Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Pontine infarction is the most common stroke subtype in the posterior circulation, while there lacks a rodent model mimicking pontine infarction. Provided ...

The First Affiliated Hospital of Soochow University

Intraluminal suture middle cerebral artery occlusion (MCAO) model is the most frequently used model for ischemic stroke. However, the success rate of this model is variable among different research studies. This study aimed to investigate the effect of postischemic temperature on the success rate. A total of 100 C57BL/6 mice were randomized into two groups: control group (n=50), body temperature was allowed to self-regulate after MCAO; temperature-controlled group (n=50), mice were kept warm in an incubator for 12 h after MCAO. The body temperature of animals was measured before, during, and for 12 h after MCAO. Neurological deficits and infarct volumes were measured at 24 h after MCAO. There was significant difference (P<0.05) of the body temperature between the two groups from 0.5 h to 3.5 h post ischemia. Moreover, there was obvious difference between the success rates of the two groups (control group: 52%, temperature-controlled group: 84%, P<0.05). In the successful models, infarct volume was significantly (P<0.05) higher in temperature-controlled group (53.44%±9.83%, n=42) than control group (45.63%±10.24%, n=26). There was significant difference of the modified neurological severity scores (P<0.05), left adhesive tests (P<0.05) between the two groups. Our data demonstrated that postischemic warming contributed to the success of mouse MCAO model.

Keep warm and get success: the role of postischemic temperature in the mouse middle cerebral artery occlusion model.

Vascular endothelial growth factor (VEGF) is a promising candidate for the treatment of ischemic stroke. However, accumulating evidence demonstrated that VEGF could exacerbate blood-brain barrier (BBB) disruption after ischemic stroke. This study was designed to investigate the underlying mechanisms. In the present study, a transient (90 min) middle cerebral artery occlusion (MCAO) model was performed to induce ischemic stroke in mice. VEGF was administered intracerebroventricularly 3h after reperfusion. A gene expression microarray was utilized to investigate the differentially expressed genes among the sham, MCAO, and VEGF groups. A total of 3381 mRNAs were significantly altered by cerebral ischemia when compared with the sham group, and 15 of them were changed in the VEGF group when compared with the MCAO group. Among the 15 genes, orosomucoid (Orm) 1 was most sharply changed, and this gene has previously been reported to maintain the permeability of microvessels and integrity of the BBB. Results of the microarray showed that the expression of Orm1 increased after cerebral ischemia, whereas it decreased in response to VEGF, which was confirmed by real-time quantitative PCR, western blotting, immunohistochemistry, and immunofluorescence. The bioinformatics analysis indicated two NF-κB binding sites on the Orm1 promoter, and a super-shift assay verified that NF-κB could bind the Orm1 promoter. Results of the electrophoretic mobility shift assay (EMSA) revealed that VEGF inhibited the DNA-binding activity of NF-κB/p65. Furthermore, the elevated expression and activation of key members in the canonical NF-κB pathway induced by cerebral ischemia were also inhibited by VEGF treatment. In conclusion, this study demonstrated that decreasing the Orm1 expression via inhibition of the NF-κB pathway could be a possible mechanism involved in the aggravation of BBB disruption after stroke by VEGF.

Orosomucoid1: Involved in vascular endothelial growth factor-induced blood-brain barrier leakage after ischemic stroke in mouse.

