Involvement of baroreflex deficiency in the age-related loss of estrogen efficacy against cerebral ischemia.

For post-menopausal women, stroke is complicated by the variable effects of estrogen therapy and the age-related therapeutic consequences involved. Estrogen therapy has been shown to have an age-dimorphic effect, which is neuroprotective in young females, but non-neuroprotective, even neurotoxic in acyclic females. We hypothesized that arterial baroreflex (ABR) and its downstream acetylcholine-α7 nicotinic acetylcholine receptor (α7nAChR) anti-inflammatory pathways are involved in estrogen efficacy toward cerebral ischemic damage. Our data showed that estrogen supplements contributed to ABR improvement and neuroprotection in adult, not aged, ovariectomized (OVX) rats. In adult rats, OVX-induced estrogen deficiency aggravated middle cerebral artery occlusion (MCAO), which induced brain infarction and reduced ABR function, with decreased α7nAChR expression of the brain and exaggerated inflammation following MCAO; these effects were significantly prevented by supplementation with estrogen. ABR impairment by sinoaortic denervation partly attenuated the estrogen effect on baroreflex sensitivity (BRS) and ischemic damage in adult rats, as well as α7nAChR expression and inflammatory response. These data suggested that ABR and acetylcholine-α7nAChR anti-inflammatory pathways are involved in the neuroprotection of estrogen in adult OVX rats. In contrast, aged rats exhibited more severe ischemic damage and inflammatory response than adult rats, as well as poorer baroreflex function and lower α7nAChR expression. Estrogen supplements did not improve BRS or confer neuroprotection in aged rats without affecting brain α7nAChR and post-ischemic inflammation. Most importantly, ketanserin restored ABR function and significantly postponed the onset of stroke in aged female strokeprone spontaneously hypertensive rats, whereas estrogen treatment failed to delay the development of stroke. Our findings reveal that estrogen is protective against ischemic stroke (IS) in adult female rats and that ABR played a role in this beneficial action. Dysfunction of ABR and unresponsiveness to estrogen in aged female rats may contribute to a reduced estrogen efficacy against cerebral ischemia.

The discovery of novel small molecule allosteric activators of aldehyde dehydrogenase 2.

Aldehyde dehydrogenase 2 (ALDH2) plays important role in ethanol metabolism, and also serves as an important shield from the damage occurring under oxidative stress. A special inactive variant was found carried by 35-45% of East Asians. The variant carriers have recently been found at the higher risk for the diseases related to the damage occurring under oxidative stress, such as cardiovascular and cerebrovascular diseases. As a result, ALDH2 activators may potentially serve as a new class of therapeutics. Herein, N-benzylanilines were found as novel allosteric activators of ALDH2 by computational virtual screening using ligand-based and structure-based screening parallel screening strategy. Then a structural optimization was performed and has led to the discovery of the compound C6. It has good activity in vitro and in vivo, which could reduce infarct size by ∼70% in ischemic stroke rat models. This study provided good lead compounds for the further development of ALDH2 activators.

Anisodamine Ameliorates Hyperkalemia during Crush Syndrome through Estradiol-Induced Enhancement of Insulin Sensitivity.

Hyperkalemia is a major cause of on-site death in crush syndrome (CS), which is more severe and common in male victims. Anisodamine is a belladonna alkaloid and widely used in China for treatment of shock through activation of α7 nicotinic acetylcholine receptor (α7nAChR). The present work was designed to study the protective effect of anisodamine in CS and the possible role of estradiol involved. Male and ovariectomized female CS mice exhibited lower serum estradiol and insulin sensitivity, and higher potassium compared to the relative female controls at 6 h after decompression. There was no gender difference in on-site mortality in CS mice within 24 h after decompression. Serum estradiol increased with similar values in CS mice of both gender compared to that in normal mice. Anisodamine decreased serum potassium and increased serum estradiol and insulin sensitivity in CS mice, and methyllycaconitine, selective antagonist of α7nAChR, counteracted such effects of anisodamine. Treatment with anisodamine or estradiol increased serum estradiol and insulin sensitivity, decreased serum potassium and on-site mortality, and eliminated the difference in these parameters between CS mice received ovariectomy or its sham operation. Anisodamine could also increase blood pressure in CS rats within 3.5 h after decompression, which could also be attenuated by methyllycaconitine, without influences on heart rate. These results suggest that activation of α7nAChR with anisodamine could decrease serum potassium and on-site mortality in CS through estradiol-induced enhancement of insulin sensitivity.

