Acute Phase Of Cerebral Ischemia Research Articles

Hypoxic postconditioning drives protective microglial responses and ameliorates white matter injury after ischemic stroke.

Ischemic stroke (IS) is a cerebrovascular disease with high incidence and mortality. White matter repair plays an important role in the long-term recovery of neurological function after cerebral ischemia. Neuroprotective microglial responses can promote white matter repair and protect ischemic brain tissue. The aim of this study was to investigate whether hypoxic postconditioning (HPC) can promote white matter repair after IS, and the role and mechanism of microglial polarization in white matter repair after HPC treatment. Adult male C57/BL6 mice were randomly divided into three groups: Sham group (Sham), MCAO group (MCAO), and hypoxic postconditioning group (HPC). HPC group were subjected to 45 min of transient middle cerebral artery occlusion (MCAO) immediately followed by 40 min of HPC. The results showed that HPC reduced the proinflammatory level of immune cells. Furthermore, HPC promoted the transformation of microglia to anti-inflammatory phenotype on the third day after the procedure. HPC promoted the proliferation of oligodendrocyte progenitors and increased the expression of myelination-related proteins on the 14th day. On the 28th day, HPC increased the expression of mature oligodendrocytes, which enhanced myelination. At the same time, the motor neurological function of mice was restored. During the acute phase of cerebral ischemia, the function of proinflammatory immune cells was enhanced, long-term white matter damage was aggravated, and motor sensory function was decreased. HPC promotes protective microglial responses and white matter repair after MCAO, which may be related to the proliferation and differentiation of oligodendrocytes.

Expanded ischemic lesion due to herniation leads to axonal injury in a site remote to the primary lesion on autopsy brain with acute focal cerebral ischemia.

Cerebral ischemia may lead to axonal injury not only at the site of the primary lesion but also in a region remote to the site of insult. In this study, we investigated the effect of herniation on the development of axonal injury at a site remote to the primary lesion during the acute phase of cerebral ischemia. We obtained postmortem brains of 13 cases with acute phase of unilateral cerebral infarction in the territory of the internal carotid artery or middle cerebral artery and seven controls. We classified the brain tissues into herniation and non-herniation groups. Then we examined whether axonal and ischemic changes existed in the corpus callosum contralateral to the ischemic hemisphere and the upper pons. In the herniation group, we detected white-matter lesions by Klüver-Barrera staining, microglial loss by immunohistochemistry for ionized calcium-binding adaptor molecule 1, and axonal injury by immunohistochemistry for amyloid precursor protein. However, none of the aforementioned findings were observed in the non-herniation group. These findings suggest the existence of regional overlap in axonal and ischemic pathologies in remote regions in the presence of herniation. We concluded that herniation may play a significant role in the development of axonal and ischemic changes in the remote region. Our results suggest that axonal injury in a remote region may result from expanded ischemic lesions due to herniation.

Development and Evaluation of Novel Metformin Derivative Metformin Threonate for Brain Ischemia Treatment.

Epidemiologic data reveal that diabetes patients taking metformin exhibit lower incidence of stroke and better functional outcomes during post-stroke neurologic recovery. We previously demonstrated that chronic post-ischemic administration of metformin improved functional recovery in experimental cerebral ischemia. However, few beneficial effects of metformin on the acute phase of cerebral ischemia were reported either in experimental animals or in stroke patients, which limits the application of metformin in stroke. We hypothesized that slow cellular uptake of metformin hydrochloride may contribute to the lack of efficacy in acute stroke. We recently developed and patented a novel metformin derivative, metformin threonate (SHY-01). Pharmacokinetic profile in vivo and in cultured cells revealed that metformin is more rapidly uptaken and accumulated from SHY-01 than metformin hydrochloride. Accordingly, SHY-01 treatment exhibited more potent and rapid activation of AMP-activated protein kinase (AMPK). Furthermore, SHY-01 elicited a stronger inhibition of microglia activation and more potent neuroprotection when compared to metformin hydrochloride. SHY-01 administration also had superior beneficial effects on neurologic functional recovery in experimental stroke and offered strong protection against acute cerebral ischemia with reduced infarct volume and mortality, as well as the improved sensorimotor and cognitive functions in rats. Collectively, these results indicated that SHY-01 had an improved pharmacokinetic and pharmacological profile and produced more potent protective effects on acute stroke and long-term neurological damage. We propose that SHY-01 is a very promising therapeutic candidate for cerebral ischemic stroke.

