Potential circadian effects on translational failure for neuroprotection.

Neuroprotectant strategies that have worked in rodent models of stroke have failed to provide protection in clinical trials. Here we show that the opposite circadian cycles in nocturnal rodents versus diurnal humans1,2 may contribute to this failure in translation. We tested three independent neuroprotective approaches-normobaric hyperoxia, the free radical scavenger α-phenyl-butyl-tert-nitrone (αPBN), and the N-methyl-D-aspartic acid (NMDA) antagonist MK801-in mouse and rat models of focal cerebral ischaemia. All three treatments reduced infarction in day-time (inactive phase) rodent models of stroke, but not in night-time (active phase) rodent models of stroke, which match the phase (active, day-time) during which most strokes occur in clinical trials. Laser-speckle imaging showed that the penumbra of cerebral ischaemia was narrower in the active-phase mouse model than in the inactive-phase model. The smaller penumbra was associated with a lower density of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive dying cells and reduced infarct growth from 12 to 72 h. When we induced circadian-like cycles in primary mouse neurons, deprivation of oxygen and glucose triggered a smaller release of glutamate and reactive oxygen species, as well as lower activation of apoptotic and necroptotic mediators, in 'active-phase' than in 'inactive-phase' rodent neurons. αPBN and MK801 reduced neuronal death only in 'inactive-phase' neurons. These findings suggest that the influence of circadian rhythm on neuroprotection must be considered for translational studies in stroke and central nervous system diseases.

Aquaporin-4 deletion leads to reduced infarct volume and increased peri-infarct astrocyte reactivity in a mouse model of cortical stroke.

Aquaporin-4 (AQP4) is the main water channel in brain and is enriched in perivascular astrocyte processes abutting brain microvessels. There is a rich literature on the role of AQP4 in experimental stroke. While its role in oedema formation following middle cerebral artery occlusion (MCAO) has been studied extensively, its specific impact on infarct volume remains unclear. This study investigated the effects of total and partial AQP4 deletion on infarct volume in mice subjected to distal medial cerebral artery (dMCAO) occlusion. Compared to MCAO, this model induces smaller infarcts confined to neocortex, and less oedema. We show that AQP4 deletion significantly reduced infarct volume as assessed 1 week after dMCAO, suggesting that the role of AQP4 in stroke goes beyond its effect on oedema formation and dissolution. The reduction in infarct volume was associated with increased astrocyte reactivity in the peri-infarct areas. No significant differences were observed in the number of microglia among the genotypes. These findings provide new insights in the role of AQP4 in ischaemic injury indicating that AQP4 affects both infarct volume and astrocyte reactivity in the peri-infarct zone. KEY POINTS: Aquaporin-4 (AQP4) is the main water channel in brain and is enriched in perivascular astrocyte processes abutting microvessels. A rich literature exists on the role of AQP4 in oedema formation following middle cerebral artery occlusion (MCAO). We investigated the effects of total and partial AQP4 deletion on infarct volume in mice subjected to distal medial cerebral artery occlusion (dMCAO), a model inducing smaller infarcts confined to neocortex and less oedema compared to MCAO. AQP4 deletion significantly reduced infarct volume 1 week after dMCAO, suggesting a broader role for AQP4 in stroke beyond oedema formation. The reduction in infarct volume was associated with increased astrocyte reactivity in the peri-infarct areas, while no significant differences were observed in the number of microglia among the genotypes. These findings provide new insights into the role of AQP4 in stroke, indicating that AQP4 affects both infarct volume and astrocyte reactivity in the peri-infarct zone.

Acquired amusia after a right middle cerebral artery infarction - a case study.

A 62-year-old musician-MM-developed amusia after a right middle-cerebral-artery infarction. Initially, MM showed melodic deficits while discriminating pitch-related differences in melodies, musical memory problems, and impaired sensitivity to tonal structures, but normal pitch discrimination and spectral resolution thresholds, and normal cognitive and language abilities. His rhythmic processing was intact when pitch variations were removed. After 3 months, MM showed a large improvement in his sensitivity to tonality, but persistent melodic deficits and a decline in perceiving the metric structure of rhythmic sequences. We also found visual cues aided melodic processing, which is novel and beneficial for future rehabilitation practice.