Diurnal macular choroidal area fluctuation in normal and primary open angle glaucoma groups.

AIM: To assess and compare the diurnal macular choroidal area fluctuation in normal and primary open angle glaucoma (POAG) groups using enhanced depth imaging optical coherence tomography (EDI-OCT). METHODS: Twenty-eight normal and 27 POAG eyes were enrolled in this study. EDI-OCT was used to measure the macular choroidal area every 3h from 9:00 a.m. to 21:00 p.m. RESULTS: Significant diurnal fluctuations of macular choroidal area were observed in both normal (P=0.003) and POAG groups (P<0.001). But no significant macular choroidal area difference has been found between the two groups at all the five measurement time-points (512778±166242 vs 455079±207278 µm2, P=0.195 at 9:00 a.m.; 501526±168953 vs 447846±211147 µm2, P=0.245 at 12:00 a.m.; 501982±173158 vs 448024±206653 µm2, P=0.239 at 15:00 p.m.; 508912±174589 vs 457783±207081 µm2, P=0.252 at 18:00 p.m.; 503787±171241 vs 453230±205955 µm2, P=0.274 at 21:00 p.m.; respectively). Furthermore, neither the fluctuation manners nor the change in macular choroidal area between the two adjacent measurement time points showed significant difference between normal and POAG groups (all P>0.05). In the meantime, significant diurnal intraocular pressure fluctuations were also observed in normal and POAG groups (both P<0.001). CONCLUSION: In diurnal time, the macular choroidal area in both normal and POAG groups fluctuated significantly; moreover, neither the value of macular choroidal area, nor the fluctuation of macular choroidal area in POAG group is significantly different from that in normal group.

SIRT1-dependent AMPK pathway in the protection of estrogen against ischemic brain injury.

AIMS: Stroke is a major cause of mortality and disability, especially for postmenopausal women. In view of the protective action of estrogen, hormone therapy remains the only effective way to limit this risk. The objective of this study was to investigate the efficiency and underlying mechanisms of estrogen neuroprotection. METHODS: Subcutaneous injection of 17ß-estradiol in rats after ovariectomy (OVX) was used to manipulate estrogen level and explore the effects of estrogen in cerebral ischemic damage both in vivo and in vitro. Silent mating type information regulation 2 homolog 1 (SIRT1) knockout mice and adenosine monophosphate (AMP)-activated kinase (AMPK) inhibitor Compound C were also used to investigate the underlying pathway of estrogen. RESULTS: Estrogen deficiency induced by OVX aggravated brain infarction in experimentally induced cerebral ischemia rats, whereas estrogen pretreatment reduced ischemia-induced cerebral injuries. Neurons of estrogen deficiency models were susceptible to apoptosis under oxygen-glucose deprivation (OGD). In contrast, neurons with estrogen-supplemented serum exhibited restored resistance to cell apoptosis. In OGD neurons, estrogen promoted AMPK activation through estrogen receptor α, and neuroprotection of estrogen was prevented by AMPK inhibition. Estrogen increased SIRT1 expression and activation, and estrogen-induced AMPK activation disappeared in SIRT1 knockout neurons. Moreover, estrogen-induced neuroprotection was abolished in SIRT1 knockout mice and AMPK-inhibited rats. CONCLUSION: Our data support that estrogen protects against ischemic stroke through preventing neuron death via the SIRT1-dependent AMPK pathway.

Involvement of arterial baroreflex and nicotinic acetylcholine receptor α7 subunit pathway in the protection of metformin against stroke in stroke-prone spontaneously hypertensive rats.

