Focal Cerebral Ischemia In Rats Research Articles

Alpha-Tocopherol Reduces Brain Edema and Protects Blood-Brain Barrier Integrity following Focal Cerebral Ischemia in Rats

Objective: This study was conducted to examine the neuroprotective effects of α-tocopherol against edema formation and disruption of the blood-brain barrier (BBB) following transient focal cerebral ischemia in rats. Materials and Methods: Ninety-six male Sprague-Dawley rats were divided into 3 major groups (n = 32 in each), namely the sham, and control and α-tocopherol-treated (30 mg/kg) ischemic groups. Transient focal cerebral ischemia (90 min) was induced by occlusion of the left middle cerebral artery. At the end of the 24-hour reperfusion period, the animals were randomly selected and used for 4 investigations (n = 8) in each of the 3 main groups: (a) assessment of neurological score and measurement of infarct size, (b) detection of brain edema formation by the wet/dry method, (c) evaluation of BBB permeability using the Evans blue (EB) extravasation technique, and (d) assessment of the malondialdehyde (MDA) and reduced glutathione (GSH) concentrations using high-performance liquid chromatography methods. Results: Induction of cerebral ischemia in the control group produced extensive brain edema (brain water content 83.8 ± 0.11%) and EB leakage into brain parenchyma (14.58 ± 1.29 µg/g) in conjunction with reduced GSH and elevated MDA levels (5.86 ± 0.31 mmol/mg and 63.57 ± 5.42 nmol/mg, respectively). Treatment with α-tocopherol significantly lowered brain edema formation and reduced EB leakage compared with the control group (p < 0.001, 80.1 ± 0.32% and 6.66 ± 0.87 µg/g, respectively). Meanwhile, treatment with α-tocopherol retained tissue GSH levels and led to a lower MDA level (p < 0.01, 10.17 ± 0.83 mmol/mg, and p < 0.001, 26.84 ± 4.79 nmol/mg, respectively). Conclusion: Treatment with α-tocopherol reduced ischemic edema formation and produced protective effects on BBB function following ischemic stroke occurrence. This effect could be through increasing antioxidant activity.

Folic acid deficiency increases brain cell injury via autophagy enhancement after focal cerebral ischemia

Folic acid (FA) deficiency is not only associated with an increased risk of ischemic stroke, but also with increased oxidative DNA damage and brain injury after cerebral ischemia–reperfusion. However, the cellular and molecular mechanisms underlying FA deficiency-associated neuropathogenesis are not completely understood. In the present study, we tested the hypothesis that neuronal autophagy in focal cerebral ischemia rats may be involved in the mechanisms of FA deficiency-induced injury to neuronal cells. The results demonstrated that, accompanied by obvious neuron damage, the expression of the autophagic markers LC3 and Beclin-1, and the formation of 8-OHdG (a marker of oxidative stress to DNA) and autophagosomes were significantly increased in the brain cortex after ischemia–reperfusion. FA deficiency further induced neuronal cell death, and significantly increased the formation of autophagosomes and the expression of LC3 and Beclin-1 in NeuN-positive cell bodies after ischemia–reperfusion. The elevated level of 8-OHdG was also observed in the ischemic cortex of FA deficiency-treated animals. Conversely, the neuronal cell injury, autophagosome accumulation and the effects of LC3 and Beclin1 overexpression caused by FA deficiency were partially blocked by an autophagic inhibitor 3-methyladenine. These results suggest that FA deficiency progresses autophagic activation and aggravates the damage in rat brain cortex following focal cerebral ischemia–reperfusion. The oxidative injury may be involved in cell morphological damage and autophagy alteration caused by FA deficiency.

Sodium ferulate and n-butylidenephthalate combined with bone marrow stromal cells (BMSCs) improve the therapeutic effects of angiogenesis and neurogenesis after rat focal cerebral ischemia