Electroacupuncture Preconditioning Reduces Oxidative Stress in the Acute Phase of Cerebral Ischemia-Reperfusion in Rats by Regulating Iron Metabolism Pathways.

Background Oxidative stress is an important mechanism of cerebral ischemia-reperfusion injury. Ferroptosis caused by iron overload after cerebral ischemia-reperfusion is considered a common cause of oxidative stress. Many recent studies have shown that electroacupuncture (EA) can regulate the expression of inflammatory factors, and the use of electroacupuncture preconditioning can produce a protective effect, which can reduce injury after cerebral ischemia and reperfusion. We aimed to assess whether EA could be used to reduce oxidative stress. Methods The oxidative stress level of rats during the acute phase of cerebral ischemia and reperfusion was assessed with and without preconditioning with EA. Molecular biology methods were used to detect iron metabolism and oxidative stress-related proteins. Results Rats that had EA preconditioning had lower infarct volumes than rats in the control group. Furthermore, western blot analysis showed that the expression of iron metabolism-related protein FPN-1 was higher in the intervention group than in the model group after reperfusion. In this regard, further investigation also demonstrated higher expression of glutathione and glutathione peroxidase-4, and lower reactive oxygen species values in the brain tissue of the EA group were compared with those of the control group rats. Conclusions Electroacupuncture preconditioning can reduce oxidative stress after cerebral ischemia-reperfusion by regulating iron overload.

Self-extracellular RNA promotes pro-inflammatory response of astrocytes to exogenous and endogenous danger signals

ObjectiveAstrocytes participate in the local innate immune response of the central nervous system. In response to stress such as ischemia, activated cells release endogenous factors known as damage-associated molecular patterns (DAMPs). Self-extracellular RNA (eRNA) is such a ubiquitous alarm signal. However, it is unclear whether eRNA is involved in the early acute phase of cerebral ischemia and is sufficient to sensitize astrocytes towards a DAMP or PAMP (pathogen-associated molecular pattern) reaction.MethodsPro-inflammatory activation upon eRNA stimulation was characterized in primary murine astrocyte cultures. In vivo, an experimental stroke model was used to localize and quantify eRNA in murine brain sections. Using primary cortical neurons and the mouse hippocampal neuronal cell line HT-22, neuronal RNA release upon stress conditions related to cerebral hypoxia/ischemia was analyzed.ResultsWhile low-dose eRNA alone did not promote pro-inflammatory activation of astrocytes in culture, it strongly enhanced the expression of pro-inflammatory cytokines in the presence of either Pam2CSK4, a synthetic PAMP molecule that mimics bacterial infection, or high mobility group box 1 (HMGB1), a prominent DAMP. Synergism of eRNA/Pam2CSK4 and eRNA/HMGB1 was prevented by blockage of the astroglial toll-like receptor (TLR)-2. Inhibition of NF-κB- and mitogen-activated protein kinase-dependent signaling pathways hampered eRNA/Pam2CSK4-mediated pro-inflammatory activation of astrocytes. In vivo, the amount of non-nuclear, presumably extracellular ribosomal RNA in close proximity to neurons significantly accumulated across the infarct core and peri-infarct areas that was accompanied by transcriptional up-regulation of various pro-inflammatory factors. Accordingly, the exposure of neurons to hypoxic/ischemic stress in vitro resulted in the release of eRNA, partly mediated by active cellular processes dependent on the cytosolic calcium level.ConclusionThe DAMP signal eRNA can sensitize astrocytes as active players in cerebral innate immunity towards exogenous and endogenous activators of inflammation (PAMPs and DAMPs) in a synergistic manner via TLR2-NF-κB-dependent signaling mechanisms. These findings provide new insights into the pathogenesis of ischemic stroke and other inflammatory neurological disorders. Further studies will clarify whether administration of RNase in vivo may serve as an effective treatment for inflammatory brain pathologies.

Lack of Neuroprotective Effects of High-Density Lipoprotein Therapy in Stroke under Acute Hyperglycemic Conditions.