Stroke is a leading cause of mortality and disability worldwide. There is growing evidence that metformin (Met) has potent neuroprotective effects; however, its mechanisms remain unclear. We examined the role of the arterial baroreflex and cholinergic-α7 nicotinic acetylcholine receptor (α7nAChR) anti-inflammory pathway in the beneficial effects of Met against stroke. Stroke-prone spontaneously hypertensive rats (SHRSP) were used to observe stroke development indicated by lifespan of SHRSP and the ischemic injury induced by permanent middle cerebral artery occlusion (MCAO). Sinoaortic denervation was used to inactivate the arterial baroreflex. MCAO were also performed in α7nAChR knockout (KO) mice. Briefly, Met increased the life span of SHRSP and reduced the infarct area induced by MCAO. Met also improved the function of arterial baroreflex. The beneficial effects of Met on stroke were markedly attenuated by blunting the arterial baroreflex. Met up-regulated the expression of vesicular acetylcholine transporter (VAChT) and α7nAChR, down-regulated the level of pro-inflammtory cytokines in serum and peri-infarct of ischemic brain. Arterial baroreflex dysfunction decreased the expression of VAchT and α7nAChR, showed upward tendency in the level of pro-inflammtory cytokines. Most importantly, arterial baroreflex dysfunction nearly abolished such effect of Met on cholinergic signaling. In addition, the α7nAChR KO mice also had significantly worse ischemic damage induced by MCAO, and neuroprotection of Met disappeared in α7nAChR KO mice. In conclusion, Met improved the arterial baroreflex function, and then enhancing cholinergic anti-inflammatory pathway in an α7nAChR-dependent manner, thereby effectively prevent ischemic induced brain injury and delayed stroke onset in SHRSP.

Activation of α7 Nicotinic Acetylcholine Receptor Decreases On-site Mortality in Crush Syndrome through Insulin Signaling-Na/K-ATPase Pathway.

On-site mortality in crush syndrome remains high due to lack of effective drugs based on definite diagnosis. Anisodamine (Ani) is widely used in China for treatment of shock, and activation of α7 nicotinic acetylcholine receptor (α7nAChR) mediates such antishock effect. The present work was designed to test whether activation of α7nAChR with Ani decreased mortality in crush syndrome shortly after decompression. Sprague-Dawley rats and C57BL/6 mice with crush syndrome were injected with Ani (20 mg/kg and 28 mg/kg respectively, i.p.) 30 min before decompression. Survival time, serum potassium, insulin, and glucose levels were observed shortly after decompression. Involvement of α7nAChR was verified with methyllycaconitine (selective α7nAChR antagonist) and PNU282987 (selective α7nAChR agonist), or in α7nAChR knockout mice. Effect of Ani was also appraised in C2C12 myotubes. Ani reduced mortality and serum potassium and enhanced insulin sensitivity shortly after decompression in animals with crush syndrome, and PNU282987 exerted similar effects. Such effects were counteracted by methyllycaconitine or in α7nAChR knockout mice. Mortality and serum potassium in rats with hyperkalemia were also reduced by Ani. Phosphorylation of Na/K-ATPase was enhanced by Ani in C2C12 myotubes. Inhibition of tyrosine kinase on insulin receptor, phosphoinositide 3-kinase, mammalian target of rapamycin, signal transducer and activator of transcription 3, and Na/K-ATPase counteracted the effect of Ani on extracellular potassium. These findings demonstrated that activation of α7nAChR could decrease on-site mortality in crush syndrome, at least in part based on the decline of serum potassium through insulin signaling-Na/K-ATPase pathway.

Heavy ethanol consumption aggravates the ischemic cerebral injury by inhibiting ALDH2.