BackgroundStudies have indicated that bone marrow stromal cell (BMSC) administration is a promising approach for stroke treatment. For our study, we chose sodium ferulate (SF) and n-butylidenephthalide (BP) combined with BMSC, and observed if the combination treatment possessed more significant effects on angiogenesis and neurogenesis post-stroke.MethodsWe established rat permanent middle cerebral artery occlusion (MCAo) model and evaluated ischemic volumes of MCAo, BMSC, SF + BP, Simvastatin + BMSC and SF + BP + BMSC groups with TTC staining on the 7th day after ischemia. Immunofluorescence staining of vascular endothelial growth factor (VEGF) and brain derived neurotrophic factor (BDNF), as well as immunohistochemistry staining of von Willebrand factor (vWF) and neuronal class III β-tubulin (Tuj1) were performed in ischemic boundary zone (IBZ), furthermore, to understand the mechanism, western blot was used to investigate AKT/mammalian target of rapamycin (mTOR) signal pathway in ischemic cortex. We also tested BMSC derived-VEGF and BDNF expressions by western blot assay in vitro.ResultsSF + BP + BMSC group obviously decreased infarction zone, and elevated the expression of VEGF and the density and perimeter of vWF-vessels as same as Simvastatin + BMSC administration; moreover, its effects on BDNF and Tuj1 expressions were superior to Simvastatin + BMSC treatment in IBZ. Meanwhile, it showed that SF and BP combined with BMSC treatment notably up-regulated AKT/mTOR signal pathway compared with SF + BP group and BMSC alone post-stroke. Western blot results showed that SF and BP treatment could promote BMSCs to synthesize VEGF and BDNF in vitro.ConclusionsWe firstly demonstrate that SF and BP combined with BMSC can significantly improve angiogenesis and neurogenesis in IBZ following stroke. The therapeutic effects are associated with the enhancement of VEGF and BDNF expressions via activation of AKT/mTOR signal pathway. Furthermore, triggering BMSC paracrine function of SF and BP might contribute to amplifying the synergic effects of the combination treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0979-5) contains supplementary material, which is available to authorized users.

Combination of Zizyphus jujuba and silymarin showed better neuroprotective effect as compared to single agent in MCAo-induced focal cerebral ischemia in rats

Ethnopharmacological relevanceTraditionally, Zizyphus jujuba is used for anticonvulsant, hypnotic-sedative, anxiolytic, tranquilizer, antioxidant and anti-inflammatory properties. Likewise silymarin is popularly used for its potent antioxidant and hepatoprotective effects. Stroke being a multifactorial disease with unsatisfactory treatment outcomes, necessitates development of multimodal therapeutic interventions. Thus, we evaluated the therapeutic benefits of herbal combination of Z. jujuba and silymarin in a focal cerebral ischemia model. Aim of the studyTo evaluate the neuroprotective potential of hydroalcoholic extract of Z. jujuba (HEZJ) fruit and silymarin alone and in combination in middle cerebral artery occlusion (MCAo) model of focal cerebral ischemia in rats. Materials and methodsMale Wistar rats were pretreated with HEZJ (100, 250 and 500mg/kg, p.o.) or silymarin (250mg/kg, p.o.) for 3 days prior to induction of MCAo. Neurological deficit score, motor impairment and cerebral infarction were assessed 24h following MCAo. HEZJ (250mg/kg) co-administered with silymarin (250mg/kg) for 3 days prior to induction of MCAo was also evaluated for above parameters and oxidative stress. Malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD) levels in the cortex, striatum and hippocampal brain regions were estimated 24h post MCAo. ResultsPretreatment with HEZJ and silymarin reduced the neurological deficit score, motor impairment and cerebral infarction volume. HEZJ and silymarin pretreatment also ameliorated the oxidative stress in different brain regions, which was evident from increased SOD levels, decreased MDA and NO levels as compared to MCAo control rats. Interestingly neuroprotective efficacy was potentiated by pretreatment with HEZJ and silymarin combination. ConclusionPretreatment with HEZJ and silymarin combination was observed to have better neuroprotection mediated via amelioration of oxidative stress in the focal cerebral ischemia model.

The expression of oligodendrocytes in the emotional disorder-related brain areas of the post-stroke depression model rats

