Song-Jie Liao

dl-3n-butylphthalide prevents stroke via improvement of cerebral microvessels in RHRSP

The purpose of the study is to establish a model of cold-induced stroke in hypertensive rats, and to study the preventive effect of dl-3n-butylphthalide ( NBP ) on stroke. Stroke-prone renovascular hypertension(RHRSP) was created in Sprague-Dawley rats. The animals were assigned randomly to NBP, aspirin treated and vehicle control group, with administration of the medications for 7 days, and then subjected to cold treatment in an environmentally controlled chamber for 3 days to induce the occurrence of stroke. The incidence of stroke, the volume of the brain lesion, patency of the microvessels by FITC-dextran perfusion and the number of microvessels by immunohisochemical detection of vwF were investigated. Cold induced different types of stroke in RHRSP. The incidence of ischemic stroke and the volume of the infarct were decreased, and the perfused microvessels were increased with NBP pretreatment. Our data suggest that NBP prevents cold-induced ischemic stroke via improvement of cerebral microvessels.

Metallothionein Abrogates GTP Cyclohydrolase I Inhibition–Induced Cardiac Contractile and Morphological Defects Role of Mitochondrial Biogenesis

One key mechanism for endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O2•− rather than NO because of deficient eNOS cofactor tetrahydrobiopterin (BH4). This study was designed to examine the effect of BH4 deficiency on cardiac morphology and function, as well as the impact of metallothionein (MT) on BH4 deficiency–induced abnormalities, if any. Friend virus B (FVB) and cardiac-specific MT transgenic mice were exposed to 2,4-diamino-6-hydroxy-pyrimidine (DAHP; 10 mmol/L, 3 weeks), an inhibitor of the BH4 synthetic enzyme GTP cyclohydrolase I. DAHP reduced plasma BH4 levels by 85% and elevated blood pressure in both FVB and MT mice. Echocardiography found decreased fractional shortening and increased end-systolic diameter in DAHP-treated FVB mice. Cardiomyocytes from DAHP-treated FVB mice displayed enhanced O2•− production, contractile and intracellular Ca2+ defects including depressed peak shortening and maximal velocity of shortening/relengthening, prolonged duration of relengthening, reduced intracellular Ca2+ rise, and clearance. DAHP triggered mitochondrial swelling/myocardial filament aberrations and mitochondrial O2•− accumulation, assessed by transmission electron microscopy and MitoSOX Red fluorescence, respectively. DAHP also promoted the NG-nitro-l-arginine methyl ester–inhibitable O2•− production and eNOS phosphorylation at Thr497. Although MT had little effect on cardiac mechanics and ultrastructure, it attenuated DAHP-induced defects in cardiac function, morphology, O2•− production, and eNOS phosphorylation (Thr497). The DAHP-induced cardiomyocyte mechanical responses were alleviated by in vitro BH4 treatment. DAHP inhibited mitochondrial biogenesis, mitochondrial uncoupling protein 2, and chaperone heat shock protein 90, and all but uncoupling protein 2 were rescued by MT. Our data suggest a role for BH4 deficiency in cardiac dysfunction and the therapeutic potential of antioxidants against eNOS uncoupling in the heart.

Enhanced angiogenesis with dl-3n-butylphthalide treatment after focal cerebral ischemia in RHRSP.

Appropriate restoration of blood flow via angiogenesis is critical for the recovery from ischemic stroke. Previously, we reported that treatment with dl-3n-butylphthalide (NBP) increases the number of local potent cerebral microvessels. However, the underlying mechanism remained unclear. The present study was conducted to test whether NBP enhances post-ischemic cerebral angiogenesis via vascular endothelial growth factor (VEGF) and hypoxia induced factor-1 alpha (HIF-1 alpha). Stroke-prone renovascular hypertensive rats (RHRSP) were used to create middle cerebral artery occlusion (MCAO) model. NBP was given 80 mg/kg per d for 10 consecutive days, starting 12, 24, 48 and 72 h respectively after MCAO. Neurological function was assessed daily and infarct volume as well as the expressions of CD31, VEGF, HIF-1 alpha and bFGF was detected 13 days after MCAO. The administration of NBP starting within 24 h after MCAO enhanced recovery of neurobehavioral function, reduced infarct volume, increased the quantity of CD31 positive vessels, and up-regulated expressions of VEGF and HIF-1 alpha. These findings suggest that treatment with NBP within 24 h post-ischemic stroke rescues brain tissue by enhancing angiogenesis associated with up-regulation of VEGF and HIF-1 alpha expressions.