Inhibition of Notch1 signal promotes brain recovery by modulating glial activity after stroke.

BACKGROUND: Notch1 signaling inhibiton with N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester] (DAPT) treatment could promote brain recovery and the intervention effect is different between striatum (STR) and cortex (CTX), which might be accounted for different changes of glial activities, but the in-depth mechanism is still unknown. The purpose of this study was to identify whether DAPT could modulate microglial subtype shifts and astroglial-endfeet aquaporin-4 (AQP4) mediated waste solute drainage. METHODS: Sprague-Dawley rats (n=10) were subjected to 90min of middle cerebral artery occlusion (MCAO) and were treated with DAPT (n=5) or act as control with no treatment (n=5). Two groups of rats underwent MRI scans at 24h and 4 week, and sacrificed at 4 week after stroke for immunofluorescence (IF). RESULTS: Compared with control rats, MRI data showed structural recovery in ipsilateral STR but not CTX. And IF showed decreased pro-inflammatory M1 microglia and increased anti-inflammatory M2 microglia in striatal lesion core and peri-lesions of STR, CTX. Meanwhile, IF showed decreased AQP4 polarity in ischemic brain tissue, however, AQP4 polarity in striatal peri-lesions of DAPT treated rats was higher than that in control rats but shows no difference in cortical peri-lesions between control and treated rats. CONCLUSIONS: The present study indicated that DAPT could promote protective microglia subtype shift and striatal astrocyte mediated waste solute drainage, that the later might be the major contributor of waste solute metabolism and one of the accounts for discrepant recovery of STR and CTX.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

RATIONALE: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE: To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

Activity in grafted human iPS cell-derived cortical neurons integrated in stroke-injured rat brain regulates motor behavior.

Stem cell transplantation can improve behavioral recovery after stroke in animal models but whether stem cell-derived neurons become functionally integrated into stroke-injured brain circuitry is poorly understood. Here we show that intracortically grafted human induced pluripotent stem (iPS) cell-derived cortical neurons send widespread axonal projections to both hemispheres of rats with ischemic lesions in the cerebral cortex. Using rabies virus-based transsynaptic tracing, we find that at 6 mo after transplantation, host neurons in the contralateral somatosensory cortex receive monosynaptic inputs from grafted neurons. Immunoelectron microscopy demonstrates myelination of the graft-derived axons in the corpus callosum and that their terminals form excitatory, glutamatergic synapses on host cortical neurons. We show that the stroke-induced asymmetry in a sensorimotor (cylinder) test is reversed by transplantation. Light-induced inhibition of halorhodopsin-expressing, grafted neurons does not recreate the impairment, indicating that its reversal is not due to neuronal activity in the graft. However, we find bilateral decrease of motor performance in the cylinder test after light-induced inhibition of either grafted or endogenous halorhodopsin-expressing cortical neurons, located in the same area, and after inhibition of endogenous halorhodopsin-expressing cortical neurons by exposure of their axons to light on the contralateral side. Our data indicate that activity in the grafted neurons, probably mediated through transcallosal connections to the contralateral hemisphere, is involved in maintaining normal motor function. This is an example of functional integration of efferent projections from grafted neurons into the stroke-affected brain's neural circuitry, which raises the possibility that such repair might be achievable also in humans affected by stroke.

TBC1Domain Family Member 25 deficiency aggravates cerebral ischemia-reperfusion injury via TAK1-JNK/p38 pathway.

