunits, are coupled to Gαq-type G protein-coupled receptors for the major classes of excitatory neurotransmitters. Sustained activation of this TRPC channel-based pathophysiological signaling hub in neurons and glia likely contributes to prodigious excitotoxicity-driven secondary brain injury expansion. This was investigated in mouse models with selective Trpc gene knockout (KO). In adult cerebellar brain slices, application of glutamate and the class I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine to Purkinje neurons expressing the GCaMP5g Ca2+ reporter demonstrated that the majority of the Ca2+ loading in the molecular layer dendritic arbors was attributable to the TRPC3 effector channels (Trpc3KO compared with wildtype (WT)). This Ca2+ dysregulation was associated with glutamate excitotoxicity causing progressive disruption of the Purkinje cell dendrites (significantly abated in a GAD67-GFP-Trpc3KO reporter brain slice model). Contribution of the Gαq-coupled TRPC channels to secondary brain injury was evaluated in a dual photothrombotic focal ischemic injury model targeting cerebellar and cerebral cortex regions, comparing day 4 post-injury in WT mice, Trpc3KO, and Trpc1/3/6/7 quadruple knockout (TrpcQKO), with immediate 2-h (primary) brain injury. Neuroprotection to secondary brain injury was afforded in both brain regions by Trpc3KO and TrpcQKO models, with the TrpcQKO showing greatest neuroprotection. These findings demonstrate the contribution of the Gαq-coupled TRPC effector mechanism to excitotoxicity-based secondary brain injury expansion, which is a primary driver for mortality and morbidity in stroke, traumatic brain injury, and epilepsy.

Group 2 innate lymphoid cells resolve neuroinflammation following cerebral ischaemia

BackgroundAcute brain ischaemia elicits pronounced inflammation, which aggravates neural injury. However, the mechanisms governing the resolution of acute neuroinflammation remain poorly understood. In contrast to regulatory T and B cells,...

Effects of the New Thrombolytic Compound LT3001 on Acute Brain Tissue Damage After Focal Embolic Stroke in Rats.

LT3001 is a novel synthetic small molecule with thrombolytic and free radical scavenging activities. In this study, we tested the effects of LT3001 as a potential alternative thrombolytic in focal embolic ischemic stroke rat model. Stroked rats received intravenous injection of 10mg/kg LT3001 or tPA at 1.5, 3, or 4.5h after stroke, respectively, and the outcomes were measured at different time points after stroke by performing multi-parametric MRI, 2,3,5-triphenyltetrazolium chloride (TTC) staining, and modified neurological severity score. Lastly, we assessed the effect of LT3001 on the tPA activity in vitro, the international normalized ratio (INR), and the serum levels of active tPA and plasminogen activator inhibitor-1 (PAI-1). LT3001 treated at 1.5h after stroke is neuroprotective by reducing the CBF lesion size and lowering diffusion and T2 lesion size measured by MRI, which is consistent with the reduction in TTC-stained infarction. When treated at 3h after stroke, LT3001 had significantly better therapeutic effects regarding reduction of infarct size, swelling rate, and hemorrhagic transformation compared to tPA. When treated at 4.5h after stroke, tPA, but not LT3001, significantly increased brain swelling and intracerebral hemorrhagic transformation. Lastly, LT3001 did not interfere with tPA activity in vitro, or significantly alter the INR and serum levels of active tPA and PAI-1 in vivo. Our data suggests that LT3001 is neuroprotective in focal embolic stroke rat model. It might have thrombolytic property, not interfere with tPA/PAI-1 activity, and cause less risk of hemorrhagic transformation compared to the conventional tPA. Taken together, LT3001 might be developed as a novel therapy for treating thrombotic ischemic stroke.

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.

Taurine-Upregulated Gene 1 Attenuates Cerebral Angiogenesis following Ischemic Stroke in Rats.