Objective To explore the expression of the CNPase positive oligodendrocytes in the frontal cortex, hippocampus and amygdala of the post-stroke depression (PSD) model rats, and to understand the role of oligodendrocytes in the pathogenesis of PSD. Methods Healthy adult SD rats were randomly divided into normal group, depression group, stroke group, and PSD group(n=5 in each group). In the stroke group, focal cerebral ischemic rat model was made with thread embolization method. In the depression group, the depression model rats were made by chronic unpredictable mild stress(CUMS) combined with separately breeding.In the PSD group, focal cerebral ischemic rat model was made with thread embolization method firstly, and then PSD rat model was established with comprehensive chronic unpredictable mild stress(CUMS) and separately breeding on this basis.After the procedure, rats were subjected to sucrose preference test(SPT) and open field test(OFT). Immunofluorescence staining was used to detect the expression of the CNPase positive oligodendrocytes in the frontal cortex, hippocampus and amygdala at day 29. Results On the 29th day after CUMS, comparing the sucrose solution consumption, horizontal movement distance of open field test and vertical movement distance of the each group, the depression((26.6±1.1)%, (63.6±2.3)cm, (26.4±1.1)cm) and the PSD groups((23.8±0.8)%, (63.0±1.2)cm, (25.0±1.0)cm) were significantly lower than normal((31.2±1.9)%, (69.8±2.3)cm, (31.0±1.6)cm)and the stroke groups((31.0±1.4)%, (70.2±2.4)cm, (30.8±1.1)cm)(P<0.05). Comparing the expression of the CNPase positive oligodendrocytes in the frontal cortex, hippocampus and amygdala of the each group, the number of the stroke((16.60±1.82), (14.60±1.82), (15.00±2.12)), depression((16.40±2.07), (14.80±2.17), (15.80±2.28)) and the PSD groups((12.40±1.52), (11.20±1.48), (10.80±1.92)) were significantly less than that in normal group((20.40±3.51), (18.20±2.59), (19.00±2.55)), and the number of expression CNPase positive oligodendrocytes in the PSD group was significantly less than that in stroke and depression groups(all P<0.05). Conclusion The number of the CNPase positive oligodendrocytes in PSD group in the emotional disorders related brain areas(the frontal lobe, hippocampus and amygdale) reduced obviously, and the oligodendrocytes may play an important role in the pathogenesis of PSD. Key words: Post-stroke depression; Oligodendrocytes; Frontal lobe; Hippocampus; Amygdala

Protective effect of delta opioid receptor agonist (D-Ala2, D-Leu5) enkephalin on permanent focal cerebral ischemia in rats

To investigate the effect of delta opioid receptor agonist (D-Ala, D-Leu) enkephalin (DADLE) on the permanent focal cerebral ischemia in rats. Thirty four male Sprague-Dawley rats were assigned randomly into three groups: sham group (group Sham, n=10), artificial cerebrospinal fluid group (group ACSF, n=12), and DADLE group (group DADLE, n=12). Permanent middle cerebral artery occlusion was performed to induce permanent focal cerebral ischemia in rats. Then, the animals in group DADLE and group ACSF were treated with DADLE or ACSF by an intracerebroventricular injection at 45 min after ischemia. Neurologic deficit scores were assessed according to the Garcia criterion at 24 h after ischemia. Infarct volume was determined using the 2,3,5-triphenyltetrazolium chloride staining method. The histological analysis was used to evaluate the extent of cerebral injury. Compared with the control group, the Garcia scores were significantly higher (P=0.000) and the infarct volumes (P=0.018) were significantly smaller in the DADLE treatment group at 24 h after ischemia. These neurologic changes were closely correlated with the outcome of the infarct volumes. In addition, the histological examination showed more intact neurons in rats treated with DADLE than those treated with ACSF at 24 h after ischemia (P=0.000). DADLE by intracerebroventricular administration at 45 min after ischemia can improve neurologic outcome and mitigate cortical neuronal injury induced by permanent focal cerebral ischemia in rats.

Inhibition of chemokine-like factor 1 improves blood-brain barrier dysfunction in rats following focal cerebral ischemia

Disruption of blood-brain barrier (BBB) and subsequent edema are major contributors to the pathogenesis of ischemic stroke, and the current clinical therapy remains unsatisfied. Chemokine-like factor 1 (CKLF1), as a novel C-C chemokine, plays important roles in immune response. The expression of CKLF1 increased after focal cerebral ischemia and inhibition of CKLF1 activity showed neuroprotective effect by alleviating infiltration of neutrophil and neuron apoptosis in cerebral ischemia. However, few studies have focused on the role of CKLF1 on BBB integrity. The objective of present study was to investigate the role of CKLF1 on BBB integrity by applying anti-CKLF1 antibodies in rat focal cerebral ischemia and reperfusion model. Brain water content, Evans blue leakage and the expression of aquaporin-4 (AQP-4), matrix metalloproteinase-9 (MMP-9), Zonula Occludens-1 (ZO-1) and Occludin were measured. After treatment with anti-CKLF1 antibody, brain water content and Evans blue leakage in ipsilateral hemisphere were decreased in a dose-dependent manner at 24h after reperfusion, but not changed in contralateral hemisphere. Anti-CKLF1 antibody reduced the expression of AQP-4 and MMP-9, and upregulated the expression of ZO-1 and Occludin. These results suggest that CKLF1 is involved in BBB disruption after reperfusion. Inhibition of CKLF1 protects against cerebral ischemia by maintaining BBB integrity, possibly via inhibiting the expression of AQP-4 and MMP-9, and increasing the expression of tight junction protein.