TBC1Domain Family Member 25 (TBC1D25) is a protein that contains a TBC/RAB-GTPase activating protein (GAP) domain, which was shown to participate in autophagy in previous studies. However, the role of TBC1D25 in cerebral ischemia-reperfusion (I/R) injury remains unknown. In this study, we found that the mRNA and protein expression levels of TBC1D25 decreased in mouse brain after I/R injury and primary cortical neurons treated with oxygen and glucose deprivation/reoxygenation (OGD/R). Then TBC1D25 knockout (KO) mice were applied to demonstrate that TBC1D25 ablation aggravated cerebral I/R-induced neuronal loss and infarct size. In addition, neuronal apoptosis and inflammation were significantly potentiated in the TBC1D25-KO group. In in vitro OGD/R model, TBC1D25 knockdown can attenuate neuronal cell viability and aggravate the process of inflammation and apoptosis. Conversely, over-expression of TBC1D25 in primary neurons ameliorated the aforementioned processes. Mechanistically, RNA-sequencing (RNA-seq) analysis revealed mitogen-activated protein kinase (MAPK) signaling pathway was the most significant pathway that contributed to TBC1D25-mediated brain I/R injury process. Through experimental verification, TBC1D25 deficiency increased the phosphorylation of the transforming growth factor-ß-activated kinase 1 (TAK1)-c-Jun N-terminal kinase (JNK)/p38 axis in neurons during the brain I/R injury. Furthermore, we found that TAK1 blockade abrogated the apoptosis and inflammatory response produced by TBC1D25 knockdown in vitro. In conclusion, this study is the first to demonstrate the functional significance of TBC1D25 in the pathophysiology of brain I/R injury, and the protective mechanism of TBC1D25 is dependent on the TAK1-JNK/p38 pathway.

Perfusion-Dependent Cerebral Autoregulation Impairment in Hemispheric Stroke.

OBJECTIVE: Loss of cerebral autoregulation (CA) plays a key role in secondary neurologic injury. However, the regional distribution of CA impairment after acute cerebral injury remains unclear because, in clinical practice, CA is only assessed within a limited compartment. Here, we performed large-scale regional mapping of cortical perfusion and CA in patients undergoing decompressive surgery for malignant hemispheric stroke. METHODS: In 24 patients, autoregulation over the affected hemisphere was calculated based on direct, 15 to 20-minute cortical perfusion measurement with intraoperative laser speckle imaging and mean arterial blood pressure (MAP) recording. Cortical perfusion was normalized against noninfarcted tissue and 6 perfusion categories from 0% to >100% were defined. The interaction between cortical perfusion and MAP was estimated using a linear random slope model and Pearson correlation. RESULTS: Cortical perfusion and CA impairment were heterogeneously distributed across the entire hemisphere. The degree of CA impairment was significantly greater in areas with critical hypoperfusion (40-60%: 0.42% per mmHg and 60-80%: 0.46% per mmHg) than in noninfarcted (> 100%: 0.22% per mmHg) or infarcted (0-20%: 0.29% per mmHg) areas (*p < 0.001). Pearson correlation confirmed greater CA impairment at critically reduced perfusion (20-40%: r = 0.67; 40-60%: r = 0.68; and 60-80%: r = 0.68) compared to perfusion > 100% (r = 0.36; *p < 0.05). Tissue integrity had no impact on the degree of CA impairment. INTERPRETATION: In hemispheric stroke, CA is impaired across the entire hemisphere to a variable extent. Autoregulation impairment was greatest in hypoperfused and potentially viable tissue, suggesting that precise localization of such regions is essential for effective tailoring of perfusion pressure-based treatment strategies. ANN NEUROL 2021;89:358-368.

Impaired vessel relaxation response and increased infarct size in smooth muscle cannabinoid receptor 1 knockout mice.

Cannabinoids are reported to regulate cardiovascular functions. Cannabinoid receptors 1 (CB1Rs) are widely expressed in both the neuronal system and vascular system, but the contribution of CB1Rs in vascular smooth muscle (CB1RSM) to cardiovascular functions is not clear yet. In this research, we analyzed the effects of CB1RSM on blood pressure, vasoconstriction, and vasodilation abilities by using conditionally CB1R knockout mice (CB1RSMKO). The results show no significant difference in basal blood pressure between the conscious CB1RSMKO and control mice, indicating that CB1RSM is not essential for basal blood pressure maintenance. The constriction of the CB1RSMKO mesenteric artery in vitro was not significantly altered compared with that of the control mice. In contrast, the relaxation to CB1R agonist 2-AG or WIN55212-2 was decreased in CB1RSMKO vessels, suggesting that activation of CB1RSM mediates the vasodilation effect of cannabinoids. Ischemia stroke mouse model was used to further identify the potential function of CB1RSM in pathological conditions, and the results showed that the infarct volume in CB1RSMKO mice is significantly increased compared with the control littermates. These results suggest that vascular CB1R may not play a central role in basal vascular health maintenance but is protective in ischemia states, such as stroke. The protection function may be mediated, at least partly, by the relaxation effect of CB1RSM-dependent activities of endocannabinoids.