Angiogenesis is one of the therapeutic targets of cerebral infarction. Long noncoding RNAs (lncRNAs) can regulate the pathological process of angiogenesis following ischemic stroke. Taurine-upregulated gene 1 (TUG1), an lncRNA, is correlated to ischemic stroke. We intended to determine the effect of TUG1 on angiogenesis following an ischemic stroke. Middle cerebral artery occlusion (MCAO) was adopted to build a focal ischemic model of the rat brain, and pcDNA-TUG1 and miR-26a mimics were injected into rats. Neurological function was estimated through modified neurological severity scores. The volume of focal brain infarction was calculated through 2,3,5-triphenyltetrazolium chloride staining. The level of TUG1 and miR-26a was measured by PCR. The expression of vascular endothelial growth factor (VEGF) and CD31 was checked using immunohistochemistry and western blot. The correlation between miR-26a and TUG1 was verified through a luciferase reporter assay. TUG1 increased noticeably while miR-26a was markedly reduced in MCAO rats. Overexpression of miR-26a improved neurological function recovery and enhanced cerebral angiogenesis in MCAO rats. TUG1 overexpression aggravated neurological deficits and suppressed cerebral angiogenesis in MCAO rats. Bioinformatics analysis revealed that miR-26a was one of the predicted targets of TUG1. Furthermore, TUG1 combined with miR-26a to regulate angiogenesis. TUG1 overexpression antagonized the role of miR-26a in neurological recovery and angiogenesis in MCAO rats. TUG1/miR-26a, which may act as a regulatory axis in angiogenesis following ischemic stroke, can be considered a potential target for cerebral infarction therapy.

Laser Speckle Flowmetry for the Prognostic Estimation Study of Permanent Focal Ischemia in Mice

The distal middle cerebral artery occlusion (dMCAO) model that mainly targets the cortex and causes low mortality is developed for the study of permanent focal ischemia, and it is highly appropriate for the study in the aged population. The two most common methods used to establish dMCAO models are dMCAO alone and dMCAO plus ipsilateral common carotid artery occlusion (CCAO). Up to now, studies on the prognosis of the two types of dMCAO models and the accuracy of cerebral blood flow (CBF) in predicting prognosis have not yet been reported. In the present study, we established permanent focal ischemia models in two groups of aged mice by dMCAO alone or by dMCAO plus ipsilateral common carotid artery occlusion (CCAO). CBF was evaluated by laser speckle flowmetry (LSF) before and after surgery. Cerebral infarction was assessed by TTC staining at day 2 after surgery and MAP2 staining at day 21 after surgery. In addition, behavioral outcomes were evaluated using the modified Garcia scoring system, adhesive removal test, and foot-fault test. Our results showed that compared with those in the dMCAO alone group, the mice in the dMCAO plus CCAO group had a larger cerebral infarct size and more severe neurological deficits. According to the results of the correlation analysis, the area of the ischemic core region on CBF imaging in the dMCAO group was helpful in predicting the infarct volume. In addition, the total CBF of the ischemic area in the dMCAO plus CCAO group showed a significant correlation with Garcia scores 3 days after surgery, but there was no significant correlation of CBF imaging with the foot-fault test 7 days after surgery. These results suggest that the total CBF of the ischemic area might be helpful to predict the severity of neurological damage at the acute stage.

Transient Intraluminal Filament Middle Cerebral Artery Occlusion Stroke Model in Rats: A Step-by-Step Guide and Technical Considerations

Stroke is a leading cause of disability and mortality worldwide. Related research, although already providing significant insights regarding the underlying pathophysiology and potential treatment strategies, has been far from conclusive. Stroke models have been proved of extreme significance for laboratories around the world. In the present report, we have described in detail the most popular to date focal stroke model, the transient intraluminal filament middle cerebral artery occlusion (tifMCAO) model in rats. This model reliably mimics stroke in humans and also approximates endovascular thrombectomy. The tifMCAO model was performed using Wistar rats weighing 300-400 g. We have described the surgical technique in a stepwise manner, with figures and/or high-definition video provided for each step. We have also introduced the use of complete arteriotomy of the external carotid artery stump during the procedure. We performed tifMCAO in 65 rats (male and female) involved in various experimental protocols. Although the initial mortality was 48%, practice reduced the rate to 10%. The mean procedural time was 53 minutes (range, 38-85 minutes). In a group of 8 rats ischemia was confirmed in 7 of them, with the stroke induction rate being 87.5%. The tifMCAO stroke model in rats is the most often used experimental model of focal ischemia because of its clinical relevance. We revisited the procedure and divided it, for instructional purposes, into 15 consecutive and distinct steps.

Establishment of the reproducible branch retinal artery occlusion mouse model and intravital longitudinal imaging of the retinal CX3CR1-GFP+ cells after spontaneous arterial recanalization.

