MCAO-induced Focal Cerebral Ischemia Research Articles

Tau-mediated iron export prevents ferroptotic damage after ischemic stroke.

Functional failure of tau contributes to age-dependent, iron-mediated neurotoxicity, and as iron accumulates in ischemic stroke tissue, we hypothesized that tau failure may exaggerate ischemia-reperfusion-related toxicity. Indeed, unilateral, transient middle cerebral artery occlusion (MCAO) suppressed hemispheric tau and increased iron levels in young (3-month-old) mice and rats. Wild-type mice were protected by iron-targeted interventions: ceruloplasmin and amyloid precursor protein ectodomain, as well as ferroptosis inhibitors. At this age, tau-knockout mice did not express elevated brain iron and were protected against hemispheric reperfusion injury following MCAO, indicating that tau suppression may prevent ferroptosis. However, the accelerated age-dependent brain iron accumulation that occurs in tau-knockout mice at 12 months of age negated the protective benefit of tau suppression against MCAO-induced focal cerebral ischemia-reperfusion injury. The protective benefit of tau knockout was revived in older mice by iron-targeting interventions. These findings introduce tau-iron interaction as a pleiotropic modulator of ferroptosis and ischemic stroke outcome.

Coenzyme Q10 ameliorates cerebral ischemia reperfusion injury in hyperglycemic rats

The purpose of this study is to investigate the effect of coenzyme Q10 (CoQ10) on focal cerebral ischemia/reperfusion (I/R) injury in hyperglycemic rats and the possible involved mechanisms. In this study, we established the transient middle cerebral artery occlusion (MCAO) for 30min in the rats with diabetic hyperglycemia. The neurological deficit score, 2,3,5-triphenyltetrazolium chloride (TTC) staining and pathohistology are applied to detect the extent of the damage. The expression of Fis1, Mfn2 and Lc3 in the brain is investigated by immunohistochemical and Western blotting techniques. The results showed that the streptozotocin-induced diabetic hyperglycemia and MCAO-induced focal cerebral ischemia were successfully prepared in rats. In the hyperglycemic group, the neurological deficit scores, infarct volumes, and number of pyknotic cells were higher than that in the normalglycemic group at 24h and/or 72h reperfusion. Pretreated with CoQ10 (10mg/kg) for four weeks could significantly reduce the neurological scores, infarct volume, and pyknotic cells at 24h and/or 72h reperfusion of the hyperglycemic rats compared with non-CoQ10 pretreated hyperglycemic animals. Immunohistochemistry and Western blotting showed that pretreatment with CoQ10 or insulin could significantly reduce the expression of Fis1 protein in the brain at 24h and 72h reperfusion. Inversely, a significantly increased expression of Mfn2 was observed in the rats CoQ10 or insulin pretreated at 24h and/or 72h reperfusion when compared with matched hyperglycemic rats. These results demonstrated that hyperglycemia could aggravate ischemic brain injury. Pretreatment with CoQ10 might ameliorate the diabetic hyperglycemia aggravated I/R brain damage in the MCAO rats by maintain the balance between mitochondrial fission and fusion.

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.

Effect of embelin in middle cerebral artery occlusion-induced focal cerebral ischemia in rats

Objective: Cerebral ischemia induced by stroke is one of the major causes of death and disability in the world and reactive oxygen species play key role in pathogenesis of cerebral ischemia. Chemical structure of embelin is similar to antioxidant coenzyme Q10 and embelin is reported to have potent antioxidant activity. Therefore, the present investigation is carried out to study the effect of embelin using the middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia model. Methods: Male Wistar rats were treated with embelin (50, 75 and 100 mg/kg, p.o.) for 20 days, followed by MCAO-induced focal cerebral ischemia. Measurement of neurological score, infarct size and malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) in brain homogenates were carried out at the end of study. Results: Embelin decreases MDA level whereas increases SOD and CAT level as compared to ischemic control group. Moreover, embelin decreases infarct size, but there was no improvement in neurological score. Conclusion: Pretreatment of embelin enhances the antioxidant defense and thereby ameliorates cerebral ischemia/reperfusion induced injury in the rat MCAO model.