Introduction: The pleiotropic protective effects of high-density lipoproteins (HDLs) on cerebral ischemia have never been tested under acute hyperglycemic conditions. The aim of this study is to evaluate the potential neuroprotective effect of HDL intracarotid injection in a mouse model of middle cerebral artery occlusion (MCAO) under hyperglycemic conditions. Methods: Forty-two mice were randomized to receive either an intracarotid injection of HDLs or saline. Acute hyperglycemia was induced by an intraperitoneal injection of glucose (2.2 g/kg) 20 min before MCAO. Infarct size (2,3,5-triphenyltetrazolium chloride (TTC)-staining), blood–brain barrier leakage (IgG infiltration), and hemorrhagic changes (hemoglobin assay by ELISA and hemorrhagic transformation score) were analyzed 24 h post-stroke. Brain tissue inflammation (IL-6 by ELISA, neutrophil infiltration and myeloperoxidase by immunohisto-fluorescence) and apoptosis (caspase 3 activation) were also assessed. Results: Intraperitoneal D-glucose injection allowed HDL- and saline-treated groups to reach a blood glucose level of 300 mg/dl in the acute phase of cerebral ischemia. HDL injection did not significantly reduce mortality (19% versus 29% in the saline-injected group) or cerebral infarct size (p = 0.25). Hemorrhagic transformations and inflammation parameters were not different between the two groups. In addition, HDL did not inhibit apoptosis under acute hyperglycemic conditions. Conclusion: We observed a nonsignificant decrease in cerebral infarct size in the HDL group. The deleterious consequences of reperfusion such as hemorrhagic transformation or inflammation were not improved by HDL infusion. In acute hyperglycemia, HDLs are not potent enough to counteract the adverse effects of hyperglycemia. The addition of antioxidants to therapeutic HDLs could improve their neuroprotective capacity.

MiRNA-132/212 Gene-Deletion Aggravates the Effect of Oxygen-Glucose Deprivation on Synaptic Functions in the Female Mouse Hippocampus.

Cerebral ischemia and its sequelae, which include memory impairment, constitute a leading cause of disability worldwide. Micro-RNAs (miRNA) are evolutionarily conserved short-length/noncoding RNA molecules recently implicated in adaptive/maladaptive neuronal responses to ischemia. Previous research independently implicated the miRNA-132/212 cluster in cholinergic signaling and synaptic transmission, and in adaptive/protective mechanisms of neuronal responses to hypoxia. However, the putative role of miRNA-132/212 in the response of synaptic transmission to ischemia remained unexplored. Using hippocampal slices from female miRNA-132/212 double-knockout mice in an established electrophysiological model of ischemia, we here describe that miRNA-132/212 gene-deletion aggravated the deleterious effect of repeated oxygen-glucose deprivation insults on synaptic transmission in the dentate gyrus, a brain region crucial for learning and memory functions. We also examined the effect of miRNA-132/212 gene-deletion on the expression of key mediators in cholinergic signaling that are implicated in both adaptive responses to ischemia and hippocampal neural signaling. miRNA-132/212 gene-deletion significantly altered hippocampal AChE and mAChR-M1, but not α7-nAChR or MeCP2 expression. The effects of miRNA-132/212 gene-deletion on hippocampal synaptic transmission and levels of cholinergic-signaling elements suggest the existence of a miRNA-132/212-dependent adaptive mechanism safeguarding the functional integrity of synaptic functions in the acute phase of cerebral ischemia.

Dynamic changes of inflammation and apoptosis incerebral ischemia‑reperfusion injury in mice investigated byferumoxytol‑enhanced magnetic resonance imaging.