Nrf2/antioxidant defense pathway is involved in the neuroprotective effects of Sirt1 against focal cerebral ischemia in rats after hyperbaric oxygen preconditioning

Sirtuin 1 (Sirt1) is a class III histone deacetylase involved in neuroprotection induced by hyperbaric oxygen preconditioning (HBO-PC) in animal models of ischemia. However, the underlying mechanisms remain to be illustrated. In the present study, rats exposed to middle cerebral artery occlusion (MCAO) were used to establish an ischemic stroke model. The infarct volume ratio, neurobehavioral score, and expressions of Sirt1, nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and superoxide dismutase 1 (SOD1) were evaluated at 7 days after reperfusion, and the level of malondialdehyde (MDA) was used to assess oxidative stress. HBO-PC increased the expression of Sirt1 and reduced infarct volume ratio and neurobehavioral deficit in MCAO rats. Meanwhile, HBO-PC also increased expression of Nrf2, HO-1, and SOD1 and decreased MDA content. Furthermore, either Sirt1 or Nrf2 knockdown by short interfering RNA (siRNA) inhibited the expression of Nrf2, HO-1, and SOD1 and eliminated the neuroprotective effects of HBO-PC. Taken together, the results suggest that the Nrf2/antioxidant defense pathway is involved in the long lasting neuroprotective effects of Sirt1 induced by HBO-PC against transient focal cerebral ischemia.

Remote limb preconditioning protects against ischemia-induced neuronal death through ameliorating neuronal oxidative DNA damage and parthanatos

Remote limb preconditioning (RPC) ameliorates ischemia-induced cerebral infarction and promotes neurological function recovery; however, the mechanism of RPC hasn't been fully understood, which limits its clinical application. The present study aimed at exploring the underlying mechanism of RPC through testing its effects on neuronal oxidative DNA damage and parthanatos in a rat focal cerebral ischemia model. Infarct volume was investigated by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, and neuronal survival was evaluated by Nissl staining. Oxidative DNA damage was investigated via analyzing the expression of 8-hydroxy-2′-deoxyguanosine (8-OHdG). Besides, terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL) and DNA laddering were utilized to evaluate neuronal DNA fragmentation. Moreover, we tested whether RPC regulated poly(ADP-ribose) polymer (PAR) and apoptosis inducing factor (AIF) pathway; thus, PAR expression, AIF translocation and AIF/histone H2AX (H2AX) interaction were investigated. The results showed that RPC exerted neuroprotective effects by ameliorating oxidative DNA damage and neuronal parthanatos; additionally, RPC suppressed PAR/AIF pathway through reducing AIF translocation and AIF/H2AX interaction. The present study further exposed neuroprotective mechanism of RPC, and provided new evidence for the research on RPC and ICS.

1H NMR-based metabonomics revealed protective effect of Naodesheng bioactive extract on ischemic stroke rats

Ethnopharmacological relevanceStroke is a leading cause of death and disability in the world. However, current therapies are limited. Naodesheng, a widely used traditional Chinese medicine prescription, has shown a good clinical curative effect on ischemic stroke. Also, Naodesheng has been suggested to have neuroprotective effect on focal cerebral ischemia rats, but the underlying molecular mechanism remains unclear. Aim of the studyThe present study was designed to evaluate the effect of Naodesheng bioactive extract on the metabolic changes in brain tissue, plasma and urine induced by cerebral ischemia perfusion injury, and explore the possible metabolic mechanisms by using a 1H NMR-based metabonomics approach. Materials and methodsA middle cerebral artery occlusion rat model was established and confirmed by the experiments of neurobehavioral abnormality evaluation, brain tissue TTC staining and pathological examination. The metabolic changes in brain tissue, plasma and urine were then assessed by a 1H NMR technique combined with multivariate statistical analysis method. ResultsThese NMR data showed that cerebral ischemia reperfusion induced great metabolic disorders in brain tissue, plasma and urine metabolisms. However, Naodesheng bioactive extract could reverse most of the imbalanced metabolites. Meanwhile, it was found that both the medium and high dosages of Naodesheng bioactive extract were more effective on the metabolic changes than the low dosage, consistent with histopathological assessments. ConclusionsThese results revealed that Naodesheng had protective effect on ischemic stroke rats and the underlying mechanisms involved multiple metabolic pathways, including energy metabolism, amino acid metabolism, oxidative stress and inflammatory injury. The present study could provide evidence that metabonomics revealed its capacity to evaluate the holistic efficacy of traditional Chinese medicine and explore the underlying mechanisms.

