The objective of this study was to investigate a possible role of mitochondrial dihydrolipoamide dehydrogenase (DLDH) as a chemical preconditioning target for neuroprotection against ischemic injury. We used 5-methoxyindole-2-carboxylic acid (MICA), a reportedly reversible DLDH inhibitor, as the preconditioning agent and administered MICA to rats mainly via dietary intake. Upon completion of 4 week's MICA treatment, rats underwent 1h transient ischemia and 24h reperfusion followed by tissue collection. Our results show that MICA protected the brain against ischemic stroke injury as the infarction volume of the brain from the MICA-treated group was significantly smaller than that from the control group. Data were then collected without or with stroke surgery following MICA feeding. It was found that in the absence of stroke following MICA feeding, DLDH activity was lower in the MICA treated group than in the control group, and this decreased activity could be partly due to DLDH protein sulfenation. Moreover, DLDH inhibition by MICA was also found to upregulate the expression of NAD(P)H-ubiquinone oxidoreductase 1(NQO1) via the Nrf2 signaling pathway. In the presence of stroke following MICA feeding, decreased DLDH activity and increased Nrf2 signaling were also observed along with increased NQO1 activity, decreased oxidative stress, decreased cell death, and increased mitochondrial ATP output. We also found that MICA had a delayed preconditioning effect four weeks post MICA treatment. Our study indicates that administration of MICA confers chemical preconditioning and neuroprotection against ischemic stroke injury.

Diffuse optical tomography (DOT) has been used by several groups to assess cerebral hemodynamics of cerebral ischemia in humans and animals. In this study, we combined DOT with an indocyanine green (ICG)-tracking method to achieve interleaved images of cerebral hemodynamics and blood flow index (BFI) using two middle cerebral artery occlusion (MCAO) rat models. To achieve volumetric images with high-spatial resolution, we first integrated a depth compensation algorithm (DCA) with a volumetric mesh-based rat head model to generate three-dimensional (3D) DOT on a rat brain atlas. Then, the experimental DOT data from two rat models were collected using interleaved strategy for cerebral hemodynamics and BFI during and after ischemic stroke, with and without a thrombolytic therapy for the embolic MCAO model. The acquired animal data were further analyzed using the integrated rat-atlas-guided DOT method to form time-evolving 3D images of both cerebral hemodynamics and BFI. In particular, we were able to show and identify therapeutic outcomes of a thrombolytic treatment applied to the embolism-induced ischemic model. This paper demonstrates that volumetric DOT is capable of providing high-quality, interleaved images of cerebral hemodynamics and blood perfusion in small animals during and after ischemic stroke, with excellent 3D visualization and quantifications.

Background and Purpose-Pyruvate is known to be cytoprotective through antioxidant and anti-inflammatory mechanisms. We tested the hypothesis that pyruvate protects the brain against ischemia-reperfusion injury by inducing endogenous erythropoietin (EPO) expression.Methods-Pyruvate's protective effect was evaluated in C6 glioma cells and HT22 neuronal cells subjected to transient oxygen glucose deprivation. Cell viability (calcein AM assay) and expression of hypoxia-inducible factor-1 alpha, EPO, Akt and Erk (immunoblot), and EPO receptor (reverse transcription-polymerase chain reaction) were analyzed. Transient focal cerebral ischemia in rats was induced by 2 hours middle cerebral artery occlusion followed by 24 hours reperfusion. Pyruvate or saline was infused from 60 minutes occlusion until 30 minutes reperfusion. Lesion volume and DNA fragmentation were assessed by 2,3,5-triphenyltetrazolium staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, respectively. Immunoblots were conducted to determine cerebral EPO contents.Results-Pyruvate increased cell viability, hypoxia-inducible factor-1 alpha, EPO, and Akt phosphorylation. Small interfering RNA suppression of hypoxia-inducible factor-1 alpha and EPO abolished pyruvate-induced cytoprotection. In the rat stroke model, pyruvate reduced lesion volume by 84% and DNA fragmentation by 77% versus controls; increased EPO content paralleled these cerebroprotective actions of pyruvate.Conclusions-Pyruvate activation of the hypoxia-inducible factor-1 alpha-EPO signaling cascade in neurons and glia could protect the brain from ischemia-reperfusion injury. (Stroke. 2012;43:1101-1107.)