Ovariectomy Reduces Vasocontractile Responses of Rat Middle Cerebral Arteries After Focal Cerebral Ischemia.

ABSTRACT: Effects of sex hormones on stroke outcome are not fully understood. A deleterious consequence of cerebral ischemia is upregulation of vasoconstrictor receptors in cerebral arteries that exacerbate stroke injury. Here, we tested the hypothesis that female sex hormones alter vasocontractile responses after experimental stroke in vivo or after organ culture in vitro, a model of vasocontractile receptor upregulation. Female rats with intact ovaries and ovariectomized (OVX) females treated with 17ß-estradiol, progesterone, or placebo were subjected to transient, unilateral middle cerebral artery occlusion followed by reperfusion (I/R). The maximum contractile response, measured my wire myography, in response to the endothelin B receptor agonist sarafotoxin 6c was increased in female arteries after I/R, but the maximum response was significantly lower in arteries from OVX females. Maximum contraction mediated by the serotonin agonist 5-carboxamidotryptamine was diminished after I/R, with arteries from OVX females showing a greater decrease in maximum contractile response. Contraction elicited by angiotensin II was similar in all arteries. Neither estrogen nor progesterone treatment of OVX females affected I/R-induced changes in endothelin B- and 5-carboxamidotryptamine-induced vasocontraction. These findings suggest that sex hormones do not directly influence vasocontractile alterations that occur after ischemic stroke; however, loss of ovarian function does impact this process.

Transcranial Doppler 6 h after Successful Reperfusion as a Predictor of Infarct Volume.

OBJECTIVES: The aim of the study is to analyze the hemodynamic changes in the middle cerebral artery (MCA) after endovascular revascularization in acute ischemic stroke (AIS) due to large vessel occlusion and its association with the infarct volume size in the control head CT. MATERIALS AND METHODS: Prospective study of patients with AIS due to internal carotid artery terminus or M1 segment of the MCA occlusion, who underwent endovascular treatment with a final TICI 2b-3 score, without concomitant stenosis ≥50% in both cervical carotid arteries. Transcranial Doppler ultrasound (TCD) of both MCAs was carried out at 6 h after the endovascular procedure. Mean flow velocities (MFV) after arterial reperfusion and its association with the infarct volume size in 24-36 h control head CT were determined. RESULTS: 91 patients (51 women) were included with a median age of 78 years and National institute of Health Stroke Scale of 18. The MCA was occluded in 76.92%, and intravenous thrombolysis was administered in 40.7%. The incidence of symptomatic intracranial hemorrhage was 5.5%. At three months, mortality was 19.8% and a 52.7% of patients achieved functional independence (modified Rankin Scale 0-2). After a multivariable logistic regression analysis, an increase in the MFV greater than 50% at 6 h in the treated MCA compared to contralateral MCA, was an independent predictor of large infarct volume in the control head CT with an OR 9.615 (95%CI: 1.908-47.620), p=0.006 CONCLUSIONS: Increased MFV assessed by TCD examination following endovascular recanalization is independently associated with larger infarct volume.

Large-Scale Multivariate Analysis to Interrogate an Animal Model of Stroke: Novel Insights Into Poststroke Pathology.