Neuroprotective effect of chondroitinase ABC on primary and secondary brain injury after stroke in hypertensive rats

Focal cerebral infarction causes secondary damage in the ipsilateral ventroposterior thalamic nucleus (VPN). Chondroitin sulfate proteoglycans (CSPGs) are a family of putative inhibitory components, and its degradation by chondroitinase ABC (ChABC) promotes post-injury neurogenesis. This study investigated the role of ChABC in the primary and secondary injury post stroke in hypertension. Renovascular hypertensive Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAO), and were subjected to continuous intra-infarct infusion of ChABC (0.12 U/d for 7 days) 24 h later. Neurological function was evaluated by a modified neurologic severity score. Neurons were counted in the peri-infarct region and the ipsilateral VPN 8 and 14 days after MCAO by Nissl staining and NeuN labeling. The expressions of CSPGs, growth-associated protein-43 (GAP-43) and synaptophysin (SYN) were detected with immunofluorescence or Western blotting. The intra-infarct infusion of ChABC, by degrading accumulated CSPGs, rescued neuronal loss and increased the levels of GAP-43 and SYN in both the ipsilateral cortex and VPN, indicating enhancd neuron survival as well as augmented axonal growth and synaptic plasticity, eventually improving overall neurological function. The study demonstrated that intra-infarct ChABC infusion could salvage the brain from both primary and secondary injury by the intervention on the neuroinhibitory environment post focal cerebral infarction.

Netrin-1 rescues neuron loss by attenuating secondary apoptosis in ipsilateral thalamic nucleus following focal cerebral infarction in hypertensive rats

Neurological deficit following cerebral infarction correlates with not only primary injury, but also secondary neuronal apoptosis in remote loci connected to the infarction. Netrin-1 is crucial for axonal guidance by interacting with its receptors, deleted in colorectal cancer (DCC) and uncoordinated gene 5H (UNC5H). DCC and UNC5H are also dependence receptors inducing cell apoptosis when unbound by netrin-1. The present study is to investigate the role of netrin-1 and its receptors in ipsilateral ventroposterior thalamic nucleus (VPN) injury secondary to stroke in hypertensive rats. Renovascular hypertensive Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAO). Continuous intracerebroventricular infusion of netrin-1 (600 ng/d for 7 days) or vehicle (IgG/Fc) was given 24h after MCAO. Neurological function was evaluated by postural reflex 8 and 14 days after MCAO. Then, immunoreactivity was determined in the ipsilateral VPN for NeuN, glial fibrillary acidic protein, netrin-1 and its receptors (DCC and UNC5H2), apoptosis was detected with Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL) assay, and the expressions of caspase-3, netrin-1, DCC, and UNC5H2 were quantified by western blot analysis. MCAO resulted in the impaired postural reflex after 8 and 14 days, with decreased NeuN marked neurons and increased TUNEL-positive cells, as well as an up-regulation in the levels of cleaved caspase-3 and UNC5H2 protein in the ipsilateral VPN, without significant change in DCC or netrin-1 expression. By exogenous netrin-1 infusion, the number of neurons was increased in the ipsilateral VPN, and both TUNEL-positive cell number and caspase-3 protein level were reduced, while UNC5H2 expression remained unaffected, simultaneously, the impairment of postural reflex was improved. Taken together, the present study indicates that exogenous netrin-1 could rescue neuron loss by attenuating secondary apoptosis in the ipsilateral VPN after focal cerebral infarction, possibly via its receptor UNC5H2, suggesting that relative insufficiency of endogenous netrin-1 be an underlying mechanism of secondary injury in the VPN post stroke.