N-Acetylcysteine and Ceftriaxone as Preconditioning Strategies in Focal Brain Ischemia: Influence on Glutamate Transporters Expression

Glutamate (Glu) plays a key role in excitotoxicity-related injury in cerebral ischemia. In the brain, Glu homeostasis depends on Glu transporters, including the excitatory amino acid transporters and the cysteine/Glu antiporter (xc-). We hypothesized that drugs acting on Glu transporters, such as ceftriaxone (CEF, 200 mg/kg, i.p.) and N-acetylcysteine (NAC, 150 mg/kg, i.p.), administered repeatedly for 5 days before focal cerebral ischemia in rats and induced by a 90-min middle cerebral artery occlusion (MCAO), may induce brain tolerance to ischemia. We compared the effects of these drugs on brain infarct volume, neurological deficits and the mRNA and protein expression of the Glu transporter-1 (GLT-1) and xc- with the effects of ischemic preconditioning and chemical preconditioning using 3-nitropropionic acid. Administration of CEF and NAC significantly reduced infarct size and neurological deficits caused by a 90-min MCAO. These beneficial effects were accompanied by changes in GLT-1 expression caused by a 90-min MCAO at both the mRNA and protein levels in the frontal cortex, hippocampus, and dorsal striatum. Thus, the results of this study suggest that the regulation of GLT-1 and xc- plays a role in the development of cerebral tolerance to ischemia and that this regulation may be a novel approach in the therapy of brain ischemia.

Histopathologic comparison of dexmedetomidine's and thiopental's cerebral protective effects on focal cerebral ischemia in rats

This study was designed to investigate whether dexmedetomidine and thiopental have cerebral protective effects after focal cerebral ischemia in rats. Thirty male Sprague Dawley rats were randomly assigned to three groups: control group (Group C, n=10), dexmedetomidine group (Group D, n=10), thiopental group (Group T, n=10). After all rats were anesthetized, they were intubated, then mechanically ventilated. A catheter was inserted into the right femoral artery for continuous mean arterial pressure, physiological parameters and blood sampling at baseline, 5min after occlusion and 20min after reperfusion. A catheter was inserted into the left femoral vein for intravenous (IV) medication administration. Right common carotid artery of each rat was isolated and clamped for 45min. At the end of the duration common carotid artery were unclamped and the brain reperfusion was achieved for 90min. Dexmedetomidine was administered for Group D IV infusion, and Group T received thiopental IV. According to histopathologic scores cerebral ischemia was documented in all rats in Group C, but no ischemia was found in three rats in Group T and in four rats in Group D. Grade 3 cerebral ischemia was documented in three rats in Group C, and in only one rat in both groups T and D. For histopathologic grades the difference between Group T and Group D was not significant (p>0.05). But the differences between Group C and Group T (p<0.05) and Group C and Group D (p<0.01) were statically significant. In conclusion, we demonstrated that dexmedetomidine and thiopental have experimental histopathologic cerebral protective effects on experimental focal cerebral ischemia in rats.

Neuroprotective Effects of Isosteviol Sodium Injection on Acute Focal Cerebral Ischemia in Rats.

Previous report has indicated that isosteviol has neuroprotective effects. However, isosteviol was administered preventively before ischemia and the inclusion criteria were limited. In the present study, a more soluble and injectable form of isosteviol sodium (STVNA) was administered intravenously hours after transient or permanent middle cerebral artery occlusion (tMCAO or pMCAO) to investigate its neuroprotective effects in rats. The rats were assessed for neurobehavioral deficits 24 hours after ischemia and sacrificed for infarct volume quantification and histology evaluation. STVNA 10 mg·kg−1 can significantly reduce the infarct volumes compared with vehicle in animals subjected to tMCAO and is twice as potent as previously reported. Additionally, the therapeutic window study showed that STVNA could reduce the infarct volume compared with the vehicle group when administered 4 hours after reperfusion. A similar effect was also observed in animals treated 4 hours after pMCAO. Assessment of neurobehavioral deficits after 24 hours showed that STVNA treatment significantly reduced neurobehavioral impairments. The number of restored NeuN-labeled neurons was increased and the number of TUNEL positive cells was reduced in animals that received STVNA treatment compared with vehicle group. All of these findings suggest that STVNA might provide therapeutic benefits against cerebral ischemia-induced injury.

