Brain Cell Senescence: A New Therapeutic Target for the Acute Treatment of Ischemic Stroke.

Aging is a major risk factor for cerebral infarction. Since cellular senescence is intrinsic to aging, we postulated that stroke-induced cellular senescence might contribute to neural dysfunction. Adult male Wistar rats underwent 60-minute middle cerebral artery occlusion and were grouped according to 3 reperfusion times: 24 hours, 3, and 7 days. The major biomarkers of senescence: 1) accumulation of the lysosomal pigment, lipofuscin; 2) expression of the cell cycle arrest markers p21, p53, and p16INK4a; and 3) expression of the senescence-associated secretory phenotype cytokines interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin-1ß (IL-1ß) were investigated in brain samples. Lipofuscin accumulation was scarce at the initial stage of brain damage (24 hours), but progressively increased until it reached massive distribution at 7 days post-ischemia. Lipofuscin granules (aggresomes) were mainly confined to the infarcted areas, that is parietal cortex and adjacent caudate-putamen, which were equally affected. The expression of p21, p53, and p16INK4a, and that of IL-6, TNF-α, and IL-1ß, was significantly higher in the ischemic hemisphere than in the non-ischemic hemisphere. These data indicate that brain cell senescence develops during acute ischemic infarction and suggest that the acute treatment of ischemic stroke might be enhanced using senolytic drugs.

Uric Acid Neuroprotection Associated to IL-6/STAT3 Signaling Pathway Activation in Rat Ischemic Stroke.

Despite the promising neuroprotective effects of uric acid (UA) in acute ischemic stroke, the seemingly pleiotropic underlying mechanisms are not completely understood. Recent evidence points to transcription factors as UA targets. To gain insight into the UA mechanism of action, we investigated its effects on pertinent biomarkers for the most relevant features of ischemic stroke pathophysiology: (1) oxidative stress (antioxidant enzyme mRNAs and MDA), (2) neuroinflammation (cytokine and Socs3 mRNAs, STAT3, NF-κB p65, and reactive microglia), (3) brain swelling (Vegfa, Mmp9, and Timp1 mRNAs), and (4) apoptotic cell death (Bcl-2, Bax, caspase-3, and TUNEL-positive cells). Adult male Wistar rats underwent intraluminal filament transient middle cerebral artery occlusion (tMCAO) and received UA (16 mg/kg) or vehicle (Locke's buffer) i.v. at 20 min reperfusion. The outcome measures were neurofunctional deficit, infarct, and edema. UA treatment reduced cortical infarct and brain edema, as well as neurofunctional impairment. In brain cortex, increased UA: (1) reduced tMCAO-induced increases in Vegfa and Mmp9/Timp1 ratio expressions; (2) induced Sod2 and Cat expressions and reduced MDA levels; (3) induced Il6 expression, upregulated STAT3 and NF-κB p65 phosphorylation, induced Socs3 expression, and inhibited microglia activation; and (4) ameliorated the Bax/Bcl-2 ratio and induced a reduction in caspase-3 cleavage as well as in TUNEL-positive cell counts. In conclusion, the mechanism for morphological and functional neuroprotection by UA in ischemic stroke is multifaceted, since it is associated to activation of the IL-6/STAT3 pathway, attenuation of edematogenic VEGF-A/MMP-9 signaling, and modulation of relevant mediators of oxidative stress, neuroinflammation, and apoptotic cell death.

The selective oestrogen receptor modulator, bazedoxifene, mimics the neuroprotective effect of 17ß-oestradiol in diabetic ischaemic stroke by modulating oestrogen receptor expression and the MAPK/ERK1/2 signalling pathway.

Because neuroprotection in stroke should be revisited in the era of recanalisation, the present study analysed the potential neuroprotective effect of the selective oestrogen receptor modulator, bazedoxifene acetate (BZA), in an animal model of diabetic ischaemic stroke that mimics thrombectomy combined with adjuvant administration of a putative neuroprotectant. Four weeks after induction of diabetes (40 mg kg-1 streptozotocin, i.p.), male Wistar rats were subjected to transient middle cerebral artery occlusion (intraluminal thread technique, 60 minutes) and assigned to one of three groups treated with either: vehicle, BZA (3 mg kg-1  day-1 , i.p.) or 17ß-oestradiol (E2 ) (100 µg kg-1  day-1 , i.p.). At 24 hours post-ischaemia-reperfusion, brain damage (neurofunctional score, infarct size and apoptosis), expression of oestrogen receptors (ER)α, ERß and G protein-coupled oestrogen receptor), and activity of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)1/2 and phosphoinositide 3-kinase/Akt pathways were analysed. At 24 hours after the ischaemic insult, both BZA- and E2 -treated animals showed lower brain damage in terms of improved neurofunctional condition, decreased infarct size and decreased apoptotic cell death. Ischaemia-reperfusion induced a significant decrease in ERα and ERß expression without affecting that of G protein-coupled oestrogen receptor, whereas BZA and E2 reversed such a decrease. The ischaemic insult up-regulated the activity of both the MAPK/ERK1/2 and phosphoinositide 3-kinase/Akt pathways; BZA and E2 attenuated the increased activity of the ERK1/2 pathway, without affecting that of the Akt pathway. The results of the present study lend further support to the consideration of BZA as an effective and safer alternative overcoming the drawbacks of E2 with respect to improving diabetic ischaemic stroke outcome after successful reperfusion.