Axon guidance factor netrin-1 and its receptors regulate angiogenesis after cerebral ischemia

Neurogenesis and angiogenesis play important roles in functional recovery after ischemic stroke. When cerebral ischemia occurs, axon regeneration can compensate for the loss of apoptotic neurons in the ischemic area. The formation of new blood vessels ameliorates the local decrease in blood supply, enhancing the supply of oxygen and nutrients to newly-formed neurons. New blood vessels also act as a scaffold for the migration of neuroblasts to the infarct area after ischemic stroke. In light of this, researchers have been actively searching for methods to treat cerebral infarction. Netrins were first identified as a family of proteins that mediate axon guidance and direct axon migration during embryogenesis. Later studies have revealed other functions of this protein family. In this review, we focus on netrin-1, which has been shown to be involved in axon migration and angiogenesis, which are required for recovery after cerebral ischemia. Thus, therapies targeting netrin-1 may be useful for the treatment of ischemic stroke.

Endothelium-targeted transgenic GTP-cyclohydrolase I overexpression inhibits neointima formation in mouse carotid artery.

1 Tetrahydrobiopterin (BH4) is an essential cofactor that maintains the normal function of endothelial nitric oxide (NO) synthase. Restenosis is a key complication after transluminal angioplasty. Guanosine 5′‐triphosphate‐cyclohydrolase I (GTPCH) is the first rate‐limiting enzyme for de novo BH4 synthesis. However, the role of GTPCH in restenosis is not fully understood. The present study tested the hypothesis that endothelial‐targeted GTPCH overexpression retards neointimal formation, a hallmark of restenosis, in mouse carotid artery. 2 Transluminal wire injury was induced in the left carotid arteries of adult male wild‐type C57BL/6 (WT) and endothelial GTPCH transgenic (Tg‐GCH) mice. Re‐endothelialization was confirmed with in vivo Evans blue staining. Endothelium‐dependent and ‐independent relaxations were measured using isometric tension recording. Morphological analysis was performed 2 and 4 weeks after carotid injury to assess neointimal formation. Fluorescence‐based high‐performance liquid chromatography (HPLC) was used to determine GTPCH activity and BH4 levels. Basal NO release following carotid injury was assessed by NG‐nitro‐l‐arginine methyl ester‐induced vascular contraction. 3 The endothelium was completely removed upon transluminal wire injury and full re‐endothelialization was achieved at Day 10. Endothelium‐dependent relaxation was impaired 10 days and 4 weeks after carotid injury, whereas endothelium‐independent relaxation remained unaffected. Morphological analysis revealed that the endothelial‐specific overexpression of GTPCH reduced neointimal formation and medial hypertrophy 2 and 4 weeks after carotid injury. Both arterial GTPCH enzyme activity and BH4 levels were significantly elevated in Tg‐GCH mice compared with WT mice and basal NO release of the injured carotid artery tended to increase in Tg‐GCH mice. 4 These findings suggest that the endothelial overexpression of GTPCH increased endothelial BH4 synthesis and played a preventive role in neointimal formation induced by endothelium denudation.

Beclin 1 knockdown retards re-endothelialization and exacerbates neointimal formation via a crosstalk between autophagy and apoptosis