Cellular therapy has provided hope for restoring neurological function post stroke through promoting endogenous neurogenesis, angiogenesis and synaptogenesis. The current study was based on the observation that transplantation of human umbilical cord mesenchymal stem cells (hUCMSCs) promoted the neurological function improvement in stroked mice and meanwhile enhanced angiogenesis in the stroked hemisphere. Grafted hUCMSCs secreted human vascular endothelial growth factor A (VEGF-A). Notch1 signaling was activated after stroke and also in the grafted hUCMSCs. To address the potential mechanism that might mediate such pro-angiogenic effect, we established a hUCMSC-neuron co-culture system. Neurons were subjected to oxygen glucose deprivation (OGD) injury before co-culturing to mimic the in vivo cell transplantation. Consistent with the in vivo data, co-culture medium claimed from hUCMSC-OGD neuron co-culture system significantly promoted the capillary-like tube formation of brain-derived endothelial cells. Moreover, coincident with our in vivo data, Notch 1 signaling activation was detected in hUCMSCs after co-cultured with OGD neurons as demonstrated by the up-regulation of key Notch1 signaling components Notch1 and Notch1 intercellular domain (NICD). In addition, OGD-neuron co-culture also increased the VEGF-A production by hUCMSCs. To verify whether Notch1 activation was involved in the pro-angiogenic effect, γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was added into the co-culture medium before co-culture. It turned out that DAPT significantly prevented the Notch1 activation in hUCMSCs after co-culture with OGD neurons. More importantly, the pro-angiogenic effect of hUCMSCs was remarkably abolished by DAPT addition as demonstrated by inhibited capillary-like tube formation and less VEGF-A production. Regarding how Notch1 signaling was linked with VEGF-A secretion, we provided some clue that Notch1 effector Hes1 mRNA expression was significantly up-regulated by OGD-neuron co-culturing and down-regulated after additional treatment of DAPT. In summary, our data provided evidence that the VEGF-A secretion from hUCMSCs after being triggered by OGD neurons is Notch1 signaling associated. This might be a possible mechanism that contributes to the angiogenic effect of hUCMSC transplantation in stroked brain. 

Enhanced angiogenesis promoted by human umbilical mesenchymal stem cell transplantation in stroked mouse is Notch1 signaling associated.

Cellular therapy with mesenchymal stem cells (MSCs) protects cortical neurons against hypoxic-ischemic injury of stroke. Although sorts of efforts have been made to confirm the neuroprotective effect of MSCs on neurons against hypoxic-ischemic injury, the mechanism is until now far away from clear. Here in this study, oxygen-glucose deprivation (OGD)-injured neuron model was applied to mimic the neuronal hypoxic-ischemic injury in vitro. Co-culturing with MSCs in a transwell co-culture system, the OGD injured neurons were rescued by 75.0 %. Further data demonstrated that co-culturing with MSCs protected the cortical neurons from the OGD-induced parthanatos by alleviating apoptosis-inducing factor (AIF) nuclear translocation; attenuated the neuronal necroptosis by down-regulating the expression of the two essential kinases in necroptosis, receptor interacting protein kinase1 (RIP1) and 3 (RIP3); rescued the neurons from apoptosis by deactivating caspase-3; whilst performed no significant influence on OGD-induced neuronal autophagy, according to its failed regulation on Beclin1. In conclusion, MSCs potentially protect the cortical neurons from OGD-injury in vitro, through rescuing neurons from the cell death of parthanatos, necroptosis, and apoptosis, but not autophagy, which could provide some evidence to the mechanism explanation on stem cell treatment for ischemic stroke.

Mesenchymal stem cells protect neurons against hypoxic-ischemic injury via inhibiting parthanatos, necroptosis, and apoptosis, but not autophagy.

Circulation inflammation markers such as high-sensitive C-reactive protein (hsCRP) and lipoprotein-associated phospholipase A2 (Lp-PLA2) are considered as predictors of cerebral and cardiac vascular diseases. However, the role of hsCRP and Lp-PLA2 in the anterior circulation cerebral infarction (ACI) is to be elaborated.

Serum lipoprotein-associated phospholipase A2 predicts the formation of carotid artery plaque and its vulnerability in anterior circulation cerebral infarction.

The correlation between platelet function and recurrent ischemic stroke or TIA remains uncertain.

Adequate Platelet Function Inhibition Confirmed by Two Inductive Agents Predicts Lower Recurrence of Ischemic Stroke/Transient Ischemic Attack.