The inflammatory response and apoptosis are key factors in cerebral ischemia-reperfusion injury. The severity of the inflammatory reaction and apoptosis has an important impact on the prognosis of stroke. The ultrasmall superparamagnetic iron oxide particle has provided an effective magnetic resonance molecular imaging method for dynamic observation of the cell infiltration process in vivo. The aims of the present study were to investigate the inflammatory response of cerebral ischemia-reperfusion injury in mice using ferumoxytol-enhanced magnetic resonance imaging, and to observe the dynamic changes of inflammatory response and apoptosis. In the present study a C57BL/6n mouse cerebral ischemia-reperfusion model was established by blocking the right middle cerebral artery with an occluding suture. Subsequently, the mice were injected with ferumoxytol via the tail vein, and magnetic resonance scanning was performed at corresponding time points to observe the signal changes. Furthermore, blood samples were used to measure the level of serum inflammatory factors, and histological staining was performed to assess the number of iron-swallowing microglial cells and apoptotic cells. The present results suggested that there was no significant difference in the serum inflammatory factors tumor necrosis factor-α and interleukin 1β between the middle cerebral artery occlusion (MCAO) and MCAO + ferumoxytol groups injected with ferumoxytol and physiological saline. The lowest signal ratio in the negative enhancement region was decreased 24 h after reperfusion in mice injected with ferumoxytol. The proportion of iron-swallowing microglial cells and TUNEL-positive cells were the highest at 24 h after reperfusion, and decreased gradually at 48 and 72 h after reperfusion. Therefore, the present results indicated that ferumoxytol injection of 18 mg Fe/kg does not affect the inflammatory response in the acute phase of cerebral ischemia and reperfusion. Ferumoxytol-enhanced magnetic resonance imaging can be used as an effective means to monitor the inflammatory response in the acute phase of cerebral ischemia-reperfusion injury. Furthermore, it was found that activation of the inflammatory response and apoptosis in the acute stage of cerebral ischemia-reperfusion injury is consistent.

Exercise preconditioning ameliorates cognitive impairment and anxiety-like behavior via regulation of dopamine in ischemia rats

Cognitive impairment and anxiety are common health problems in acute ischemic stroke patients. Meanwhile, dopamine in the striatal brain region is significantly increased during the acute phase of cerebral ischemia. Besides, the studies shown that striatum and change of striatal dopamine are associated with learning and memory and anxiety. Further, physical exercise has been shown to improve neurocognitive and emotional function in animal models and patients with cerebral ischemia. However, the exact mechanism underlying this effect is unclear. The purpose of this research is to explore the effect of pre-ischemic voluntary wheel running on levels of striatal dopamine, cognition and anxiety in cerebral ischemia rats. Methods: 48 adult male Sprague-Dawley rats were enrolled in this study and divided randomly in following 6 groups: sham group (S group, n = 8), ischemia group (I group, n = 8), 1 week wheel running group (1R group), 4 weeks wheel running group (4R group), 1 week pre-ischemia wheel running group (1RI group, n = 8) and 4 weeks pre-ischemia wheel running group (4RI group, n = 8). After training, cerebral ischemia was induced by permanent bilateral common carotid artery ligation (2-VO) operation. Microdialysis was used to collect dialysates from the striatum immediately from 30 min before ischemia to 90 min after ischemia. High-performance liquid chromatography-electrochemical detection system (HPLC) was used to determine the content of dopamine in the dialysates. Passive avoidance and elevated plus maze test were used to test neurocognitive function 24 h after 2-VO cerebral ischemia. Results: As compare with the constant striatal dopamine level of S group, the striatal dopamine level in I group after ischemia showed a trend of rapid increasing and reached maximum value at the 20 min (P<0.001), then decreased gradually. The striatal dopamine level in 1RI and 4RI group showed the trend were similar to I group, but the increasing magnitude was attenuated. A comparison of the basal striatal dopamine level in 4 groups found that the basal dopamine level in 1RI and 4RI group were higher than S and I group (P<0.001). In passive avoidance task, the retention latency of I group was significantly shorter than S group (P<0.001), and the retention latency of the 1RI, 1R and 4R, 4RI group were longer than I group (P<0.001), there was no significant difference in S, 1RI, 1R, 4R and 4RI group (P>0.05). In elevated plus maze test, the time and entrance numbers of open arms in I group were significantly less than S group (P<0.05), but these indices were no significant difference in S, 1RI, 1R, 4RI and 4RI group. Conclusion: According to our results, 1 or 4 weeks pre-ischemia wheel running can significantly increase the basal dopamine level, attenuate the increase of striatal dopamine induced by cerebral ischemia and improve neurocognitive function in ischemia rats.

Dynamic changes and mislocalizations of neurodegenerative disease-related proteins in mice stroke model

The aggregation and cellular mislocalization of several RNA-binding proteins (RBPs) have been identified as the major hallmarks of neurodegenerative diseases such as frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). However, it remains obscure whether these pathological changes also occur during cerebral ischemia. In this study, we report that RBPs increased significantly compared with the sham group (*p < 0.05 and **p < 0.01 vs sham), with nuclear depletion and cytoplasmic deposition in neurons in the acute phase of cerebral ischemia. On the other hand, such nucleocytoplasmic mislocalization were not observed in astrocytes. We provide evidence of the alteration of these neurodegeneration-related RBPs after cerebral ischemia, suggesting a potential association between cerebral ischemia and neurodegenerative diseases.