OBJECTIVE Endothelial regeneration is an essential process for the prevention of excessive neointimal formation following endothelial denudation. Beclin 1, a mammalian autophagy gene, is a link between autophagy and apoptosis. We hypothesized that the interference of Beclin 1 can influence re-endothelialization and ultimately affect neointimal formation by regulating autophagy and apoptosis. METHODS A rat carotid injury model of endothelial denudation was used, and small interfering RNA of Beclin 1 was perivascularly administered. Neointima was evaluated by morphological analysis. von Willebrand factor, Beclin 1, LC3, autophagic substrate p62 and caspase-3 levels were detected by immunofluorescence or Western blotting. Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling assay was performed to evaluate apoptosis. RESULTS Carotid injury induced an upregulation of Beclin 1 protein which was down regulated by more than 50% with small RNA interference. Beclin 1 knockdown significantly retarded re-endothelialization 7 days after injury and subsequently augmented neointima by more than 2 folds at 14 and 21 days. Autophagy and apoptosis were detected to reveal the regulatory effect of Beclin 1. The injury-activated autophagy, shown by the increased levels of punctate LC3 and LC3II as well as decreased p62 expression, was significantly inhibited by Beclin 1 knockdown. Meanwhile, the apoptotic endothelial cell number was increased and caspase-3 was up-regulated, though the expression of truncated BID was not significantly influenced. CONCLUSION Beclin 1 knockdown exacerbated neointimal formation after rat carotid injury, associated with retarded re-endothelialization due to enhanced apoptosis, while simultaneously prohibiting autophagic activation. The data suggested an essential role of Beclin 1 as a regulator between autophagy and apoptosis in the setting of neointimal formation.

Stroke-prone renovascular hypertensive rat as an animal model for stroke studies: From artery to brain

High blood pressure is a main risk factor for both initial and recurrent stroke. Compared to the post stroke situation in normotension, the brain lesion is larger in hypertension, and the treatments may not be as effective. Thus, the results from healthy individuals may not be directly applied to the hypertensive. In fact, the high prevalence of hypertension in stroke patients and its devastating effect urge the necessity to integrate arterial hypertension in the study of stroke in order to better mimic the clinical situations. The first step to do so is to have an appropriate hypertensive animal model for stroke studies. Stroke-prone renovascular hypertensive rat (RHRSP) introduced in 1998, is an animal model with acquired hypertension independent of genetic deficiency. The blood pressure begins to increase during the first week after constriction of bilateral renal arteries, and becomes sustained since around the 3rd month. Because the morphological and physiological changes of cerebral arteries are similar to those in hypertensive patients, the rats represent a higher than 60% incidence of spontaneous stroke. The animal model has several advantages: one hundred percent development of hypertension without gene modification, high similarity to human hypertension in cerebrovascular pathology and physiology, and easy establishment with low cost. Thus, the model has been extensively used in the investigation of ischemic stroke, and has been shown as a reliable animal model. This paper reviewed the features of RHRSP and its applications in the treatment and prevention of stroke, as well as the investigations of secondary lesions postischemic stroke.

Hypoxia inducible factor-1alpha mediates protection of DL-3-n-butylphthalide in brain microvascular endothelial cells against oxygen glucose deprivation-induced injury

Studies have demonstrated that DL-3-n-butylphthalide can significantly alleviate oxygen glucose deprivation-induced injury of human umbilical vein endothelial cells at least partly associated with its enhancement on oxygen glucose deprivation -induced hypoxia inducible factor-1α expression. In this study, we hypothesized that DL-3-n-butylphthalide can protect against oxygen glucose deprivation-induced injury of newborn rat brain microvascular endothelial cells by means of upregulating hypoxia inducible factor-1α expression. MTT assay and Hoechst staining results showed that DL-3-n-butylphthalide protected brain microvascular endothelial cells against oxygen glucose deprivation-induced injury in a dose-dependent manner. Western blot and immunofluorescent staining results further confirmed that the protective effect was related to upregulation of hypoxia inducible factor-1α. Real-time RT-PCR reaction results showed that DL-3-n-butylphthalide reduced apoptosis by inhibiting downregulation of pro-apoptotic gene caspase-3 mRNA expression and upregulation of apoptosis-executive protease bcl-2 mRNA expression; however, DL-3-n-butylphthalide had no protective effects on brain microvascular endothelial cells after knockdown of hypoxia inducible factor-1α by small interfering RNA. These findings suggest that DL-3-n-butylphthalide can protect brain microvascular endothelial cells against oxygen glucose deprivation-induced injury by upregulating bcl-2 expression and downregulating caspase-3 expression though hypoxia inducible factor-1α pathway.