BACKGROUND: Heavy ethanol consumption is widely accepted as a risk for ischemic stroke. The molecular mechanisms of ethanol-induced brain injury have not been fully understood. AIM: This study aims to find out the mechanism of the ischemic cerebral injury. METHODS: We used Sprague-Dawley rats with transient middle cerebral artery occlusion for acute experiment and stroke-prone spontaneously hypertensive rats for long-term experiment in vivo, and oxygen-glucose deprivation model in vitro to define a detrimental effect of different doses of ethanol on ischemic stroke injury. We also used mitochondrial aldehyde dehydrogenase 2 knockdown/overexpression or inhibitor/activator to investigate mechanism of the adverse effects of ethanol. RESULTS: High-dose ethanol (36% of calorie derived from ethanol) significantly increased the infarct size in rats (P < 0·01) and decreased the survival time of stroke-prone spontaneously hypertensive rats by about 20%. Six-week treatment with high-dose ethanol changed a distribution of isoelectric point of aldehyde dehydrogenase 2 and inhibited aldehyde dehydrogenase 2 activity in brain. High dose of ethanol increased the cerebral acetaldehyde level, and increased 4-hydroxy-2-nonenal and malondialdehyde in serum of rats with middle cerebral artery occlusion. The activator of aldehyde dehydrogenase 2, Alda-1 abolished neuronal cells death and ischemic injury induced by ethanol and the inhibitor reversed the injurious effects. An overexpression of aldehyde dehydrogenase 2 completely abolished the increased infarct size and neurological deficit score by ethanol. Conversely, knockdown of aldehyde dehydrogenase 2 increased the infarct size and exaggerated the cerebral injury induced by ethanol. CONCLUSIONS: High concentrations of ethanol aggravate cerebral injury by inhibiting of aldehyde dehydrogenase 2 and inducing excess accumulation of aldehydes.

ALDH2 protects against stroke by clearing 4-HNE.

Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that metabolizes ethanol and toxic aldehydes such as 4-hydroxy-2-nonenal (4-HNE). Using an unbiased proteomic search, we identified ALDH2 deficiency in stroke-prone spontaneously hypertensive rats (SHR-SP) as compared with spontaneously hypertensive rats (SHR). We concluded the causative role of ALDH2 deficiency in neuronal injury as overexpression or activation of ALDH2 conferred neuroprotection by clearing 4-HNE in in vitro studies. Further, ALDH2-knockdown rats revealed the absence of neuroprotective effects of PKCε. Moderate ethanol administration that is known to exert protection against stroke was shown to enhance the detoxification of 4-HNE, and to protect against ischemic cerebral injury through the PKCε-ALDH2 pathway. In SHR-SP, serum 4-HNE level was persistently elevated and correlated inversely with the lifespan. The role of 4-HNE in stroke in humans was also suggested by persistent elevation of its plasma levels for at least 6 months after stroke. Lastly, we observed that 21 of 1 242 subjects followed for 8 years who developed stroke had higher initial plasma 4-HNE levels than those who did not develop stroke. These findings suggest that activation of the ALDH2 pathway may serve as a useful index in the identification of stroke-prone subjects, and the ALDH2 pathway may be a potential target of therapeutic intervention in stroke.

Involvement of arterial baroreflex in the protective effect of dietary restriction against stroke.

Dietary restriction (DR) protects against neuronal dysfunction and degeneration, and reduces the risk of ischemic stroke. This study examined the role of silent information regulator T1 (SIRT1) and arterial baroreflex in the beneficial effects of DR against stroke, using two distinct stroke models: stroke-prone spontaneously hypertensive rats (SP-SHRs) and Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO). Sirt1 knockout (KO) mice were used to examine the involvement of sirt1. Sinoaortic denervation was used to inactivate arterial baroreflex. Dietary restriction was defined as 40% reduction of dietary intake. Briefly, DR prolonged the life span of SP-SHRs and reduced the infarct size induced by MCAO. Dietary restriction also improved the function arterial baroreflex, decreased the release of proinflammatory cytokines, and reduced end-organ damage. The beneficial effect of DR on stroke was markedly attenuated by blunting arterial baroreflex. Lastly, the infarct area in sirt1 KO mice was significantly larger than in the wild-type mice. However, the beneficial effect of DR against ischemic injury was still apparent in sirt1 KO mice. Accordingly, arterial baroreflex, but not sirt1, is important in the protective effect of DR against stroke.

Baroreflex deficiency hampers angiogenesis after myocardial infarction via acetylcholine-α7-nicotinic ACh receptor in rats.