Background and Purpose: Preclinical stroke studies endeavor to model the pathophysiology of clinical stroke, assessing a range of parameters of injury and impairment. However, poststroke pathology is complex and variable, and associations between diverse parameters may be difficult to identify within the usual small study designs that focus on infarct size. Methods: We have performed a retrospective large-scale big data analysis of records from 631 C57BL/6 mice of either sex in which the middle cerebral artery was occluded by 1 of 5 surgeons either transiently for 1 hour followed by 23-hour reperfusion (transient middle cerebral artery occlusion [MCAO]; n=435) or permanently for 24 hours without reperfusion (permanent MCAO; n=196). Analyses included a multivariate linear mixed model with random intercept for different surgeons as a random effect to reduce type I and type II errors and a generalized ordinal regression model for ordinal data when random effects are low. Results: Analyses indicated that brain edema volume was associated with infarct volume at 24 hours (ß, 0.52 [95% CI, 0.45­0.59]) and was higher after permanent MCAO than after transient MCAO (P<0.05). A more severe clinical score was associated with a greater infarct volume but not with the animal's age or edema volume. Further, a more severe clinical score was observed for a given brain infarct volume after transient MCAO versus permanent MCAO. Remarkably the animal's age, which corresponded with the period of young adulthood (6­40 weeks; equivalent to ≈18­35 years in humans), was positively associated with severity of lung infection (ß, 0.65 [95% CI, 0.42­0.88]) and negatively with spleen weight (ß, −0.36 [95% CI, −0.63 to −0.09]). Conclusions: Large-scale analysis of preclinical stroke data can provide researchers in our field with insight into relationships between variables not possible if individual studies are analyzed in isolation and has identified hypotheses for future study.

Microglial Calcium Waves During the Hyperacute Phase of Ischemic Stroke.

BACKGROUND AND PURPOSE: Ischemic injury triggers multiple pathological responses in the brain tissue, including spreading depolarizations across the cerebral cortex (cortical spreading depolarizations [CSD]). Microglia have been recently shown to play a significant role in the propagation of CSD. However, the intracellular responses of myeloid cells during ischemic stroke have not been investigated. METHODS: We have studied intracellular calcium activity in cortical microglia in the stroke model of the middle cerebral artery occlusion, using the murine Polr2a-based and Cre-dependent GCaMP5 and tdTomato reporter (PC::G5-tdT). High-speed 2-photon microscopy through cranial windows was employed to record signals from genetically encoded indicators of calcium. Inflammatory stimuli and pharmacological inhibition were used to modulate microglial calcium responses in the somatosensory cortex. RESULTS: In vivo imaging revealed periodical calcium activity in microglia during the hyperacute phase of ischemic stroke. This activity was more frequent during the first 6 hours after occlusion, but the amplitudes of calcium transients became larger at later time points. Consistent with CSD nature of these events, we reproducibly triggered comparable calcium transients with microinjections of potassium chloride (KCl) into adjacent cortical areas. Furthermore, lipopolysaccharide-induced peripheral inflammation, mimicking sterile inflammation during ischemic stroke, produced significantly greater microglial calcium transients during CSD. Finally, in vivo pharmacological analysis with CRAC (calcium release-activated channel) inhibitor CM-EX-137 demonstrated that CSD-associated microglial calcium transients after KCl microinjections are mediated at least in part by the CRAC mechanism. CONCLUSIONS: Our findings demonstrate that microglia participate in ischemic brain injury via previously undetected mechanisms, which may provide new avenues for therapeutic interventions.

Characterizing Diaschisis-Related Thalamic Perfusion and Diffusion After Middle Cerebral Artery Infarction.