Aspirin Resistance Affects Medium-Term Recurrent Vascular Events after Cerebrovascular Incidents: A Three-Year Follow-up Study

Background: The aim of this prospective, a three-year follow-up study, was to establish the role of high on-treatment platelet reactivity (HTPR) in predicting the recurrence of vascular events in patients after cerebrovascular incidents, particularly in the aspect of stroke etiology. Methods: The study included 101 subjects with non-embolic cerebral ischemia (69 patients with ischemic stroke and 32 patients with transient ischemic attack) treated with 150 mg of acetylsalicylic acid (aspirin) a day. The platelet reactivity was tested in the first 24 h after the onset of cerebral ischemia by impedance aggregometry. Recurrent vascular events, including recurrent ischemic stroke, transient ischemic attack, myocardial infarction, systemic embolism, or sudden death of vascular reason, were assessed 36 months after the onset of cerebral ischemia. Results: Recurrent vascular events occurred between 3 and 9 months after onset in 8.5% of all subjects; in the HTPR subgroup, recurrent vascular events occurred in 17.9%; in the normal on-treatment platelet reactivity (NTPR) subgroup, they occurred in 4.6%. We did not notice early or long-term recurrent events. Aspirin resistant subjects had a significantly higher risk of recurrent vascular events than did aspirin sensitive subjects (Odds ratio (OR) = 4.57, 95% Confidence interval (CI) 1.00–20.64; p = 0.0486). Cox proportional hazard models showed that large-vessel disease (Hazard ratio (HR) 12.04, 95% CI 2.43–59.72; p = 0.0023) and high on-treatment platelet reactivity (HR 4.28, 95% CI 1.02–17.93; p = 0.0465) were independent predictors of recurrent vascular events. Conclusion: Aspirin resistance in the acute phase of cerebral ischemia was associated with a higher risk of recurrent medium-term vascular events, coexisting with large-vessel etiology of stroke. Platelet function-guided personalized antiplatelet treatment should be considered for patients with recurrent strokes, especially when due to large-vessel disease.

Temporal effect of acupuncture on amino acid neurotransmitters in rats with acute cerebral ischaemia.

Acupuncture stimulation at GV26 during the acute phase of cerebral ischaemia can effectively reduce brain damage induced by ischaemic injury. However, the time course of the effects of acupuncture stimulation has not yet been thoroughly studied. To investigate the effects of manual acupuncture (MA) on glutamic acid (Glu) and γ-aminobutyric acid (GABA) expression in the cerebrospinal fluid of rats with middle cerebral artery occlusion (MCAO) and determine whether there is a temporal effect of acupuncture on the treatment of cerebral ischaemia. We performed thread occlusion of the right middle cerebral artery in rats to establish an animal model of MCAO. Simultaneously, during acupuncture treatment, microdialysis was used to continuously and dynamically observe immediate alterations in amino acid metabolism with acupuncture stimulation after cerebral ischaemia in vivo in this rat model of MCAO. We found that, in comparison with an untreated MCAO group, Glu content was significantly decreased during the first acupuncture stimulation and during the course of the acupuncture treatment in the MCAO+MA group (MCAO vs MCAO+MA: day 1, P=0.032; day 2, P=0.021; day 3, P=0.017). These findings were also seen after the end of treatment when acupuncture was no longer applied (MCAO vs MCAO+MA: day 7, P=0.009). Measurements of GABA content following cerebral ischaemic injury showed that GABA peaks 24 hours after damage, falls thereafter and decreases to baseline levels on day 7. In the MCAO+MA group, GABA content on days 1 to day 2 was lower than in the MCAO group (MCAO+MA vs MCAO: day 1, P=0.003; day 2, P=0.001), although it was higher than in the control group (MCAO+MA vs control: day 1, P=0.024; day 2, P=0.009). GABA content on day 3 and day 7 was higher in the MCAO+MA group than in the MCAO group and the control group (MCAO+MA vs MCAO: day 3, P=0.008; day 7, P=0.013; MCAO+MA vs control: day 3, P=0.002; day 7, P=0.009). Acupuncture stimulation at GV26 can effectively decrease excessive release of Glu induced by ischaemia and maintain the endogenous inhibitory activity of GABA. This phenomenon was seen during the entire course of acupuncture treatment and continued for some time after the end of acupuncture treatment.