AIMS: Angiogenesis is critical for re-establishing blood supply to ischaemic myocardium after myocardial infarction (MI). Human studies have associated arterial baroreflex (ABR) deficiency with higher rate of sudden death after MI. The present work was designed to examine whether ABR deficiency affects angiogenesis in MI rats. METHODS AND RESULTS: Baroreflex sensitivity (BRS) was determined in conscious rats at 1 month after occlusion of the left anterior descending coronary artery. The survival time was significantly shorter in Sprague-Dawley rats with BRS <0.60 ms/mmHg vs. those with BRS ≥0.60 ms/mmHg. Sinoaortic denervation destroyed ABR, and decreased capillary density, regional blood flow and vascular endothelial growth factor (VEGF) concentration after MI. Ketanserin (0.6 mg/kg/day) enhanced BRS, and increased capillary density, regional blood flow, and VEGF. Sinoaortic denervation also reduced the expression of vesicular acetylcholine (ACh) transporter and α7-nicotinic ACh receptor (α7-nAChR). Angiogenesis after MI was significantly attenuated in α7-nAChR knockout mice. In contrast, increase in endogenous ACh with cholinesterase inhibitor pyridostigmine (30 mg/kg/day) increased angiogenesis after MI. In cultured cardiac microvascular endothelial cells, ACh stimulated the expression of VEGF, phosphorylation of VEGF receptor 2, and tube formation in a manner dependent upon α7-nAChR. CONCLUSION: Our results demonstrated that ABR deficiency could attenuate angiogenesis in ischaemic myocardium. These findings provide further mechanistic basis for enhancing baroreflex function in the treatment of MI.

Involvement of acetylcholine-α7nAChR in the protective effects of arterial baroreflex against ischemic stroke.

AIMS: Decreased baroreflex sensitivity is associated with poor outcome in many cardiovascular diseases including stroke, but the molecular mechanism underlying this relationship is unclear. This work was designed to test the hypothesis that acetylcholine (ACh) and α7 nicotinic ACh receptor (α7nAChR) mediate the protection of arterial baroreflex against stroke. METHODS: Sinoaortic denervation (SAD) was used to impair the function of arterial baroreflex, and anticholinesterase agents were used to activate the cholinergic system and increase endogenous ACh. Middle cerebral artery occlusion (MCAO) was performed in the α7nAChR knockout (KO) mice and Sprague-Dawley rats. RESULTS: We found decreased expression of vesicular ACh transporter (VAChT) and α7nAChR in rat brain after SAD. In rats subjected to MCAO, neostigmine significantly reduced the infarct size. The protective effects of neostigmine were abolished by selective nAChR antagonist vecuronium but not by mAChR antagonist anisodamine. In addition, the effect of neostigmine disappeared in α7nAChR KO mice. In cultured neurons, ACh inhibited cell death induced by H(2) O(2) . In cultured microglial cells, ACh decreased the release of proinflammatory cytokines induced by lipopolysaccharide. These in vitro effects were blocked by selective α7nAChR antagonists. CONCLUSION: Taken together, these findings indicate that the ACh-α7nAChR involved in the protective effects of arterial baroreflex against ischemic stroke.

Genetics of stroke.

Stroke is the second most common cause of death and the most common cause of disability in developed countries. Stroke is a multi-factorial disease caused by a combination of environmental and genetic factors. Numerous epidemiologic studies have documented a significant genetic component in the occurrence of strokes. Genes encoding products involved in lipid metabolism, thrombosis, and inflammation are believed to be potential genetic factors for stroke. Although a large group of candidate genes have been studied, most of the epidemiological results are conflicting. Studies of stroke as a monogenic disease have made huge progress, and animal models serve as an indispensable tool to dissect the complex genetics of stroke. In the present review, we provide insight into the role of in vivo stroke models for the study of stroke genetics.

New strategies for the prevention of stroke.

1. Stroke is a major cause of disability and death worldwide. It is preferable to prevent stroke rather than to treat it and, for the prevention of stroke, all risk factors relating to stroke need to be understood. The present paper reviews potential new strategies for the prevention of stroke based on findings of new risk factors, as well as classical risk factors. 2. Recently, new risk factors related to stroke were reported, including dysfunction of the arterial baroreflex, pro-inflammatory cytokines, vitamins and hormone deficiency. Correspondingly, therapies targeting these risk factors where shown to significantly reduce the incidence and/or severity of stroke. 3. Because the genesis of stroke is multifactorial, the prevention of stroke should not target one risk factor only. Combination therapies with drugs acting on different risk factors may be more effective in the prevention of stroke.