Notch1 maturation participates in apoptosis and inflammation following intracerebral hemorrhage (ICH). It has been reported that Botch bound to and blocked Notch1 maturation. Here we estimated the role of Botch in ICH-induced secondary brain injury and underlying mechanisms. Experimental ICH model was induced by autologous arterial blood injection in Sprague-Dawley rats, and cultured primary rat cortical neurons were exposed to oxyhemoglobin to mimic ICH . Specific small interfering RNAs and expression plasmids encoding wild type Botch and Botch with Glu115Ala mutation were exploited. The protein levels of Botch and Notch1 transmembrane intracellular domain (Notch1-TMIC) were increased within brain tissue around hematoma. Botch overexpression led to an increase in unprocessed Notch1 full-length form accompanied by a significant decrease in Notch1-TMIC, while Botch knockdown resulted in an approximately 1.5-fold increase in Notch1-TMIC. There were increased cell apoptosis, necrosis and neurobehavioral deficits after ICH, which was inhibited by Botch overexpression and enhanced by Botch knockdown. Double immunofluorescence showed a colocalization of Botch and Notch1 in the trans-Golgi. Overexpression of wild type Botch, but not Botch E115A mutant, led to an increase in the interaction between Botch and Notch1, reduced the formation and the nuclear localization of Notch1 intracellular domain, and attenuated cell apoptosis and inflammation. In conclusion, Botch exerts neuroprotection against neuronal damage via antagonizing the maturation of Notch1 in Glu115-denpendent manner. However, neuroprotection mediated by endogenous Botch is not enough to reverse ICH-induced secondary brain injury.

Neuroprotection of Botch in experimental intracerebral hemorrhage in rats.

Owing to the continued high morbidity and high mortality rate after stroke, it is important to seek treatments other than conventional thrombolysis. Notch1 up-regulation participates in inflammatory responses after cerebral ischemia-reperfusion (I/R) injury, and it has been reported that Botch binds to and blocks Notch1 maturation. In this study, we investigated the role of Botch during cerebral (I/R) injury and explored its potential mechanisms. A middle-cerebral-artery occlusion/reperfusion (MCAO/R) model was established in adult male Sprague-Dawley rats in vivo, and cultured neurons and microglia were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic I/R injury in vitro. The results showed that protein levels of Botch and the Notch1 intracellular domain (NICD) were increased after MCAO/R. Furthermore, after overexpression of Botch, the generation of the activated form of Notch1, NICD, was decreased, while Botch knockdown or mutation led to an increase in NICD generation. As a result, Botch overexpression exhibited neuroprotective effects by significantly decreasing neurobehavioral phenotypes, improving infiltration of activated microglia, ameliorating inflammatory cytokine release, and inhibiting neuronal cell death. Conversely, Botch knockdown and mutation induced opposite effects. In addition, NICD was found to translocate to the mitochondria after OGD/R in neurons and microglia, which stimulated accumulation of reactive oxygen species in mitochondria and resulted in neuronal cell death and microglial activation. Botch overexpression inhibited the generation of NICD and decreased the translocation of NICD to the mitochondria, which inhibited neuronal cell death and ameliorated neuroinflammation. In conclusion, we found that Botch exerts neuroprotective effects via antagonizing the maturation of Notch1-induced neuronal injury and neuroinflammation, which may provide insights into novel therapeutic targets for the treatment of I/R injury.

Botch protects neurons from ischemic insult by antagonizing Notch-mediated neuroinflammation.

The topographic location of acute pontine infarction is associated with clinical syndromes and prognosis. Previous studies focused on isolated pontine infarction, but the topographic location of unisolated pontine infarction has remained unclear.

Topographic location of unisolated pontine infarction.

Renal dysfunction (RD) is prevalent in patients with acute ischemic stroke requiring intravenous thrombolysis. The relationship between renal function and thrombolysis related intracranial hemorrhagic (ICH) complications is contradictory according to previous studies. The current study is to clarify whether RD could increase the risk of symptomatic intracranial hemorrhage (SICH) after recombinant tissue plasminogen activator (IV rtPA) in acute ischemic stroke patients.

Juehua Zhu

Department of Neurology

The First Affiliated Hospital of Soochow University

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Juehua Zhu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neurology

The First Affiliated Hospital of Soochow University

Establishment of Acute Pontine Infarction in Rats by Electrical Stimulation

Department of Neurology