Inhibition of MicroRNA-383 Ameliorates Injury After Focal Cerebral Ischemia via Targeting PPARγ

Background: Peroxisome proliferator-activated receptor gamma (PPARγ) plays a critical role in protecting against distinct brain damages, including ischemia. Our previous data have shown that the protein level of PPARγ is increased in the cortex after middle cerebral artery occlusion (MCAO); PPARγ up-regulation contributes to PPARγ activation and is effective in reducing ischemic damage to brain. However, the regulatory mechanism of PPARγ after focal cerebral ischemia in rats is still unclear. In this study, we evaluated the effect of microRNA on PPARγ in rats subjected to MCAO. Methods: Focal cerebral ischemia was established by surgical middle cerebral artery occlusion; the protein level of PPARγ was detected by Western blotting; the level of microRNA-383 (miR-383) was quantified by real-time PCR; the neurological outcomes were defined by infarct volume and neurological deficits. Luciferase assay was used to identify the luciferase activities of PPARγ and miR-383. Results: We showed here that miR-383 level was down-regulated in the ischemic hemisphere of rats 24h after MCAO. Overexpression of miR-383 by miR-383 agomir increased infarct volume and aggravated neurological damage. Administration of miR-383 antagomir had the opposite effects. Furthermore, we found that PPARγ protein was down-regulated by miR-383 overexpression, and up-regulated by miR-383 inhibition both in rat model of MCAO and in primary culture cells. Finally, we found that miR-383 suppressed the luciferase activity of the vector carrying the 3'UTR of PPARγ, whereas mutation of the binding sites relived the repressive effect of miR-383. Conclusion: Our study demonstrated that miR-383 may play a key role in focal cerebral ischemia by regulating PPARγ expression at the post-transcriptional level, and miR-383 may be a potential therapeutic target for stroke.

Effects of erythropoietin combined with tissue plasminogen activator on the rats following cerebral ischemia and reperfusion.

OBJECTIVES:Exogenously administered recombinant human erythropoietin (rhEPO) has been reported to exhibit neuroprotective effects in animal models. However, there are still have some controversies that combination of EPO and tissue plasminogen activator (tPA) in acute ischemic stroke. In the present study, we investigated the effects of local intra-arterial infusion of low-dose EPO in combination with tPA on focal cerebral ischemic stroke.MATERIALS AND METHODS:Sixty adult male Sprague–Dawley rats were randomly divided into five groups, including sham, vehicle, EPO, tPA, and EPO+tPA groups. Rats were subjected to middle cerebral artery occlusion (MCAO) and administrated with EPO (800 U/kg, middle cerebral artery injection), tPA (10 mg/kg, tail vein injection), EPO+tPA, or saline (vehicle) onset of reperfusion. Neurobehavioral deficits, infarct volume, brain edema, the expression of tight junction proteins (Claudin-5, Occludin), and AQP4 were assessed following 2 h ischemia and 24 h reperfusion. The number of apoptotic cells in the periinfarct region was detected by the terminal deoxyribonucleotide transferase dUTP nick end labeling (TUNEL) staining.RESULTS:The neurobehavioral deficits, brain infarct volume, edema volume, TUNEL-positive cells and downregulation of Claudin-5 and Occludin were alleviated by EPO or EPO plus tPA, following the ischemia/reperfusion (I/R) in rats. The EPO and EPO plus tPA both reduced the upregulation of AQP4 in the ischemic brain tissue.CONCLUSION:Our data demonstrate local intra-arterial infusion of low-dose EPO in combination with tPA protected against focal cerebral ischemia in rats manifested by a decrease in brain edema and blood-brain barrier (BBB) disruption after 2 h ischemia and 24 h reperfusion.

LXW7 ameliorates focal cerebral ischemia injury and attenuates inflammatory responses in activated microglia in rats.