Hinokitiol, a Natural Tropolone Derivative, Offers Neuroprotection from Thromboembolic StrokeIn Vivo

Hinokitiol (β-thujaplicin), a tropolone-related compound found in the heartwood cupressaceous plants, is widely used in hair tonics, tooth pastes, cosmetics, and food as an antimicrobial agent. Increasing evidence has confirmed that hinokitiol exhibits anticancer activity in a variety of cancers through inhibition of cell proliferation. In the present study, we have investigated the neuroprotective effect and mechanisms of hinokitiol in rats against middle cerebral artery occlusion (MCAO)-induced thromboembolic stroke. Treatment with hinokitiol (0.2 and 0.5 mg/kg; intraperitoneally) 30 min before MCAO dose dependently attenuated cerebral ischemia and improved neurobehavioral deficits in cerebral ischemic rats. Intraperitoneal administration of hinokitiol significantly reduced infarct size compared to that in control rats. MCAO-induced focal cerebral ischemia was associated with increased expressions of hypoxia-inducible factor (HIF)-1α, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and active caspase-3 in ischemic regions. However, these expressions were obviously inhibited by hinokitiol (0.2 and 0.5 mg/kg) treatment. This study demonstrates for the first time that in addition to being originally considered as an agent against microbes and variety of cancers, hinokitiol possesses potent neuroprotective activity. This activity is mediated, at least in part, by inhibition of inflammatory responses (i.e., HIF-1α, iNOS expression) and apoptosis (i.e., TNF-α, active caspase-3), resulting in a reduction of infarct volume and improvement in neurobehavior in rats with cerebral ischemia. Therefore, the therapeutic potential of hinokitiol may lead to novel role for treatment or prevention of ischemia/reperfusion injury-related disorders.

Neuroprotective Effects of Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus), in Ischemic Stroke of Rats

Xanthohumol is the principal prenylated flavonoid in hops (Humulus lupulus L.), an ingredient of beer. Xanthohumol was found to be a potent chemopreventive agent; however, no data are available concerning its neuroprotective effects. In the present study, the neuroprotective activity and mechanisms of xanthohumol in rats with middle cerebral artery occlusion (MCAO)-induced cerebral ischemia were examined. Treatment with xanthohumol (0.2 and 0.4 mg/kg; intraperitoneally) 10 min before MCAO dose-dependently attenuated focal cerebral ischemia and improved neurobehavioral deficits in cerebral ischemic rats. Xanthohumol treatment produced a marked reduction in infarct size compared to that in control rats. MCAO-induced focal cerebral ischemia was associated with increases in hypoxia-inducible factor (HIF)-1α, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), and active caspase-3 protein expressions in ischemic regions. These expressions were obviously inhibited by treatment with xanthohumol. In addition, xanthohumol (3-70 μM) concentration-dependently inhibited platelet aggregation stimulated by collagen (1 μg/mL) in human platelet-rich plasma. An electron spin resonance (ESR) method was used to examine the scavenging activity of xanthohumol on free radicals which had formed. Xanthohumol (1.5 and 3 μM) markedly reduced the ESR signal intensity of hydroxyl radical (OH•) formation in the H₂O₂/NaOH/DMSO system. In conclusion, this study demonstrates for the first time that in addition to its originally being considered an agent preventing tumor growth, xanthohumol possesses potent neuroprotective activity. This activity is mediated, at least in part, by inhibition of inflammatory responses (i.e., HIF-1α, iNOS expression, and free radical formation), apoptosis (i.e., TNF-α, active caspase-3), and platelet activation, resulting in a reduction of infarct volume and improvement in neurobehavior in rats with cerebral ischemia. Therefore, this novel role of xanthohumol may represent high therapeutic potential for treatment or prevention of ischemia-reperfusion injury-related disorders.

Neuroprotection by the Traditional Chinese Medicine, Tao‐Hong‐Si‐Wu‐Tang, against Middle Cerebral Artery Occlusion‐Induced Cerebral Ischemia in Rats