Ischemic Postconditioning Recovers Cortex Ascorbic Acid during Ischemia/Reperfusion Monitored with an Online Electrochemical System.

As a promising therapeutic treatment, ischemic postconditioning has recently received considerable attention. Although the neuroprotection effect of postconditioning has been observed, a reliable approach that can evaluate the neuroprotective efficiency of postconditioning treatment during the acute period after ischemia remains to be developed. This study investigates the dynamics of cortex ascorbic acid during the acute period of cerebral ischemia before and after ischemic postconditioning with an online electrochemical system (OECS). The cerebral ischemia/reperfusion injury and the neuronal functional outcome are evaluated with triphenyltetrazolium chloride staining, immunohistochemistry, and electrophysiological recording techniques. Electrochemical recording results show that cortex ascorbic acid sharply increases 10 min after middle cerebral artery occlusion and then reaches a plateau. After direct reperfusion following ischemia (i.e., without ischemic postconditioning), the cortex ascorbic acid further increases and then starts to decrease slowly at a time point of about 40 min after reperfusion. In striking contrast, the cortex ascorbic acid drops and recovers to its basal level after ischemic postconditioning followed by reperfusion. With the recovery of cortex ascorbic acid, ischemic postconditioning concomitantly promotes the recovery of neural function and reduces the oxidative damage. These results demonstrate that our OECS for monitoring cortex ascorbic acid can be used as a platform for evaluating the neuroprotective efficiency of ischemic postconditioning in the acute phase of cerebral ischemia, which is of great importance for screening proper postconditioning parameters for preventing ischemic damages.

Abstract TMP29: Microglia Are Essential for Neuronal Replacement and Improved Functional Recovery Following Cerebral Ischemia

Introduction: Microglia are the first responders after stroke, mounting the immune response to CNS injury. While this response has been shown to be both neuroprotective and neurodegenerative following brain injury, it has been generally accepted that activation of microglia during the acute response to stroke is detrimental for neurogenesis and neuronal replacement. The vast majority of studies report that microglial activation reduces the survival of newborn neurons following stoke. However, our findings indicate that proinflammatory signaling is essential for survival of newborn neurons in juveniles. Remarkably, neurogenesis appears to require prolonged activation of microglia during the acute phase of cerebral ischemia. We utilized a juvenile stroke model to study the paracrine signaling triggered by ischemia in activated microglia and its consequence on newborn neuron survival. Methods: Electrocorticography (ECoG) was recorded from cortical surface electrodes and striatal depth electrodes implanted prior to middle cerebral artery occlusion (MCAO). Microglia were attenuated with Ibudilast (10 mg/kg), a glial cell activation inhibitor. Neurogenesis and inflammatory markers were examined with immunohistochemistry, and functional outcomes with neurobehavioral measures and ECoG recordings. Results and Conclusions: Acute inhibition of microglial activation after stroke resulted in reduced neurogenesis and long-lasting neurodegenerative effects, evidenced by increased astrogliosis, infiltrating macrophages, and glial scarring at 30d post-ischemia in the lateral striatum of MCAO+Ibudilast treated compared to MCAO+Vehicle treated juvenile mice. Behavioral tests and ECoG recordings revealed deficits in affected limb use, general motor functioning, and total ECoG power (μV 2 ) in both Ibudilast and vehicle treated MCAO-injured juveniles at 7d post-ischemia, but only MCAO+Vehicle treated juveniles returned to near sham-operated/baseline levels on neurobehavioral tasks and ECoG power in striatum and primary motor cortex at 30d. These surprising and striking findings provide evidence for a novel, supportive role for microglia in neuronal replacement and enhanced functional recovery following stroke in juveniles.

