Ruxun Huang

A study on additional early physiotherapy after stroke and factors affecting functional recovery

Objective: To investigate whether additional early physiotherapy after stroke improved functional recovery in stroke patients. Design: A prospective, randomized, controlled study. Setting: One stroke ward and an acute stroke unit in a large teaching hospital, southern China. Subjects: Patients with first-onset stroke consecutively admitted to the stroke centre. Interventions: One group (n = 78) received additional early physiotherapy (AEP) for 45 minutes, five days a week for four weeks starting within the first week since stroke onset; the routine therapy (RT) group (n = 78) received no professional rehabilitation therapy. Main outcome measures: Glasgow Coma Scale, Mini-Mental State Examination, Fugl-Meyer Assessment of Motor Recovery, Clinical Neurological Deficit Scale and Modified Barthel Index (MBI). Results: Patients from the AEP group had a high drop-out rate (n = 28), but those remaining made relatively better functional recovery at 30 days than those from the RT group if measured by MBI. Multiple linear regression analysis revealed that cognitive disturbance, aphasia, double incontinence, site of lesion and sensory impairment might affect functional recovery after stroke. Conclusions: Additional early physiotherapy might improve independence of patients after stroke but failed to show benefit in other aspects in our study. Cognitive disturbance, aphasia, double incontinence, site of lesion as well as sensory impairment might affect functional outcome after stroke.

DL-3-n-butylphthalide protects endothelial cells against oxidative/nitrosative stress, mitochondrial damage and subsequent cell death after oxygen glucose deprivation in vitro

DL-3-n-butylphthalide (NBP) has been used for stroke treatment in China for years. Recently, we found that NBP can reduce the incidence of stroke and have protective action on cerebral microvessels, suggesting a direct action of NBP on endothelial cells. However, it is difficult to evaluate the direct action of NBP on endothelial cells in vivo because of the interactions of endothelial cells with other types of neuronal cells. Therefore, we investigated whether NBP protects against oxygen glucose deprivation (OGD)-induced cell injury in an immortalized human umbilical vein endothelial cells (HUVEC) in vitro. Cells were exposed to OGD, leading to endothelial damage. Endothelial injury was assessed by measuring MTT and the changes in chromatin morphology. Mitochondrial superoxide, mitochondrial membrane potential and mitochondrial morphology were assessed using MitoSOX Red. Rhodamine 123 and MitoTracker, respectively. Nitrosative stress was assessed by measuring the production of peroxynitrite. The activity of superoxide dismutase (SOD) is evaluated using SOD assay kit-WST. The expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was assessed at the protein level by immunofluorescence and Western blotting. NBP at doses between 0.01 and 100 micromol/L dose-dependently protected against OGD-induced cell death. In addition, NBP attenuated OGD-induced mitochondria superoxide, cellular formation of peroxynitrite, and decrease in SOD activity, mitochondria fragmentation and loss of mitochondrial membrane potential. In parallel, NBP enhanced OGD-induced HIF-1alpha expression. This study demonstrates that NBP can protect HUVEC against OGD-induced oxidative/nitrosative stress, mitochondrial damage and subsequent cell death. This protective effect is, at least in part, associated with its enhancement on OGD-induced HIF-1alpha expression.

Marine compound Xyloketal B protects PC12 cells against OGD-induced cell damage.

Xyloketal B is a novel marine compound with unique chemical structure isolated from mangrove fungus Xylaria sp. (no. 2508). Recently, we have demonstrated that Xyloketal B is an antioxidant and can protect against oxidized low density lipoprotein (LDL)-induced cell injury. In the present study, we investigated whether Xyloketal B can protect against ischemia-induced cell injury in an in vitro oxygen glucose deprivation (OGD) model of ischemic stroke in PC12 cells. We found that Xyloketal B could directly scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and protect PC12 cells against OGD insult. Furthermore, Xyloketal B alleviated OGD-induced mitochondria superoxide, mitochondria fragmentation and GTPase dynamin-related protein 1 (Drp1) overexpression as well as reduction of mitochondrial membrane potential. All together, the present study demonstrates that Xyloketal B protects PC12 cells against OGD-induced cell injury and that the anti-oxidative property and protective action on mitochondria may account for its neuroprotective actions.

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.

dl-3n-Butylphthalide promotes angiogenesis via the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase/Akt-endothelial nitric oxide synthase signaling pathways.