Background and Purpose: Ipsilateral thalamic diaschisis (ITD) initially describes functional depression of the thalamus ipsilateral to a supratentorial lesion, but accumulating evidence has shown morphological changes also occur. Therefore, we aimed to characterize thalamic perfusion and diffusion related to ITD over time and their inter-relationships after middle cerebral artery infarction. Methods: Eighty-five patients with middle cerebral artery infarction who underwent diffusion kurtosis imaging and arterial spin labeling were retrospectively included. ITD was diagnosed as ipsilateral thalamic hypoperfusion present on ≥2 cerebral blood flow maps. The thalamic asymmetrical index was calculated as (ipsilateral value−contralateral value)/contralateral value×100%. Finally, the inter-relationships of thalamic perfusion and diffusion were analyzed. Results: ITD was present in 56/85 patients (65.9%, ITD+). In ITD+ patients, larger abnormal perfusion volume, higher perfusion-infarct mismatch and lower rates of focal hyperperfusion were observed than ITD− patients. Infarction affecting the corona radiata were more frequent among ITD+ patients. Mean kurtosis were slightly but significantly increased within the ipsilateral thalamus compared with the contralateral one in ITD+ patients of subacute and chronic groups, while fractional anisotropy was significantly increased in subacute group but decreased in chronic group for both ITD+ and ITD− patients. Mean diffusivity was significantly increased in ITD+ patients of chronic group. Furthermore, the AICBF was negatively and significantly correlated with AIMK and AIFA in ITD+ patients in subacute group, and AIMD, even after adjustment for abnormal perfusion volume and days from symptoms onset, in chronic group. ITD+ patients had significantly higher National Institutes of Health Stroke Scale and modified Rankin Scale scores at admission and discharge and also showed a trend to independent association with clinical outcome at discharge. Conclusions: The combination of arterial spin labeling and diffusion kurtosis imaging can reveal early, time-specific thalamic perfusion and diffusion changes after middle cerebral artery infarction. ITD-related hypoperfusion was significantly correlated with underlying microstructural alterations.

Evidence for interhemispheric imbalance in stroke patients as revealed by combining transcranial magnetic stimulation and electroencephalography.

Interhemispheric interactions in stroke patients are frequently characterized by abnormalities, in terms of balance and inhibition. Previous results showed an impressive variability, mostly given to the instability of motor-evoked potentials when evoked from the affected hemisphere. We aim to find reliable interhemispheric measures in stroke patients with a not-evocable motor-evoked potential from the affected hemisphere, by combining transcranial magnetic stimulation (TMS) and electroencephalography. Ninteen stroke patients (seven females; 61.26 ± 9.8 years) were studied for 6 months after a first-ever stroke in the middle cerebral artery territory. Patients underwent four evaluations: clinical, cortical, corticospinal, and structural. To test the reliability of our measures, the evaluations were repeated after 3 weeks. To test the sensitivity, 14 age-matched healthy controls were compared to stroke patients. In stroke patients, stimulation of the affected hemisphere did not result in any inhibition onto the unaffected. The stimulation of the unaffected hemisphere revealed a preservation of the inhibition mechanism onto the affected. This resulted in a remarkable interhemispheric imbalance, whereas this mechanism was steadily symmetric in healthy controls. This result was stable when cortical evaluation was repeated after 3 weeks. Importantly, patients with a better recovery of the affected hand strength were the ones with a more stable interhemispheric balance. Finally, we found an association between microstructural integrity of callosal fibers, suppression of interhemispheric TMS-evoked activity and interhemispheric connectivity. We provide direct and sensitive cortical measures of interhemispheric imbalance in stroke patients. These measures offer a reliable means of distinguishing healthy and pathological interhemispheric dynamics.

Arbutin protects brain against middle cerebral artery occlusion-reperfusion (MCAo/R) injury.

Focal ischemia causes irreversible brain damage if cerebral blood flow is not restored promptly. Acute phase excitotoxicity and pro-oxidant and inflammatory events in the sub-chronic phase elicit coagulative necrosis, vascular injury, cerebral oedema, and neurobehavioral deficits. Earlier, in pre-clinical studies arbutin protected behavioral functions and improved therapeutic outcomes in different models of brain and metabolic disorders. Arbutin is natural hydroquinone that might protect against ischemia-reperfusion (I/R) injury. In this study, cerebro-protective effects of arbutin were evaluated in the middle cerebral artery occlusion-reperfusion (MCAo/R) mouse model. Mice were administered arbutin (50, 100 mg/kg, i.p.) for 21 days, and subjected to MCAo/R or sham surgery on day 14. Results showed brain infarction, blood-brain barrier dysfunction, oedema, and neurological deficits 24 h post-MCAo/R injury that were prevented by arbutin. Behavioral evaluations over the sub-chronic phase revealed MCAo/R triggered spatial and working memory deficits. Arbutin protected the memory against MCAo/R injury and decreased hydroxy-2'-deoxyguanosine, protein carbonyls, inflammatory cytokines (tumor necrosis factor-α, myeloperoxidase, matrix metalloproteinase-9, inducible nitric oxide synthase), and enhanced glutathione levels in the ischemia ipsilateral hemisphere. Arbutin decreased brain acetylcholinesterase activity, glutamate, and enhanced GABA levels against MCAo/R. Arbutin can alleviate I/R pathogenesis and protects neurobehavioral functions in the MCAo/R mouse model.

Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice.

Obtained from the right cell-type, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) promote stroke recovery. Within this process, microvascular remodeling plays a central role. Herein, we evaluated the effects of MSC-sEVs on the proliferation, migration, and tube formation of human cerebral microvascular endothelial cells (hCMEC/D3) in vitro and on post-ischemic angiogenesis, brain remodeling and neurological recovery after middle cerebral artery occlusion (MCAO) in mice. In vitro, sEVs obtained from hypoxic (1% O2), but not 'normoxic' (21% O2) MSCs dose-dependently promoted endothelial proliferation, migration, and tube formation and increased post-ischemic endothelial survival. sEVs from hypoxic MSCs regulated a distinct set of miRNAs in hCMEC/D3 cells previously linked to angiogenesis, three being upregulated (miR-126-3p, miR-140-5p, let-7c-5p) and three downregulated (miR-186-5p, miR-370-3p, miR-409-3p). LC/MS-MS revealed 52 proteins differentially abundant in sEVs from hypoxic and 'normoxic' MSCs. 19 proteins were enriched (among them proteins involved in extracellular matrix-receptor interaction, focal adhesion, leukocyte transendothelial migration, protein digestion, and absorption), and 33 proteins reduced (among them proteins associated with metabolic pathways, extracellular matrix-receptor interaction, focal adhesion, and actin cytoskeleton) in hypoxic MSC-sEVs. Post-MCAO, sEVs from hypoxic MSCs increased microvascular length and branching point density in previously ischemic tissue assessed by 3D light sheet microscopy over up to 56 days, reduced delayed neuronal degeneration and brain atrophy, and enhanced neurological recovery. sEV-induced angiogenesis in vivo depended on the presence of polymorphonuclear neutrophils. In neutrophil-depleted mice, MSC-sEVs did not influence microvascular remodeling. sEVs from hypoxic MSCs have distinct angiogenic properties. Hypoxic preconditioning enhances the restorative effects of MSC-sEVs.

Glucagon-like peptide-1 (GLP-1) receptor activation dilates cerebral arterioles, increases cerebral blood flow, and mediates remote (pre)conditioning neuroprotection against ischaemic stroke.

Stroke remains one of the most common causes of death and disability worldwide. Several preclinical studies demonstrated that the brain can be effectively protected against ischaemic stroke by two seemingly distinct treatments: remote ischaemic conditioning (RIC), involving cycles of ischaemia/reperfusion applied to a peripheral organ or tissue, or by systemic administration of glucagon-like-peptide-1 (GLP-1) receptor (GLP-1R) agonists. The mechanisms underlying RIC- and GLP-1-induced neuroprotection are not completely understood. In this study, we tested the hypothesis that GLP-1 mediates neuroprotection induced by RIC and investigated the effect of GLP-1R activation on cerebral blood vessels, as a potential mechanism of GLP-1-induced protection against ischaemic stroke. A rat model of ischaemic stroke (90 min of middle cerebral artery occlusion followed by 24-h reperfusion) was used. RIC was induced by 4 cycles of 5 min left hind limb ischaemia interleaved with 5-min reperfusion periods. RIC markedly (by ~ 80%) reduced the cerebral infarct size and improved the neurological score. The neuroprotection established by RIC was abolished by systemic blockade of GLP-1R with a specific antagonist Exendin(9-39). In the cerebral cortex of GLP-1R reporter mice, ~ 70% of cortical arterioles displayed GLP-1R expression. In acute brain slices of the rat cerebral cortex, activation of GLP-1R with an agonist Exendin-4 had a strong dilatory effect on cortical arterioles and effectively reversed arteriolar constrictions induced by metabolite lactate or oxygen and glucose deprivation, as an ex vivo model of ischaemic stroke. In anaesthetised rats, Exendin-4 induced lasting increases in brain tissue PO2, indicative of increased cerebral blood flow. These results demonstrate that neuroprotection against ischaemic stroke established by remote ischaemic conditioning is mediated by a mechanism involving GLP-1R signalling. Potent dilatory effect of GLP-1R activation on cortical arterioles suggests that the neuroprotection in this model is mediated via modulation of cerebral blood flow and improved brain perfusion.

