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Background: Lipid metabolism has a crucial role in neural repair in neurodegenerative diseases. We recently revealed that lipogenesis-mediated interleukin-33 (IL-33) upregulation lead to blood-brain barrier (BBB) repair after ischemic stroke. However, manipulating the key enzyme fatty acid synthase (FASN) to enhance lipogenesis was very challenging. Glyceryl triacetate (GTA) was used as a donor of acetate and precursor of acetyl coenzyme A, the key substrate for de novo lipogenesis catalyzed by FASN. Therefore, we hypothesized that GTA would promote lipogenesis the peri-infarct after ischemic stroke and contribute to the BBB repair through IL-33., Methods: Middle cerebral artery occlusion (MCAO) was performed on C57BL mice and GTA was gavage administrated (4 g/kg) on day 2 and 4 after MCAO. Lipogenesis was evaluated by assessment of the protein level of FASN, lipid droplets, and fatty acid products through liquid chromatography-mass spectrometry in the peri-infarct area on day 3 after MCAO, respectively. BBB permeability was determined by extravasation of Evans blue, IgG and dextran, and levels of tight junction proteins in the peri-infarct area on day 7 after MCAO, respectively. Infarct size and neurological defects were assessed on day 7 after MCAO. Brain atrophy on day 30 and long-term sensorimotor abilities after MCAO were analyzed as well. The inhibitor of FASN, C75 and the virus-delivered FASN shRNA were used to evaluate the role of FASN-driven lipogenesis in GTA-improved BBB repair. Finally, the therapeutic potential of recombinant IL-33 on BBB repair and neurological recovery was evaluated., Results: We found that treatment with GTA increased the lipogenesis as evidenced by lipid droplets level and lauric acid content, but not the FASN protein level. Treatment with GTA increased the IL-33 level in the peri-infarct area and decreased the BBB permeability after MCAO. However, infarct size and neurological defect score were unchanged on day 7 after MCAO, while the long-term recovery of sensorimotor function and brain atrophy were improved by GTA. Inhibition of lipogenesis using C75 or FASN shRNA reversed the beneficial effect of GTA. Finally, exogenous IL-33 improved BBB repair and long-term functional recovery after stroke., Conclusion: Collectively, we concluded that treatment with GTA improved the BBB repair and functional recovery after ischemic stroke, probably by the enhancement of lipogenesis and IL-33 expression., (© 2023. The Author(s).)

Background: Stroke is a major health concern and a leading cause of mortality and morbidity. We and other groups have documented that hyperbaric oxygen preconditioning could significantly alleviate neuronal damage in ischemia‒reperfusion models through various mechanisms. However, we found that some of the subjects did not benefit from preconditioning with hyperbaric oxygen. The preconditioning phenomenon is similar to vaccination, in which the endogenous survival system is activated to fight against further injuries. However, with vaccine inoculations, we could test for specific antibodies against the pathogens to determine if the vaccination was successful. Likewise, this experiment was carried out to explore a biomarker that can reveal the effectiveness of the preconditioning before neuronal injury occurs., Methods: Middle cerebral artery occlusion (MCAO) was used to induce focal cerebral ischemia-reperfusion injury. 2D-DIGE-MALDI-TOF-MS/MS proteomic technique was employed to screen the differentially expressed proteins in the serum of rats among the control (Con) group (MCAO model without hyperbaric oxygen (HBO) preconditioning), hyperbaric oxygen protective (HBOP) group (in which the infarct volume decreased after HBO preconditioning vs. Con), and hyperbaric oxygen nonprotective (HBOU) group (in which the infarct volume remained the same or even larger after HBO preconditioning vs. Con). Candidate biomarkers were confirmed by western blot and enzyme linked immunosorbent assay (ELISA), and the relationship between the biomarkers and the prognosis of cerebral injury was further validated., Results: Among the 15 differentially expressed protein spots detected in the HBOP group by Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), 3 spots corresponding to 3 different proteins (haptoglobin, serum albumin, and haemopexin) products were identified by MALDI-TOF-MS/MS. Serum albumin and haemopexin were upregulated, and haptoglobin was downregulated in the HBOP group ( p < 0.05 vs. Con and HBOU groups). After the western blot study, only the changes in haemopexin were validated and exhibited similar changes in subjects from the HBOP group in accordance with MALDI-TOF-MS/MS proteomic analysis and enzyme linked immunosorbent assay (ELISA) analysis. The serum level of the hemopexin (HPX) at 2 h after HBO preconditioning was correlated with the infarct volume ratio after MCAO., Conclusions: Haemopexin may be developed as a predictive biomarker that indicated the effectiveness of a preconditioning strategy against cerebral ischaemic injury., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)

Ischemic stroke is a common cause of morbidity and mortality worldwide. The current treatment fails to achieve satisfactory results, because interventional therapy as first-line treatment management has a strict time window. In recent years, a large number of studies have confirmed that adenosine, as an inhibitory neurotransmitter, has a protective effect on cerebral ischemic injury. Nevertheless, direct administration of adenosine has many side effects. Previous studies showed that adenosine exerted neuroprotective effects mainly through adenosine receptor A1 (A1 receptor). Therefore, further study on the mechanism of A 1 receptor induced neuroprotection may find new targets for stroke treament. Mitochondrial biogenesis (MB) is a therapeutic target for ischemic stroke, and the nuclear-encoded peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) is a major regulator of MB. However, the influence of A1 receptor on MB and PGC-1α is unclear. In this study, using the middle cerebral artery occlusion (MCAO) model of mice, we evaluated the temporal and spatial effects of A1 receptor after ischemic stroke and verified the neuroprotection of A1 receptor. Neurological scores were used to assess functional changes in mice. At the same time, we observed the effect of activating A1 receptor on MB and PGC-1α, and the effect of knockdown PGC-1α on A1 receptor induced MB in vitro. WB and immunofluorescence were used to detect relevant indicators of MB. In addition, we downregulated PGC-1α in vivo to observe the effects on A1 receptor induced MB and neuroprotection. The findings indicated that A1 receptor was increased and mainly expressed on neurons in the penumbra, further activated A1 receptor after stroke had neuroprotection. In vitro, activation of A1 promotes MB and increases the expression level of PGC-1α, while downregulation of PGC-1α partially reverses the effect of A1 receptor after OGD/R. Down regulation of PGC-1α in the penumbra neurons can reverse the effects of activation of A1 receptor on MB and neuroprotection. Taken together, these findings indicated that A1receptor promotes MB and improves neurological function after ischemic stroke via PGC-1α., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Newborn neurons from the subventricular zone (SVZ) are essential to functional recovery following ischemic stroke. However, the number of newly generated neurons after stroke is far from enough to support a potent recovery. Adiponectin could increase neurogenesis in the dentate gyrus of hippocampus in neurodegenerative diseases. However, the effect of adiponectin on the neurogenesis from SVZ and the functional recovery after ischemic stroke was unknown, and the underlying mechanism was not specified either. The middle cerebral artery occlusion model of mice was adopted and adiponectin was administrated once a day from day 3 to 7 of reperfusion. The levels of BDNF and p-STAT3 were detected by western blotting on day 7 of reperfusion. The virus-encoded BDNF shRNA with GFAP promoter and a STAT3 inhibitor Stattic were used, respectively. Neurogenesis was evidenced by the expression of doublecortin and 5-bromo-2'-deoxyuridine (BrdU) labelling and brain atrophy was revealed by Nissl staining on day 28 of reperfusion. Neurological functional recovery was assessed by the adhesive removal test and the forepaw grip strength. We found that adiponectin increased both the doublecortin-positive cells and NeuN/BrdU double-positive cells around the injured area on day 28 of reperfusion, along