Reverse triiodothyronine (rT3) attenuates ischemia-reperfusion injury

Hypothyroidism has been associated with better recovery from cerebral ischemia-reperfusion (IR) injury in humans. However, any therapeutic advantage of inducing hypothyroidism for mitigating IR injury without invoking the adverse effect of whole body hypothyroidism remains a challenge. We hypothesize that a deiodinase II (D2) inhibitor reverse triiodothyronine (rT3) may render brain specific hypometabolic state to ensue reduced damage during an acute phase of cerebral ischemia without affecting circulating thyroid hormone levels. Preclinical efficacy of rT3 as a neuroprotective agent was determined in rat model of middle cerebral artery occlusion (MCAO) induced cerebral IR and in oxygen glucose deprivation/reoxygenation (OGD/R) model in vitro. rT3 administration in rats significantly reduced neuronal injury markers, infarct size and neurological deficit upon ischemic insult. Similarly, rT3 increased cellular survival in primary cerebral neurons under OGD/R stress. Based on our results from both in vivo as well as in vitro models of ischemia reperfusion injury we propose rT3 as a novel therapeutic agent in reducing neuronal damage and improving stroke outcome.

The membrane mesenchymal stem cell derived conditioned medium exerts neuroprotection against focal cerebral ischemia by targeting apoptosis

ObjectiveThe mesenchymal stem cells derived from human amniotic membrane have the ability to secrete and release some factors that can promote the repair of damaged tissues. This secretome contains proteins and factors that reduce apoptosis and increase angiogenesis in the ischemia/reperfusion models. The present study was conducted to determine whether this secretome provides protection against transient focal cerebral ischemia. Materials and MethodsA rat model of focal cerebral ischemia was established through middle cerebral artery occlusion (MCAO) for 60 min and 24 h reperfusion. The amniotic mesenchymal stem cells-conditioned medium (AMSC-CM) at the dose of 0.5 μl was injected intracerebroventriculary (ICV) 30 min after reperfusion. Infarct volume, brain edema, neurobehavioral functions, and blood brain barrier (BBB) integrity were assessed 24 h after reperfusion. Neuronal loss and expression of caspase-3, Bax and Bcl-2 in motor cortex were evaluated by nissl staining and immunohistochemistry assay respectively. ResultsICV administration of AMSC-CM markedly reduced infarct volume, brain edema and the evans blue penetration rate compared with MCAO group (P < 0.05). Additionally, post-treatment with AMSC-CM significantly reduced neuronal loss, neurological motor disorders and expression of caspase-3, Bax and Bcl-2 in motor cortex compared with MCAO group (P < 0.05). ConclusionThe results of this study indicate that treatment with AMSC-CM improves the pathological effects in the acute phase of cerebral ischemia. These findings establish a substantial foundation for stroke therapy and future research.

Cerebral Ischemia Changed the Effect of Metabosensitive Muscle Afferents on Somatic Reflex Without Affecting Thalamic Activity.

The purpose of the present study was to examine the contribution of group III and IV metabosensitive afferents at spinal and supraspinal levels in rats subjected to middle cerebral artery occlusion (MCAO) with reperfusion during the acute phase. Animals were randomized in Control (n = 23), SHAM (n = 18), MCAO-D1 (n = 10), and MCAO-D7 (n = 20) groups. Rats performed the Electrical Von Frey and the Adhesive removal tests before the surgery and at day 1 (D1), D3, and D7 after MCAO. Animals were subjected to electrophysiological recordings including the responses of group III/IV metabosensitive afferents to combinations of chemical activators and the triceps brachii somatic reflex activity at D1 or D7. The response of ventral posterolateral (VPL) thalamic nuclei was also recorded after group III/IV afferent activation. Histological measurements were performed to assess the infarct size and to confirm the location of the recording electrodes into the VPL. Behavioral results indicated that MCAO induced disorders of both mechanical sensibility and motor coordination of paretic forepaw during 7 days. Moreover, injured animals exhibited an absence of somatic reflex inhibition from the group III/IV afferents at D1, without affecting the response of both these afferents and the VPL. Finally, the regulation of the central motor drive by group III/IV afferents was modified at spinal level during the acute phase of cerebral ischemia and it might contribute to the observed behavioral disturbances.