Animal models of retinal artery occlusion (RAO) have been widely used in many studies. However, most of these studies prefer using a central retinal artery occlusion (CRAO) which is a typical global ischemia model of the retina, due to the technical limitation of producing single vessel targeted modeling with real-time imaging. A focal ischemia model, such as branch retinal artery occlusion (BRAO), is also needed for explaining interactions, including the immunological reaction between the ischemic retina and adjacent healthy retina. Accordingly, a relevant model for clinical RAO patients has been demanded to understand the pathophysiology of the RAO disease. Herein, we establish a convenient BRAO mouse model to research the focal reaction of the retina. As a photo-thrombotic agent, Rose bengal was intravenously injected into 7 week-old transgenic mice (CX3CR1-GFP) for making embolism occlusion, which causes pathology similarly to clinical cases. In an optimized condition, a 561 nm laser (13.1 mw) was projected to a targeted vessel to induce photo-thrombosis for 27 s by custom-built retinal confocal microscopy. Compared to previous BRAO models, the procedures of thrombosis generation were naturally and minimal invasively generated with real-time retinal imaging. In addition, by utilizing the self-remission characteristics of Rose bengal thrombus, a reflow of the BRAO with immunological reactions of the CX3CR1-GFP+ inflammatory cells such as the retinal microglia and monocytes was monitored and analyzed. In this models, reperfusion began on day 3 after modeling. Simultaneously, the activation of CX3CR1-GFP+ inflammatory cells, including the increase of activation marker and morphologic change, was confirmed by immunohistochemical (IHC) staining and quantitative real-time PCR. CD86 and Nox2 were prominently expressed on day 3 after the modeling. At day 7, blood flow was almost restored in the large vessels. CX3CR1-GFP+ populations in both superficial and deep layers of the retina also increased around even in the BRAO peri-ischemic area. In summary, this study successfully establishes a reproducible BRAO modeling method with convenient capabilities of easily controllable time points and selection of a specific single vessel. It can be a useful tool to analyze the behavior of inflammatory cell after spontaneous arterial recanalization in BRAO and further investigate the pathophysiology of BRAO.

Connexin 43 Mediated the Angiogenesis of Buyang Huanwu Decoction via Vascular Endothelial Growth Factor and Angiopoietin-1 after Ischemic Stroke

Buyang Huanwu decoction (BYHWD), a classical prescription for ischemic stroke, has been reported to promote angiogenesis after focal ischemia. However, the mechanisms of the contribution of BYHWD on angiogenesis are still unclear. Connexin 43 (Cx43) played important roles in the functions of neurogliovascular unit. Therefore, the aim of this study was to explore the potential role of Cx43 in angiogenesis of the ischemic brain after BYHWD treatment. Middle cerebral artery occlusion (MCAO) was used to establish the model of focal ischemia. BYHWD was administrated intragastrically twice a day after MCAO with or without Gap26 (a specific Cx43 inhibitor). Western blot, neurological deficits, immunofluorescent staining, and Evans blue dye were used to confirm the role of Cx43 in angiogenesis after BYHWD treatment. The expression levels of total Cx43 and phosphorylated Cx43 were upregulated by BYHWD and peaked at 7 days post MCAO. Inhibition of Cx43 with Gap26 significantly attenuated the protective role of BYHWD in neurological behavior. BYHWD treatment promoted angiogenesis demonstrated by increased microvascular density, upregulated vascular endothelial growth factor (VEGF), and angiopoietin-1 (Ang-1), while inhibition of Cx43 with Gap26 attenuated these effects of BYHWD. In addition, Gap26 inhibited the beneficial effect of BYHWD on blood-brain barrier (BBB) integrity. These results suggested that Cx43 mediated the angiogenesis of BYHWD via VEGF and Ang-1 after focal ischemic stroke.

Effect of Neurorepair for Motor Functional Recovery Enhanced by Total Saponins From Trillium tschonoskii Maxim. Treatment in a Rat Model of Focal Ischemia.

Trillium tschonoskii Maxim. (TTM), is a perennial herb from Liliaceae, that has been widely used as a traditional Chinese medicine treating cephalgia and traumatic hemorrhage. The present work was designed to investigate whether the total saponins from Trillium tschonoskii Maxim. (TSTT) would promote brain remodeling and improve gait impairment in the chronic phase of ischemic stroke. A focal ischemic model of male Sprague-Dawley (SD) rats was established by permanent middle cerebral artery occlusion (MCAO). Six hours later, rats were intragastrically treated with TSTT (120, 60, and 30 mg/kg) and once daily up to day 30. The gait changes were assessed by the CatWalk-automated gait analysis system. The brain tissues injuries, cerebral perfusion and changes of axonal microstructures were detected by multimodal magnetic resonance imaging (MRI), followed by histological examinations. The axonal regeneration related signaling pathways including phosphatidylinositol 3-kinases (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3 (GSK-3)/collapsin response mediator protein-2 (CRMP-2) were measured by western blotting. TSTT treatment significantly improved gait impairment of rats. MRI analysis revealed that TSTT alleviated tissues injuries, significantly improved cerebral blood flow (CBF), enhanced microstructural integrity of axon and myelin sheath in the ipsilesional sensorimotor cortex and internal capsule. In parallel to MRI findings, TSTT preserved myelinated axons and promoted oligodendrogenesis. Specifically, TSTT interventions markedly up-regulated expression of phosphorylated GSK-3, accompanied by increased expression of phosphorylated PI3K, AKT, but reduced phosphorylated CRMP-2 expression. Taken together, our results suggested that TSTT facilitated brain remodeling. This correlated with improving CBF, encouraging reorganization of axonal microstructure, promoting oligodendrogenesis and activating PI3K/AKT/GSK-3/CRMP-2 signaling, thereby improving poststroke gait impairments.