Tao-Hong-Si-Wu-Tang (THSWT) is a famous traditional Chinese medicine (TMC). In the present study, oral administration of THSWT (0.7 and 1.4 g kg−1day−1) for 14 days before MCAO dose-dependently attenuated focal cerebral ischemia in rats. MCAO-induced focal cerebral ischemia was associated with increases in hypoxia-inducible factor (HIF)-1α, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and active caspase-3 expressions in ischemic regions. These expressions were obviously inhibited by 0.7 g kg−1day−1 THSWT treatment. In addition, THSWT inhibited platelet aggregation stimulated by collagen in washed platelets. In an in vivo study, THSWT (16 g kg−1) significantly prolonged platelet plug formation in mice. However, THSWT (20 and 40 μg mL−1) did not significantly reduce the electron spin resonance (ESR) signal intensity of hydroxyl radical (OH•) formation. In conclusion, the most important findings of this study demonstrate for the first time that THSWT possesses potent neuroprotective activity against MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by the inhibition of both HIF-1α and TNF-α activation, followed by the inhibition of inflammatory responses (i.e., iNOS expression), apoptosis formation (active caspase-3), and platelet activation, resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury.

Neuroprotective mechanisms of puerarin in middle cerebral artery occlusion-induced brain infarction in rats

Puerarin, a major isoflavonoid derived from the Chinese medical herb Radix puerariae (kudzu root), has been reported to be useful in the treatment of various cardiovascular diseases. In the present study, we examined the detailed mechanisms underlying the inhibitory effects of puerarin on inflammatory and apoptotic responses induced by middle cerebral artery occlusion (MCAO) in rats. Treatment of puerarin (25 and 50 mg/kg; intraperitoneally) 10 min before MCAO dose-dependently attenuated focal cerebral ischemia in rats. Administration of puerarin at 50 mg/kg, showed marked reduction in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in hypoxia-inducible factor-1α (HIF-1α), inducible nitric oxide synthase (iNOS), and active caspase-3 protein expressions as well as the mRNA expression of tumor necrosis factor-α (TNF-α) in ischemic regions. These expressions were markedly inhibited by the treatment of puerarin (50 mg/kg). In addition, puerarin (10~50 μM) concentration-dependently inhibited respiratory bursts in human neutrophils stimulated by formyl-Met-Leu-Phe. On the other hand, puerarin (20~500 μM) did not significantly inhibit the thiobarbituric acid-reactive substance reaction in rat brain homogenates. An electron spin resonance (ESR) method was conducted on the scavenging activity of puerarin on the free radicals formed. Puerarin (200 and 500 μM) did not reduce the ESR signal intensity of hydroxyl radical formation. In conclusion, we demonstrate that puerarin is a potent neuroprotective agent on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by the inhibition of both HIF-1α and TNF-α activation, followed by the inhibition of inflammatory responses (i.e., iNOS expression), apoptosis formation (active caspase-3), and neutrophil activation, resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, puerarin treatment may represent a novel approach to lowering the risk of or improving function in ischemia-reperfusion brain injury-related disorders.

Neuroprotective Effects of Acetylsalicylic Acid in Middle Cerebral Artery Occlusion–Induced Brain Ischemia in Rats: Suppression of iNOS, HIF-1α, TNF-α, and Active Caspase-3 Expression

Acetylsalicylic acid (ASA) is neuroprotective through various pharmacological action sites. The aim of this study was to examine the detailed mechanisms underlying the inhibitory effect of ASA in inflammatory and apoptotic responses induced by middle cerebral artery occlusion (MCAO) in rats. In this study, ASA significantly attenuated MCAO-induced focal cerebral ischemia in rats. Administration of ASA at 10–20 mg/kg showed marked reductions in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in iNOS, HIF-1α, active caspase-3, and TNF-α mRNA expressions in ischemic regions. These expressions were markedly inhibited by treatment with ASA (20 mg/kg). In conclusion, the neuroprotective effect of ASA may mediate at least a portion of the inhibition of HIF-1α and TNF-α activations, followed by inhibition of apoptosis formation (active caspase-3) and inflammatory response (iNOS), resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, ASA treatment may represent an ideal approach to lowering the risk of or improving function in ischemia-reperfusion brain injury–related disorders.