with the improved long-term neurological recovery. Mechanistically, adiponectin increased the protein levels of p-STAT3 and BDNF in astrocytes on day 7 of reperfusion, while silencing BDNF diminished the adiponectin-induced neurogenesis and functional recovery. Moreover, inhibition of STAT3 not only prevented the increase of BDNF but also the improved neurogenesis and functional recovery after stroke. In conclusion, adiponectin enhances neurogenesis and functional recovery after ischemic stroke via STAT3/BDNF pathway in astrocytes., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Diabetes can cause vascular remodelling and is associated with worse outcome after ischaemic stroke. Pioglitazone is a commonly used anti-diabetic agent. However, it is not known whether pioglitazone use before ischaemia could reduce brain ischaemic injury. Pioglitazone was administered to 5-week-old db + or db/db mice. Cerebral vascular remodelling was examined at the age of 9 weeks. Expression of peroxisome proliferator-activated receptor-γ (PPARγ), p-PPARγ (S112 and S273), nucleotide-binding domain (NOD)-like receptor protein 3 (Nlrp3), interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) was evaluated in the somatosensory cortex of mice. Neurological outcome was evaluated 24 h after brain ischaemia. Results showed that early pioglitazone treatment provided a long-lasting effect of euglycaemia but enhanced hyperlipidaemia in the db/db mice. Diabetic mice exhibited increased vascular tortuosity, narrower middle cerebral artery (MCA) width and IgG leakage in the brain. These changes were blocked by early pioglitazone treatment. In diabetic animals, PPARγ expression was reduced, and p-PPARγ at S273 but not S112, Nlrp3, IL-1β and TNF-α were increased in the somatosensory cortex. PPARγ decrease and Nlrp3 increase were mainly in the neurons of the diabetic brain, which was reversed by early pioglitazone treatment. Pioglitazone attenuated the aggravated neurological outcome after stroke in diabetic mice. But this protective effect was abolished through restoring cerebral inflammation by intracerebroventricular administration of IL-1β and TNF-α in pioglitazone-treated diabetic mice before MCAO. In summary, early pioglitazone treatment attenuates cerebral vascular remodelling and ischaemic brain injury possibly via blocking chronic neuroinflammation in the db/db mice., (© 2022 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Estrogen replacement therapy (ERT) is potentially beneficial for the prevention and treatment of postmenopausal cerebral ischemia but inevitably increases the risk of cerebral hemorrhage and breast cancer when used for a long period of time. Genistein, a natural phytoestrogen, has been reported to contribute to the recovery of postmenopausal ischemic stroke with reduced risks. However, the underlying mechanism of genistein-mediated neuroprotection remains unclear. We reported that genistein exerted significant neuroprotective effects by enhancing the expression of neuronal G protein-coupled estrogen receptor (GPER) in the ischemic penumbra after cerebral reperfusion in ovariectomized (OVX) mice, and this effect was achieved through GPER-mediated inhibition of nod-like receptor protein 3 (NLRP3) inflammasome activation. In addition, we found that peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) was the pivotal molecule that participated in GPER-mediated inhibition of NLRP3 inflammasome activation in OVX mice after ischemia/reperfusion (I/R) injury. Our data suggest that the neuronal GPER/PGC-1α pathway plays an important role in genistein-mediated neuroprotection against I/R injury in OVX mice., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Studies have failed to translate more than 1000 experimental treatments from bench to bedside, leaving stroke as the second leading cause of death in the world. Thrombolysis within 4.5 hours is the recommended therapy for stroke and cannot be performed until neuroimaging is used to distinguish ischemic stroke from hemorrhagic stroke. Therefore, finding a common and critical therapeutic target for both ischemic and hemorrhagic stroke is appealing. Here, we report that the expression of myeloid differentiation protein 2 (MD2), which is traditionally regarded to be expressed only in microglia in the normal brain, was markedly