Inflammation plays a pivotal role in ischemic stroke, when activated microglia release excessive pro-inflammatory mediators. The inhibition of integrin αvβ3 improves outcomes in rat focal cerebral ischemia models. However, the mechanisms by which microglia are neuroprotective remain unclear. This study evaluated whether post-ischemic treatment with another integrin αvβ3 inhibitor, the cyclic arginine-glycine-aspartic acid (RGD) peptide-cGRGDdvc (LXW7), alleviates cerebral ischemic injury. The anti-inflammatory effect of LXW7 in activated microglia within rat focal cerebral ischemia models was examined. A total of 108 Sprague-Dawley rats (250–280 g) were subjected to middle cerebral artery occlusion (MCAO). After 2 h, the rats were given an intravenous injection of LXW7 (100 μg/kg) or phosphate-buffered saline (PBS). Neurological scores, infarct volumes, brain water content (BWC) and histology alterations were determined. The expressions of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)], and Iba1-positive activated microglia, within peri-ischemic brain tissue, were assessed with ELISA, western blot and immunofluorescence staining. Infarct volumes and BWC were significantly lower in LXW7-treated rats compared to those in the MCAO + PBS (control) group. The LXW7 treatment lowered the expression of pro-inflammatory cytokines. There was a reduction of Iba1-positive activated microglia, and the TNF-α and IL-1β expressions were attenuated. However, there was no difference in the Zea Longa scores between the ischemia and LXW7 groups. The results suggest that LXW7 protected against focal cerebral ischemia and attenuated inflammation in activated microglia. LXW7 may be neuroprotective during acute MCAO-induced brain damage and microglia-related neurodegenerative diseases.

Single photon emission computed tomography imaging of cerebral blood flow, blood-brain barrier disruption, and apoptosis time course after focal cerebral ischemia in rats.

Cerebral ischemia is a leading cause of disability worldwide and no other effective therapy has been validated to date than intravenous thrombolysis. In this context, many preclinical models have been developed and recent advances in preclinical imaging represent promising tools. Thus, we proposed here to characterize invivo time profiles of cerebral blood flow, blood-brain barrier disruption and apoptosis following a transient middle cerebral artery occlusion in rats using SPECT/CT imaging. Rats underwent a 1-h middle cerebral artery occlusion followed by reperfusion. Cerebral blood flow, blood-brain barrier disruption and apoptosis were evaluated by SPECT/CT imaging using respectively (99m)Tc-HMPAO, (99m)Tc-DTPA and the experimental (99m)Tc-Annexin V-128, up to 14 days after middle cerebral artery occlusion. Histological evaluation of apoptosis has been performed using TUNEL method to validate the (99m)Tc-Annexin V-128 uptake. (99m)Tc-HMPAO cerebral blood flow evaluation showed hypoperfusion during occlusion, partially restored on days 4 and 7 and sustained up to 14 days after middle cerebral artery occlusion. (99m)Tc-DTPA SPECT/CT showed a blood-brain barrier disruption starting on day 1 post-middle cerebral artery occlusion, peaking on day 2, with barrier integrity totally restored on day 7. (99m)Tc-Annexin V-128 SPECT/CT imaging showed significant positive correlation with TUNEL immunohistochemistry and allowed ischemic-induced apoptosis to be detected from day 2 to day 7, peaking on day 3 after middle cerebral artery occlusion. Using SPECT/CT imaging, we showed that after transient middle cerebral artery occlusion in rat there was a sustained decrease in cerebral blood flow followed by blood-brain barrier disruption preceding meanwhile apoptosis. Rodent SPECT/CT imaging of cerebral blood flow, blood-brain barrier disruption and apoptosis appears to be an efficient tool for evaluating neuroprotective drugs and regenerative therapies against cerebral ischemia and time-windows for therapeutic intervention.

Neuroprotective effect of ethyl acetate extract from gastrodia elata against transient focal cerebral ischemia in rats induced by middle cerebral artery occlusion

ObjectiveTo investigate the protective effect of neuroprotection against transient focal cerebral ischemia of the extract from Tianma (Rhizoma Gastrodiae) and the possible mechanisms underlying the action. MethodsCerebral ischemia-reperfusion injury was induced through middle cerebral artery occlusion (MCAO). Adult male Sprague-Dawley rats were randomly divided into four groups: sham-operated, ischemia-reperfusion model, 102.6 mg/kg extract treated and 11.4 mg/kg extract treated groups. The extract was prepared from gastrodia elata with ethyl acetate. The effect of the extract tested on rat neurological deficits and Cerebral index, cerebral infarct volume, brain injury, terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and B-cell lymphoma-2 (Bcl-2) positive cells. ResultsThe extract was able to reduce neurological scores, cerebral index and cerebral infarction rate. The brain injury was also relieved by the extract. The results of immunofluorescence staining analysis indicated that the extract increased the expression of Bcl-2 and reduced TUNEL-positive cells significantly in the extract treated groups. ConclusionThese results suggested that the extract relieved ischemic injury induced by transient focal cerebral ischemia in rats, and this neuroprotective effect might be partially due to the attenuated apoptosis pathway.