Diet-induced weight loss in obese/diabetic mice normalizes glucose metabolism and promotes functional recovery after stroke.

BACKGROUND: Post-stroke functional recovery is severely impaired by type 2 diabetes (T2D). This is an important clinical problem since T2D is one of the most common diseases. Because weight loss-based strategies have been shown to decrease stroke risk in people with T2D, we aimed to investigate whether diet-induced weight loss can also improve post-stroke functional recovery and identify some of the underlying mechanisms. METHODS: T2D/obesity was induced by 6 months of high-fat diet (HFD). Weight loss was achieved by a short- or long-term dietary change, replacing HFD with standard diet for 2 or 4 months, respectively. Stroke was induced by middle cerebral artery occlusion and post-stroke recovery was assessed by sensorimotor tests. Mechanisms involved in neurovascular damage in the post-stroke recovery phase, i.e. neuroinflammation, impaired angiogenesis and cellular atrophy of GABAergic parvalbumin (PV)+ interneurons were assessed by immunohistochemistry/quantitative microscopy. RESULTS: Both short- and long-term dietary change led to similar weight loss. However, only the latter enhanced functional recovery after stroke. This effect was associated with pre-stroke normalization of fasting glucose and insulin resistance, and with the reduction of T2D-induced cellular atrophy of PV+ interneurons. Moreover, stroke recovery was associated with decreased T2D-induced neuroinflammation and reduced astrocyte reactivity in the contralateral striatum. CONCLUSION: The global diabetes epidemic will dramatically increase the number of people in need of post-stroke treatment and care. Our results suggest that diet-induced weight loss leading to pre-stroke normalization of glucose metabolism has great potential to reduce the sequelae of stroke in the diabetic population.

Ischemia-reperfusion injury of brain induces endothelial-mesenchymal transition and vascular fibrosis via activating let-7i/TGF-ßR1 double-negative feedback loop.

Let-7i modulates the physical function and inflammation in endothelial cells (ECs). However, whether the let-7i of ECs involves in brain vasculature and ischemic stroke is unknown. Using inducible Cadherin5-Cre lineage-tracking mice, a loxp-RNA-sponge conditional knockdown of let-7 in ECs- induced increase of transforming growth factor-ß receptor type 1 (TGF-ßR1), endothelial-mesenchymal transition (endMT), vascular fibrosis, and opening of the brain-blood barrier (BBB). By this lineage-tracking mice, we found that ECs underwent endMT after transient middle cerebral artery occlusion (MCAO). Through specifically overexpressed let-7i in ECs, we found that it reduced TGF-ßR1, endMT, and vascular fibrosis. Furthermore, this overexpression reduced the infarct volume and leakage of the BBB, and improved the neurological function. Further, the expression of let-7i decreased after MCAO, but was reversed by antagonist of TGF-ßR1 or inhibition of Mek phosphorylation. And the inhibition of Mek attenuated the vascular fibrosis after MCAO. In summary, we concluded that ischemic stroke activates a let-7i/TGF-ßR1 double-negative feedback loop, thereby inducing endMT and vascular fibrosis. These results suggest that endMT is a potential target for the treatment of cerebral vascular fibrosis.