increased in cortical neurons after stroke. We synthesized a small peptide, Trans-trans-activating (Tat)-cold-inducible RNA binding protein (Tat-CIRP), which perturbed the function of MD2 and strongly protected neurons against excitotoxic injury in vitro. In addition, systemic administration of Tat-CIRP or genetic deletion of MD2 induced robust neuroprotection against ischemic and hemorrhagic stroke in mice. Tat-CIRP reduced the brain infarct volume and preserved neurological function in rhesus monkeys 30 days after ischemic stroke. Tat-CIRP efficiently crossed the blood-brain barrier and showed a wide therapeutic index for stroke because no toxicity was detected when high doses were administered to the mice. Furthermore, we demonstrated that MD2 elicited neuronal apoptosis and necroptosis via a TLR4-independent, Sam68-related cascade. In summary, Tat-CIRP provides robust neuroprotection against stroke in rodents and gyrencephalic nonhuman primates. Further efforts should be made to translate these findings to treat both ischemic and hemorrhagic stroke in patients., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background and Purpose: Endogenous neuroprotection can be induced by ischemic and nonischemic preconditioning. However, not all subjects that undergo preconditioning exhibit similar favorable outcome. This study is to explore the molecules responsible for this phenomenon and find new therapeutic targets for stroke., Methods: Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion. High-throughput proteomic technique, isobaric tag for relative and absolute quantification, was used to screen differentially expressed proteins in the rats that developed ischemic tolerance from hyperbaric oxygen (HBO) preconditioning. The proteomic results were verified by Western blot and ELISA. Then, short interfering RNA and gene knockout rats were used to further determine the pivotal role of candidate proteins in HBO preconditioning-induced endogenous neuroprotection. Finally, lysosomal permeability was tested to elaborate the mechanism underlying this intrinsic neuroprotective effect., Results: Nine proteins differentially expressed in the serum of rats, which acquired benefits from HBO preconditioning, were screened and identified. Western blot and ELISA revealed that cystatin C (CysC) and mannose-binding lectin protein C were uniquely changed in rats with smaller infarction after HBO preconditioning and cerebral ischemia. Knockdown and knockout of CysC abolished HBO-induced neuroprotection. Moreover, HBO-induced endogenous CysC elevation preserved lysosomal membrane integrity after stroke in wild-type rats but not in CysC siRNA infusion or CysC - /- rats. Most importantly, exogenous CysC also induced neuroprotection against ischemic/reperfusion injury., Conclusions: CysC is a crucial determinant contributing to endogenous neuroprotection. It is also a novel candidate for stroke treatment through maintaining lysosomal membrane integrity., (© 2016 American Heart Association, Inc.)

Stroke is the major cause of permanent disability and mortality in China. Apigenin-7-O-β-D-(-6''-p-coumaroyl)-glucopyranoside (APG) is a glycoside subtype of apigenin and has the antioxidant activity; however, whether and how it plays a neuroprotective role following cerebral ischemia remains unknown. In present study, we adopted the oxygen glucose/reperfusion model in PC12 cells, bilateral common carotid artery occlusion model in C57B6 mice and middle cerebral artery occlusion model in SD rats to observe the therapeutic effects of APG on ischemic stroke. We also discussed the underlying mechanism. Treatment with 0.4 μg/ml or 0.8 μg/ml APG promoted cell viability and proliferation, reduced LDH release and apoptotic cell death levels in PC12 cells. Treatment with 50 mg/kg or 100 mg/kg APG at 30 minutes after reperfusion improved neurological outcomes in vivo, as demonstrated by elevation of neurological scores in both mice and rats. It also increased the number of survival neurons in mice and reduced infarct volume in rats. APG also increased the contents of Mn-SOD and the phosphorylation level of STAT3, elevated the antioxidant activity and reduced oxidative productions. These findings revealed a neuroprotective effect of APG, which possibly induced by the STAT3 phosphorylation-mediated Mn-SOD up-regulation.