Neuroprotective Effects of Brain-Derived Neurotrophic Factor and Noggin-Modified Bone Mesenchymal Stem Cells in Focal Cerebral Ischemia in Rats

Administration of bone marrow stromal cells (BMSCs) has been reported to ameliorate functional deficits in rat ischemia models. In the present study, we tried to reveal the underlying mechanism of the improvement of neurological function after stroke by BMSCs transfected with brain-derived neurotrophic factor (BDNF) and/or Noggin. BMSCs were transfected with BDNF or/and Noggin using the adenovirus method. Middle cerebral artery occlusion (MCAO) rat models were treated with different types of transfected BMSCs. The treatment effect was assessed by measuring the modified Neurological Severity Score and the expression levels of different stroke-related molecules using Western blot, immunohistochemistry assay (IHC), and enzyme-linked immunosorbent assay (ELISA). The injection of BDNF or/and Noggin-modified BMSCs could significantly improve the neurological function of MCAO animals. Western blot and IHC staining showed that the expression levels of vascular endothelial growth factor, BCL-2, p-GSK3β, and p-Akt were significantly upregulated, while the expressions of Bax, TLR4, and MyD88 were significantly downregulated. Moreover, ELISA assay revealed that the level of matrix metallopeptidase 9 (MMP-9) and reactive oxygen species were also significantly decreased. These results suggested that the treatment of BDNF or/and Noggin-modified BMSCs may suppress the ischemia-induced apoptosis and inflammation in the model animals, which might be through the Akt/GSK3β and TLR4/MyD88 pathways. BDNF or/and Noggin-modified BMSCs may exert neuroprotective effects through the Akt/GSK3β and TLR4/MyD88 pathways. Transplantation of BDNF or/and Noggin-modified BMSCs might be a potential therapeutic method for ischemic stroke in clinics.

Bone marrow mesenchymal stem cell transplantation enhances angiogenesis and functional recovery after cerebral ischemia in rats

Objective To investigate the effect of bone marrow mesenchymal stem cells(BMSCs) transplantation on angiogenesis and functional recovery after focal cerebral ischemia in rats. Methods BMSCs were isolated and cultured using the whole bone marrow adherent method, and conducted phenotypic identification using flow cytometry analysis of surface positive antigen of CD29, CD90 and the negative antigen of CD34, CD45.Rats were subjected to middle cerebral artery occlusion (MCAO) for 90 minutes, and divided into three groups randomly, the sham group, model group and BMSCs group.24 hours after cerebral ischemia, rats were injected with 1ml BMSCs solution (1×106 cells/ml) or PBS via the tail vein. The modified neurological severity score( mNSS ) test, the corner test and the adhesive tape test were used to evaluate sensorimotor function on the 1, 7, 14 and 28 days after ischemia. Infarcted volume was detected by toluidine blue staining, and the numbers of vWF positive microvessels and vascular endothelial growth factor (VEGF) positive cells in the ischemic boundary were determined by immunofluorescence. Results By flow cytometric analysis, the cell phenotype of passage 3 BMSCs showed that CD29, CD90, CD34 and CD45 were 98.3%, 97.4%, 0.2% and 4.8%, respectively . Compared with the model group, BMSCs significantly reduced the score of mNSS(P<0.01), the number of right turn of corner test(P<0.05), latency of removal adhesive tape(P<0.05) and the infarcted volume (P<0.01). The numbers of vWF positive vessels and the VEGF positive cells were (42.97±8.64)/mm2 and (54.83±10.66)/mm2 at the boundary zone in model group 14 days after ischemia, respectively. BMSCs significantly increased the numbers of vWF positive vessels ((69.43±7.29)/mm2) and VEGF positive cells ((78.70±6.16)/mm2 , P<0.01). Conclusion BMSCs can improve the functions of cerebral lesions after cerebral ischemia, which may be associated with the enhanced angiogenesis and VEGF expression in the ischemic boundary. Key words: Bone marrow mesenchymal stem cells; Cerebral ischemia; Angiogenesis; Vascular endothelial growth factor