Effect of Lithium Chloride Concentration on Its Neuroprotective Properties in Ischemic Stroke in Rats

Currently, a number of experimental studies have demonstrated compelling evidence of neuro-, cardio-, and nephroprotective properties of medications containing lithium chloride.Aim of the study. To evaluate the effect of various concentrations of lithium chloride on ischemic stroke volume and perifocal edema in rats after cerebral ischemia.Material and methods. Male mongrel rats weighing 315±13.5 g were used in the study. The focal ischemia model according to Longa et al. was employed. The animals (n=35) were divided into 5 groups: sham-operated, control group (ischemic stroke model with NaCl 0.9% administration) and three groups who received lithium chloride in different concentrations (4.2 mg/kg, 21 mg/kg and 63 mg/kg). Lithium chloride was administered immediately after cessation of middle cerebral artery occlusion and then every 24 h until euthanasia. To assess the degree of brain damage, the animals underwent magnetic resonance imaging (MRI) on day 2, and brain sections stained with 2,3,5-triphenyltetrazolium chloride were evaluated after euthanasia on day 7. Intergroup differences were assessed using the Mann-Whitney criterion.Results. According to MRI data, lithium chloride at a dose of 4.2 mg/kg had no significant effect on ischemic stroke volume and perifocal edema versus the control group on day 2 (P=0.9). With lithium chloride at 21 mg/kg, stroke volume and perifocal edema were significantly lower than in the control group (by 25%, P=0.04 and 18%, P=0.03, respectively). Lithium chloride at a dose of 63 mg/kg was more likely to reduce stroke volume (by 45%, P=0.004) and perifocal edema (by 35%, P=0.007). When determining lesion volume on day 7, the data were comparable to those obtained on day 2. With the 21 mg/kg dose, stroke volume was 20% lower than in the control group (P=0.04). Lithium chloride, 63 mg/kg, reduced stroke volume by 40% (P=0.004).Conclusion. Lithium chloride dose affects necrotic focus formation and manifestations of perifocal cerebral edema after middle cerebral artery occlusion. The maximum reduction in the volume of ischemic stroke and perifocal edema was observed when the 63 mg/kg dose was used.

Intranasal Delivery of RGD-Containing Osteopontin Heptamer Peptide Confers Neuroprotection in the Ischemic Brain and Augments Microglia M2 Polarization.

Osteopontin (OPN), a phosphorylated glycoprotein, is induced in response to tissue damage and inflammation in various organs, including the brain. In our previous studies, we reported the robust neuroprotective effects of the icosamer OPN peptide OPNpt20, containing arginine-glycine-aspartic acid (RGD) and serine-leucine-alanine-tyrosine (SLAY) motifs, in an animal model of transient focal ischemia and demonstrated that its anti-inflammatory, pro-angiogenic, and phagocytosis inducing functions are responsible for the neuroprotective effects. In the present study, we truncated OPNpt20 to 13 or 7 amino acid peptides containing RGD (R) and/or SLAY (S) motifs (OPNpt13RS, OPNpt7R, OPNpt7RS, and OPNpt7S), and their neuroprotective efficacy was examined in a rat middle cerebral artery occlusion (MCAO) model. Intranasal administration of all four peptides significantly reduced infarct volume; OPNpt7R (VPNGRGD), the 7-amino-acid peptide containing an RGD motif, was determined to be the most potent, with efficacy comparable to that of OPNpt20. Additionally, sensory–motor functional deficits of OPNpt7R-administered MCAO animals were significantly improved, as indicated by the modified neurological severity scores and rotarod test. Notably, the expression of M1 markers was suppressed, whereas that of M2 markers (Arginase 1, CD206, and VEGF) was significantly enhanced in OPNpt7R-treated primary microglia cultures. Inflammation resolution by OPNpt7R was further confirmed in MCAO animals, in which upregulation of anti-inflammatory cytokines (Arg1, IL-10, IL-4, and CD36) and enhanced efferocytosis were detected. Moreover, studies using three mutant peptides (OPNpt7R-RAA or OPNpt7R-RAD, where RGD was replaced with RAA or RAD, respectively, and OPNpt7R-sc containing scrambled sequences) revealed that the RGD motif plays a vital role in conferring neuroprotection. In conclusion, the RGD-containing OPN heptamer OPNpt7R exhibits neuroprotective effects in the post-ischemic brain by suppressing M1 markers and augmenting M2 polarization of microglia and the RGD motif plays a critical role in these activities.