Objective: To investigate the cerebral infarct volume 24 hours after transient middle cerebral artery occlusion (tMCAO) and the proportion of CD4⁺;CD25⁺;Foxp3⁺; regulatory T cells (Tregs) in splenocytes in diverse periods after all-trans retinoic acid (ATRA) treatment in mice, so as to explore whether ATRA have the protection against cerebral ischemia damage in mice through intervening Treg differentiation., Methods: Sixty male Kunming mice were randomly divided into two groups, i.e. pretreatment (n=40) and post-treatment (n=20) groups. Each group was against divided into two subgroups, i.e. tMCAO combined with ATRA treatment group, tMCAO combined with DMSO control group. Pretreatment groups: mice were treated intraperitoneally with ATRA (10 mg/kg) dissolved in 100 mL/L DMSO or equivalent volume of 100 mL/L DMSO daily for 7 days (n=20/group). Ten mice in each group were sacrificed and the proportion of Tregs in splenocytes was analyzed by flow cytometry (FCM) after 7-day pretreatment. The other 10 mice in each group were subjected to tMCAO by modified monofilament method. Neurologic deficit score (NDS) was recorded and the infarct volume was assessed by 2, 3, 5-triphenyltetrazolium chloride(TTC) staining 24 hours after tMCAO. The mice in post-treatment groups were treated intraperitoneally with ATRA (10 mg/kg) or equivalent volume of 100 mL/L DMSO immediately after the reperfusion of tMCAO modeling (n=10/group). NDS and infarct volume were assessed and the proportion of Tregs in splenocytes was analyzed 24 hours after tMCAO., Results: ATRA pretreatment for 7 days failed to improve neurologic function deficit (P>0.05) and to reduce the cerebral infarct volume (P>0.05) 24 hours after tMCAO in mice. ATRA post-treatment could markedly improve neurologic function (P<0.05) and reduce the cerebral infarct volume (P<0.05) 24 hours after tMCAO. However, neither ATRA pretreatment nor post-treatment had effect on the proportion of Tregs in the splenocytes of mice (P>0.05)., Conclusion: ATRA administered before tMCAO for 7 days failed to protect brain against ischemic damage. ATRA administered immediately following tMCAO induced cerebral protective effect 24 hours after tMCAO. The results suggest that Tregs change is not involved in the neuroprotection mechanism of ATRA.

Stroke has severe consequences in postmenopausal women. As replacement therapy of estrogen have various adverse effects and the undermined outcomes. Genistein, a natural phytoestrogen, has been suggested to be a potential neuroprotective agent for such stroke patients. However, the role of genistein and its underlying mechanism in ovariectomized mice has not yet been evaluated. In the present study, ovariectomized mice were treated with genistein (10 mg/kg) or vehicle daily for two weeks before developing transient cerebral ischemia (middle cerebral artery occlusion). The neurological manifestation was evaluated, and infarct volumes were demonstrated by 2,3,5-triphenyltetrazolium chloride staining at 24 h after reperfusion. In addition, phosphorylation of extracellular signal-regulated kinase (ERK) was detected by Western blotting and immunofluorescence staining, and cellular apoptosis was evaluated in the ischemic penumbra. We found that treatment with genistein reduced infarct volumes, improved neurological outcomes and attenuated cellular apoptosis at 24 h after reperfusion. ERK1/2 showed increased phosphorylation by genistein treatment after reperfusion, and an ERK1/2 inhibitor U0126 abolished this protective effect of genistein in terms of infarct volumes, neurological scores and cellular apoptosis. Our findings indicate that treatment with genistein can reduce the severity of subsequent stroke episodes, and that this beneficial function is associated with ERK activation.

We have previously shown that electroacupuncture (EA) pretreatment produces neuroprotective effects, which were mediated through an endocannabinoid signal transduction mechanism. Herein, we have studied the possible contribution of the phosphorylated form of glycogen synthase kinase-3β (GSK-3β) in EA pretreatment-induced neuroprotection via the cannabinoid CB1 receptor (CB1R). Focal transient cerebral ischemia was induced by middle cerebral artery occlusion in rats. Phosphorylation of GSK-3β at Ser-9 [p-GSK-3β (Ser-9)] was evaluated in the penumbra tissue following reperfusion. Infarct size and neurological score were assessed in the presence of either PI3K inhibitors or a GSK-3β inhibitor 72 h after reperfusion. Cellular apoptosis was evidenced by TUNEL staining and determination of the Bax/Bcl-2 ratio 24 h after reperfusion. The present study showed that EA pretreatment increased p-GSK-3β(Ser-9) 2 h after reperfusion in the ipsilateral penumbra. Augmented phosphorylation of GSK-3β induced similar neuroprotective effects as did EA pretreatment. By contrast, inhibition of PI3K dampened the levels of p-GSK-3β(Ser-9), and reversed not only the neuroprotective effect but also the anti-apoptotic effect following EA pretreatment. Regulation of GSK-3β by EA pretreatment was abolished following treatment with a CB1R antagonist and CB1R knockdown, whereas two CB1R agonists enhanced the phosphorylation of GSK-3β. Therefore we conclude that EA pretreatment protects against cerebral ischemia/reperfusion injury through CB1R-mediated phosphorylation of GSK-3β.