Tetramethylpyrazine suppresses HIF-1?, TNF-?, and activated caspase-3 expression in middle cerebral artery occlusion-induced brain ischemia in rats

To examine the detailed mechanisms underlying the inhibitory effect of tetramethylpyrazine (TMPZ) in inflammatory and apoptotic responses induced by middle cerebral artery occlusion (MCAO) in rats. MCAO-induced focal cerebral ischemia in rats was used in this study. The hypoxia-inducible factor-1alpha(HIF-1alpha), activation of caspase-3, and TNF-alpha mRNA transcription in ischemic regions were detected by immunoblotting and RT-PCR, respectively. Anti-oxidative activity was investigated using a thiobarbituric acid-reactive substance (TBARS) test in rat brain homogenate preparations. We showed the statistical results of the infarct areas of solvent- and TMPZ (20 mg/kg)-treated groups at various distances from the frontal pole in MCAO-induced focal cerebral ischemia in rats. Treatment with TMPZ (20 mg/kg) markedly reduced the infarct area in all regions, especially in the third to fifth sections. MCAO-induced focal cerebral ischemia was associated with increases in HIF-1alpha and the activation of caspase-3, as well as TNF-alpha transcription in ischemic regions. These expressions were markedly inhibited by treatment with TMPZ (20 mg/kg). However, TMPZ (0.5-5 mmol/L) did not significantly inhibit TBARS reaction in rat brain homogenates. The neuroprotective effect of TMPZ may be mediated at least by a portion of the inhibition of HIF-1alpha and TNF-alpha activations, followed by the inhibition of apoptosis formation (active caspase-3), resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, TMPZ treatment may represent an ideal approach to lowering the risk of or improving function in ischemia- reperfusion brain injury-related disorders.

Neuroprotective effects of PMC, a potent α-tocopherol derivative, in brain ischemia-reperfusion: Reduced neutrophil activation and anti-oxidant actions

2,2,5,7,8-Pentamethyl-6-hydroxychromane (PMC) is the most potent analogue of α-tocopherol for anti-oxidation. It is more hydrophilic than other α-tocopherol derivatives and has potent free radical-scavenging activity. In the present study, PMC significantly attenuated middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Administration of PMC at 20 mg/kg, showed marked reductions in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in HIF-1α, active caspase-3, iNOS, and nitrotyrosine expressions in ischemic regions. These expressions were markedly inhibited by treatment with PMC (20 mg/kg). In addition, PMC (4–12 μM) inhibited respiratory bursts in human neutrophils stimulated by fMLP (800 nM) and PMA (320 nM). Furthermore, PMC (6, 12, and 60 μM) also significantly inhibited neutrophil migration stimulated by leukotriene B 4 (160 nM). An electron spin resonance (ESR) method was conducted on the scavenging activity of PMC on the free radicals formed. PMC (12 μM) greatly reduced the ESR signal intensities of superoxide anion, hydroxyl radical, and methyl radical formation. In conclusion, we demonstrate a potent neuroprotective effect of PMC on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by inhibition of free radical formation, followed by inhibition of HIF-1α activation, apoptosis formation (active caspase-3), neutrophil activation, and inflammatory responses (i.e., iNOS and nitrotyrosine expressions), resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, PMC treatment may represent a novel approach to lowering the risk or improving function in ischemia-reperfusion brain injury-related disorders.

Inhibitory Mechanisms of Tetramethylpyrazine in Middle Cerebral Artery Occlusion (MCAO)-Induced Focal Cerebral Ischemia in Rats

Tetramethylpyrazine (TMPZ) is an active ingredient isolated from a commonly used Chinese herb, Ligusticum wallichii Franchat, which has long been used in China for the treatment of vascular diseases. In the present study, TMPZ significantly attenuated middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Administration of TMPZ at 10 and 20 mg/kg produced concentration-dependent reductions in infarct size compared to that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in both nitrotyrosine and inducible nitric oxide synthase (iNOS) expression in ischemic regions. The expressions of nitrotyrosine and iNOS were markedly inhibited by TMPZ (20 mg/kg) treatment. Furthermore, TMPZ (100-250 microM) concentration-dependently inhibited respiratory bursts in human neutrophils stimulated by fMLP (800 nM) and PMA (320 nM). TMPZ (100-250 microM) also significantly inhibited neutrophil migration stimulated by fMLP (800 nM) and LTB4 (160 nM). An electron spin resonance (ESR) method was used to further study the scavenging activity of TMPZ on free radicals formed in human neutrophils. TMPZ (100 and 200 microM) greatly reduced the ESR signal intensity of hydroxyl radical formation. In conclusion, we demonstrate a neuroprotective effect of TMPZ in MCAO-induced focal cerebral ischemia in vivo. TMPZ mediates at least part of the free radical-scavenging activity and inhibits neutrophil activation, resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, TMPZ treatment may represent an ideal approach to lowering the risk of or improving function in ischemia-reperfusion brain injury-related disorders.