Frequency selective neuronal modulation triggers spreading depolarizations in the rat endothelin-1 model of stroke

Ischemia is one of the most common causes of acquired brain injury. Central to its noxious sequelae are spreading depolarizations (SDs), waves of persistent depolarizations which start at the location of the flow obstruction and expand outwards leading to excitotoxic damage. The majority of acute stage of stroke studies to date have focused on the phenomenology of SDs and their association with brain damage. In the current work, we investigated the role of peri-injection zone pyramidal neurons in triggering SDs by optogenetic stimulation in an endothelin-1 rat model of focal ischemia. Our concurrent two photon fluorescence microscopy data and local field potential recordings indicated that a ≥ 60% drop in cortical arteriolar red blood cell velocity was associated with SDs at the ET-1 injection site. SDs were also observed in the peri-injection zone, which subsequently exhibited elevated neuronal activity in the low-frequency bands. Critically, SDs were triggered by low- but not high-frequency optogenetic stimulation of peri-injection zone pyramidal neurons. Our findings depict a complex etiology of SDs post focal ischemia and reveal that effects of neuronal modulation exhibit spectral and spatial selectivity.

Effects of intranasal guanosine administration on brain function in a rat model of ischemic stroke.

Ischemic stroke is a major cause of morbidity and mortality worldwide and only few affected patients are able to receive treatment, especially in developing countries. Detailed pathophysiology of brain ischemia has been extensively studied in order to discover new treatments with a broad therapeutic window and that are accessible to patients worldwide. The nucleoside guanosine (Guo) has been shown to have neuroprotective effects in animal models of brain diseases, including ischemic stroke. In a rat model of focal permanent ischemia, systemic administration of Guo was effective only when administered immediately after stroke induction. In contrast, intranasal administration of Guo (In-Guo) was effective even when the first administration was 3 h after stroke induction. In order to validate the neuroprotective effect in this larger time window and to investigate In-Guo neuroprotection under global brain dysfunction induced by ischemia, we used the model of thermocoagulation of pial vessels in Wistar rats. In our study, we have found that In-Guo administered 3 h after stroke was capable of preventing ischemia-induced dysfunction, such as bilateral suppression and synchronicity of brain oscillations and ipsilateral cell death signaling, and increased permeability of the blood-brain barrier. In addition, In-Guo had a long-lasting effect on preventing ischemia-induced motor impairment. Our data reinforce In-Guo administration as a potential new treatment for brain ischemia with a more suitable therapeutic window.

Neurotrophin-3 upregulation associated with intravenous transplantation of bone marrow mononuclear cells induces axonal sprouting and motor functional recovery in the long term after neocortical ischaemia

Bone marrow mononuclear cells (BMMCs) have been identified as a relevant therapeutic strategy for the treatment of several chronic diseases of the central nervous system. The aim of this work was to evaluate whether intravenous treatment with BMMCs facilitates the reconnection of lesioned cortico-cortical and cortico-striatal pathways, together with motor recovery, in injured adult Wistar rats using an experimental model of unilateral focal neocortical ischaemia. Animals with cerebral cortex ischaemia underwent neural tract tracing for axonal fibre analysis, differential expression analysis of genes involved in apoptosis and neuroplasticity by RT-qPCR, and motor performance assessment by the cylinder test. Quantitative and qualitative analyses of axonal fibres labelled by an anterograde neural tract tracer were performed. Ischaemic animals treated with BMMCs showed a significant increase in axonal sprouting in the ipsilateral neocortex and in the striatum contralateral to the injured cortical areas compared to untreated rodents. In BMMC-treated animals, there was a trend towards upregulation of the Neurotrophin-3 gene compared to the other genes, as well as modulation of apoptosis by BMMCs. On the 56th day after ischaemia, BMMC-treated animals showed significant improvement in motor performance compared to untreated rats. These results suggest that in the acute phase of ischaemia, Neurotrophin-3 is upregulated in response to the lesion itself. In the long run, therapy with BMMCs causes axonal sprouting, reconnection of damaged neuronal circuitry and a significant increase in motor performance.