We investigated hyper-acute cerebral ischemia using diffuse optical tomography with the depth compensation algorithm (DCA) in pre-clinical rat models. Results suggest that this methodology has a great potential to monitor the effect of anti-stroke therapies.

Constriction and dilation of large arteries of the brain regulates cerebral vascular resistance and cerebral microvascular pressure, which play key roles in regulation of cerebral circulation. We investigated the effect of ischemic stroke on vascular reactivity of the middle cerebral artery (MCA) using a rat transient focal cerebral ischemia model. Focal cerebral ischemia was induced by 1 h of MCA occlusion followed by reperfusion. MCAs were dissected from ischemic or contralateral hemisphere at 2 days or 2 weeks postreperfusion and mounted on two glass micropipettes for assessment of vascular reactivity. MCAs from the brains of sham surgeries were used as control. At 2 days postreperfusion, a significant alteration of myogenic reactivity was found in MCAs dissected from both ischemic and nonischemic hemispheres, which could still be identified at 2 weeks after reperfusion. Phenylephrine (PE) induced a remarkable vasoconstriction in MCAs from animals that underwent sham surgery. No significant alteration of vasoconstrictive response to PE was found in MCAs isolated from either ischemic or contralateral hemisphere at 2 days or 2 weeks after ischemic stroke, as compared with MCAs from sham animals. Acetylcholine (ACh) induced mild dilation in normal MCAs, which was reversed in MCAs from both ischemic and nonischemic hemispheres at 2 weeks after ischemic stroke. Sodium nitroprusside (SNP) induced vasodilation in MCAs from animals with sham operation, which was diminished in MCAs from both ischemic and nonischemic hemispheres at 2 days and 2 weeks after ischemic stroke. These results demonstrated that focal cerebral ischemia could induce long-term global cerebral vasculature dysfunction.

Ischemic stroke accounts for over 80% in total human stroke which mostly affect middle cerebral artery (MCA) territory. Embolic stroke models induced by injection of homologous clots into the internal carotid artery and MCA closely mimic human stroke and have been commonly used in stroke research. Studies indicate that the size and composition of clots are critical for the reproducibility of the stroke model. In the present study, we modified the homologous clots formation by addition of thrombin and fibrinogen which produced even distribution of fibrin with tight cross linkage of red blood cells. We optimized the embolic MCA occlusion model in rats using different size of the mixed clots. A precise lodgment of the clots at the MCA bifurcation and highly reproducible ischemic lesion in the MCA territory were demonstrated in the embolic MCA occlusion model induced by injection of 10 pieces of 1-mm long mixed clots made in PE-60 catheter. We further tested the effect of recombinant tissue plasminogen activator (rtPA) in this embolic MCA occlusion model. rtPA induced thrombolysis, improved neurological outcome, and significantly reduced ischemic lesion volume when administered at 1h after embolism as compared with control. In summary, we have established a reproducible embolic MCA occlusion model using clots made of homologous blood, thrombin and fibrinogen. The mixed clots enable precise lodgment at the MCA bifurcation which is responsive to thrombolytic therapy of rtPA.

在世界范围内,卒中是导致成年人死亡的主要疾病之一,并且是导致患者致残的首要因素,卒中的治疗也越来越为医学界所重视.直到19 95年,卒中的治疗一直局限于预防性治疗.基因重组组织型纤溶酶原激活物(recombinant tissue plasminogen activator,rt-PA)的临床应用使我们首次对缺血性卒中急性期的治疗有了相对可靠的手段[1],但由于rt-PA的治疗时间窗非常狭窄,卒中患者可以接受溶栓治疗的比例至今仍然很低.