Abstract: We have previously demonstrated that dl-3n-butylphthalide (NBP) has a potential angiogenic activity. In this study, we investigated the angiogenic effect of NBP and the molecular mechanisms underlying NBP-mediated angiogenesis. Zebrafish embryos and human umbilical vein endothelial cells were treated with various doses of NBP and several signaling pathway inhibitors. NBP induced ectopic subintestinal vessel production in zebrafish embryos and induced invasion, migration, and endothelial cell tube formation of human umbilical vein endothelial cells in a dose-dependent manner. These NBP-induced angiogenic effects were partially suppressed by SU5402, a fibroblast growth factor receptor 1 inhibitor; U0126, an extracellular signal–regulated kinase 1/2 (ERK1/2) inhibitor; LY294002, a phosphatidylinositol 3-kinase inhibitor; 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate, an Akt inhibitor; cavtratin, an endothelial nitric oxide synthase (eNOS) inhibitor and completely inhibited by a combination of U0126 and LY294002. NBP enhanced phosphorylation of ERK1/2 and fibroblast growth factor receptor 2 expression, which were inhibited by U0126. NBP increased the phosphorylation of Akt and eNOS at serine 1177, which was blocked by LY294002. NBP-stimulated nitric oxide production, which was reduced by LY294002. Our data demonstrated that (1) NBP promoted angiogenesis and (2) the angiogenic effects of NBP were mediated by the ERK1/2 and phosphatidylinositol 3-kinase/Akt-eNOS signaling pathways. Our findings suggest that NBP could be a novel agent for therapeutic angiogenesis in ischemic diseases.

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.

Peroxisome proliferator activated receptor (PPAR)-γ co-activator 1-α and hypoxia induced factor-1α mediate neuro- and vascular protection by hypoxic preconditioning in vitro.

Preconditioning-induced cellular adaptation is a new therapeutic strategy for ischemic stroke. This research aims to examine the role of peroxisome proliferator activated receptor (PPAR)-γ co-activator 1-α (PGC-1α) and hypoxia induced factor-1α (HIF-1α) in hypoxic preconditioning-induced protection. In this study, rat artery endothelial cells and neuronal PC12 cells were preconditioned with hypoxia before oxygen-glucose deprivation (OGD) insult. Cell viability, protein expression and oxidative stress were then evaluated. PGC-1α and HIF-1α were knocked down by RNA interference. We found that hypoxic preconditioning significantly reduced cell damage, enhanced the expression of PGC-1α, HIF-1α and VEGF and attenuated oxidative stress in endothelial and PC12 cells in OGD model. The protective effects of hypoxic preconditioning were hardly detected in HIF-1α or PGC-1α deficit cells. The loss of protection was accompanied with a significant loss of VEGF expression in HIF-1α or PGC-1α deficit PC12 cells and PGC-1α deficit endothelial cells as well as a considerable decrease of anti-oxidative effects in PGC-1α knocked-down endothelial cells. The present study demonstrated that both PGC-1α and HIF-1α played crucial roles in hypoxic preconditioning in endothelial and neuronal cells.

INTRACEREBROVENTRICULAR INJECTION OF EPIDERMAL GROWTH FACTOR REDUCES NEUROLOGICAL DEFICIT AND INFARCT VOLUME AND ENHANCES NESTIN EXPRESSION FOLLOWING FOCAL CEREBRAL INFARCTION IN ADULT HYPERTENSIVE RATS

1 Studies have documented the proliferative effects of epidermal growth factor (EGF) on neural progenitor cells in the normal or injured brain. The effect of EGF on post‐stroke cerebral expression of nestin, a marker of neural progenitor cells, has not been examined in hypertensive rats. 2 In the present study, adult renovascular hypertensive Sprague‐Dawley rats underwent either real or sham middle cerebral artery occlusion (MCAO). Intracerebroventricular injections of either 1 µg EGF or vehicle (0.01 mol/L phosphate‐buffered saline containing 0.1 mg/mL rat serum albumin) were made 24 and 48 h after MCAO. Then, 1, 2, 3 and 4 weeks after MCAO, the postural reflex was evaluated in a blinded fashion before rat brains were processed to determine the infarct volume plus immunoreactivity for nestin and/or glial fibrillary acidic protein (GFAP). Another group of rats was used to quantify nestin expression using western blot analysis. 3 Middle cerebral artery occlusion resulted in a focal infarct that was largest at 1 week and diminished gradually over the time. The impaired postural reflex followed a similar time‐course. In addition, MCAO induced a marked increase in nestin expression in both hemispheres, with a higher expression in the right hemisphere; this change was maximal at 1 week and largely subsided at 3 or 4 weeks. Within the right hemisphere, nestin expression was most pronounced in the subventricular and peri‐infarct zones. Most nestin‐immunoreactive cells were also positive for GFAP. 4 Thus, EGF treatment significantly increases nestin expression, reduces infarct volume and ameliorates postural reflex impairment in adult hypertensive rats.

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.