Protective Mechanisms of Inosine in Platelet Activation and Cerebral Ischemic Damage

Inosine is a naturally occurring nucleoside degraded from adenosine. Recent studies have demonstrated that inosine has potent immunomodulatory and neuroprotective effects. In the present study, we further investigated the inhibitory effects of inosine on platelet activation in vitro and in vivo, as well as in attenuating middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Inosine concentration-dependently (0.5 to 6.0 mmol/L) inhibited platelet aggregation stimulated by agonists. Inosine (1.5 and 3.0 mmol/L) inhibited phosphoinositide breakdown, [Ca+2]i, and TxA2 formation in human platelets stimulated by collagen (1 microg/mL). In addition, inosine (1.5 and 3.0 mmol/L) markedly increased levels of cyclic guanylate monophosphate (GMP) and cyclic GMP-induced vasodilator-stimulated phosphoprotein Ser157 phosphorylation. Rapid phosphorylation of a platelet protein of molecular weight 47,000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/mL). This phosphorylation was markedly inhibited by inosine (3.0 mmol/L). Inosine (1.5 and 3.0 mmol/L) markedly reduced hydroxyl radical in collagen (1 microg/mL)-activated platelets. In in vivo studies, inosine (400 mg/kg) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules of mice, and administration of 2 doses (100 mg/kg) or a single dose (150 mg/kg) of inosine significantly attenuated MCAO-induced focal cerebral ischemia in rats. Platelet aggregation contributes significantly to MCAO-induced focal cerebral ischemia. The most important findings of this study suggest that inosine markedly inhibited platelet activation in vitro and in vivo, as well as cerebral ischemia. Thus, inosine treatment may represent a novel approach to lowering the risk of or improving function in thromboembolic-related disorders and ischemia-reperfusion brain injury.

Hemodilution during cardiopulmonary bypass increases cerebral infarct volume after middle cerebral artery occlusion in rats.

Although the optimal hematocrit during cardiopulmonary bypass (CPB) is not defined, excessive hemodilution may lead to organ ischemia via a reduction in oxygen-carrying capacity uncompensated by autoregulatory and/or rheologic increases in organ blood flow. As a result, the consequences of hemodilution in patients at risk for cerebral ischemia are not clearly understood. We designed this study to evaluate the effects of hemodilution in the setting of focal cerebral ischemia during CPB. Wistar rats surgically prepared for CPB were randomized to either hemodilution (hemoglobin (Hb), 6 g/dL; n = 9) or control (Hb, 11 g/dL; n = 8) groups and subsequently exposed to focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO). Immediately after the onset of MCAO (maintained for 90 min), 65 min of hypothermic (28 degrees C) CPB was initiated. Twenty-four hours later, functional neurological outcome and cerebral infarct volume were determined. Compared with controls, the hemodilution group had worse neurological performance (new score = 8 [2], hemodilution; versus 10 [2], control; P = 0.030) and larger total cerebral infarct volumes (182 +/- 84 mm(3), hemodilution; versus 103 +/- 58 mm(3), control; P = 0.043). In this experimental model of CPB with reversible MCAO-induced focal cerebral ischemia, hemodilution worsened neurological function and increased cerebral infarct volume.

Inhibition of middle cerebral artery occlusion-induced focal cerebral ischemia by carboxyfullerene

The neuroprotective effects of a water-soluble trimalonic acid derivative of fullerene, carboxyfullerene, were evaluated in mice subjected to permanent middle cerebral artery occlusion (MCAO). The ischemic disruption of the blood–brain barrier (BBB) was shown by its permeability to the peripheral M4 tracer and the damage of endothelial cells. Cytokines TNF-a and IL-1 p were expressed on arterioles. Relatively to vehicle- treated controls, mice treated with carboxyfullerene (40 mg/kg) at the time of ischemia showed a 75% reduction in brain infarction. This inhibition was dose- and time-dependent. There was still significant inhibition 6 h post-occlusion. With anti-carboxyfullerene antibody immunohistochemical staining, carboxyfullerene was detectable on the neurons and ventricle in the ipsilateral hemisphere of the carboxyfullerene-treated mice. This suggests that carboxyfullerene can be a potential therapeutic agent for MCAO-induced focal cerebral ischemia treatment.