Alpha-2-antiplasmin Arg407Lys polymorphism and cryptogenic ischemic cerebrovascular events: Association with neurological deficit

ObjectiveGenetic background of cryptogenic ischemic stroke (IS) and transient ischemic attack (TIA) remains uncertain. Alpha-2-antiplasmin (α2AP) Arg407Lys polymorphism has been shown to be less common in patients with abdominal aortic aneurysm (AAA) compared with healthy controls. We investigated associations of α2AP Arg407Lys polymorphism with cryptogenic IS and TIA. MethodsWe studied 165 consecutive Caucasian patients who experienced cryptogenic IS (n=123) or TIA (n=42). Neurological outcomes were assessed using the modified Rankin Scale (mRS) in the acute phase of cerebral ischemia and 8 (6–12) months after the index episode. Patients were genotyped for α2AP Arg407Lys polymorphism (rs1057335) using real time PCR technique. ResultsThe allele frequency of Arg407Lys polymorphism was: 0.82/0.18. The 407Lys allele was more frequent in TIA patients compared to the IS group (0.29 vs. 0.14, p=0.003). In the whole group, as well as in IS and TIA patients analyzed separately, possession of the 407Lys allele was associated with excellent outcome (mRS 0–1) during follow-up (p<0.05) but not in the acute phase of ischemic events both in thrombolyzed and nonthrombolyzed IS patients.The multivariate logistic regression model showed that the excellent outcome (mRS 0–1) assessed after 8 (6–12) months since the index cerebral ischemia was predicted by the occurrence of Lys407 allele (OR 6.18, 95% CI, 2.01–18.98, p=0.001). ConclusionThe presence of 407Lys allele is associated with better prognosis in cryptogenic cerebrovascular events. Our findings suggest that the α2AP Arg407Lys polymorphism could be involved in the pathogenesis of cerebral ischemia and its outcomes.

Recent Progress in Therapeutic Strategies for Ischemic Stroke.

Possible strategies for treating stroke include neuroprotection in the acute phase of cerebral ischemia and stem cell therapy in the chronic phase of cerebral ischemia. Previously, we have studied the temporal and spatial expression patterns of c-fos, hypoxia inducible factor-1α (HIF-1α), heat shock protein 70 (HSP70), and annexin V after 90 min of transient middle cerebral occlusion in rats and concluded that there is a time window for neuroprotection from 12 to 48 h after ischemia. In addition, we have estimated the neuroprotective effect of glial cell line-derived neurotrophic factor (GDNF) by injecting Sendai viral vector containing the GDNF gene into the postischemic brain. This Sendai virus-mediated gene transfer of GDNF showed a significant neuroprotective effect in the ischemic brain. Additionally, we have administered GDNF and hepatocyte growth factor (HGF) protein into the postischemic rat brain and estimated the infarct size and antiapoptotic and antiautophagic effects. GDNF and HGF significantly reduced infarct size, the number of microtubule-associated protein 1 light chain 3 (LC3)-positive cells, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick-end labeling (TUNEL)-positive cells, indicating that GDNF and HGF were greatly associated with not only the antiapoptotic effect but also the antiautophagic effects. Finally, we have previously transplanted undifferentiated iPSCs into the ipsilateral striatum and cortex at 24 h after cerebral ischemia. Histological analysis was performed at 14 and 28 days after cell transplantation, and we found that iPSCs could supply a great number of doublecortin-positive neuroblasts but also formed tridermal teratoma in the ischemic brain. Our results suggest that iPSCs have a potential to provide neural cells after ischemic brain injury if tumorigenesis is properly controlled. In the future, we will combine these strategies to develop more effective therapies for the treatment of strokes.

Analysis of Potential Amino Acid Biomarkers in Brain Tissue and the Effect of Galangin on Cerebral Ischemia.

Galangin, a potent scavenger of free radicals, has been used as an herbal medicine for various ailments for centuries in Asia. With complex pathophysiology, ischemic stroke is one of the most frequent causes of death and disability worldwide. We have reported that galangin provides direct protection against ischemic injury as a potential neuroprotective agent and has potential therapeutic effects on the changes of serum amino acids in ischemic stroke; however, the mechanism of the changes of amino acids in the ischemic brain tissue has not yet been clarified. In this paper, we explored brain tissue amino acid biomarkers in the acute phase of cerebral ischemia and the effect of galangin on those potential biomarkers. Finally, we identified that glutamic acid, alanine and aspartic acid showed significant changes (p < 0.05 or p < 0.01) in galangin-treated groups compared with vehicle-treated rats and the four enzymes associated with these three AAs’ metabolic pathways; GLUD1, SLC16A10, SLC1A1 and GPT were identified by multiplex interactions with the three amino acids. By metabolite-protein network analysis and molecular docking, six of 28 proteins were identified and might become potential galangin biomarkers for acute ischemic stroke. The data in our study provides thoughts for exploring the mechanism of disease, discovering new targets for drug candidates and elucidating the related regulatory signal network.