Molecular mechanisms underlying the neuroprotective role of atrial natriuretic peptide in experimental acute ischemic stroke.

Along with its role in regulating blood pressure and fluid homeostasis, the natriuretic peptide system could be also part of an endogenous protective mechanism against brain damage. We aimed to assess the possibility that exogenous atrial natriuretic peptide (ANP) could protect against acute ischemic stroke, as well as the molecular mechanisms involved. Three groups of rats subjected to transient middle cerebral artery occlusion (tMCAO, intraluminal filament technique, 60 min) received intracerebroventricular vehicle, low-dose ANP (0.5 nmol) or high-dose ANP (2.5 nmol), at 30 min reperfusion. Neurofunctional condition, and brain infarct and edema volumes were measured at 24 h after tMCAO. Apoptotic cell death and expression of natriuretic peptide receptors (NPR-A and NPR-C), K+ channels (KATP, KV and BKCa), and PI3K/Akt and MAPK/ERK1/2 signaling pathways were analyzed. Significant improvement in neurofunctional status, associated to reduction in infarct and edema volumes, was shown in the high-dose ANP group. As to the molecular mechanisms analyzed, high-dose ANP: 1) reduced caspase-3-mediated apoptosis; 2) did not modify the expression of NPR-A and NPR-C, which had been downregulated by the ischemic insult; 3) induced a significant reversion of ischemia-downregulated KATP channel expression; and 4) induced a significant reversion of ischemia-upregulated pERK2/ERK2 expression ratio. In conclusion, ANP exerts a significant protective role in terms of both improvement of neurofunctional status and reduction in infarct volume. Modulation of ANP on some molecular mechanisms involved in ischemia-induced apoptotic cell death (KATP channels and MAPK/ERK1/2 signaling pathway) could account, at least in part, for its beneficial effect. Therefore, ANP should be considered as a potential adjunctive neuroprotective agent improving stroke outcome after successful reperfusion interventions.

Molecular mechanisms mediating the neuroprotective role of the selective estrogen receptor modulator, bazedoxifene, in acute ischemic stroke: A comparative study with 17ß-estradiol.

As the knowledge on the estrogenic system in the brain grows, the possibilities to modulate it in order to afford further neuroprotection in brain damaging disorders so do it. We have previously demonstrated the ability of the selective estrogen receptor modulator, bazedoxifene (BZA), to reduce experimental ischemic brain damage. The present study has been designed to gain insight into the molecular mechanisms involved in such a neuroprotective action by investigating: 1) stroke-induced apoptotic cell death; 2) expression of estrogen receptors (ER) ERα, ERß and the G-protein coupled estrogen receptor (GPER); and 3) modulation of MAPK/ERK1/2 and PI3K/Akt signaling pathways. For comparison, a parallel study was done with 17ß-estradiol (E2)-treated animals. Male Wistar rats subject to transient right middle cerebral artery occlusion (tMCAO, intraluminal thread technique, 60min), were distributed in vehicle-, BZA- (20.7±2.1ng/mL in plasma) and E2- (45.6±7.8pg/mL in plasma) treated groups. At 24h from the onset of tMCAO, RT-PCR, Western blot and histochemical analysis were performed on brain tissue samples. Ischemia-reperfusion per se increased apoptosis as assessed by both caspase-3 activity and TUNEL-positive cell counts, which were reversed by both BZA and E2. ERα and ERß expression, but not that of GPER, was reduced by the ischemic insult. BZA and E2 had different effects: while BZA increased both ERα and ERß expression, E2 increased ERα expression but did not change that of ERß. Both MAPK/ERK1/2 and PI3K/Akt pathways were stimulated under ischemic conditions. While BZA strongly reduced the increased p-ERK1/2 levels, E2 did not. Neither BZA nor E2 modified ischemia-induced increase in p-Akt levels. These results show that modulation of ERα and ERß expression, as well as of the ERK1/2 signaling pathway accounts, at least in part, for the inhibitory effect of BZA on the stroke-induced apoptotic cell death. This lends mechanistic support to the consideration of BZA as a potential neuroprotective drug in acute ischemic stroke treatment.

Relaxant Effects of the Selective Estrogen Receptor Modulator, Bazedoxifene, and Estrogen Receptor Agonists in Isolated Rabbit Basilar Artery.

We have previously shown that the selective estrogen receptor modulator, bazedoxifene, improves the consequences of ischemic stroke. Now we aimed to characterize the effects and mechanisms of action of bazedoxifene in cerebral arteries. Male rabbit isolated basilar arteries were used for isometric tension recording and quantitative polymerase chain reaction. Bazedoxifene relaxed cerebral arteries, as 17-ß-estradiol, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [estrogen receptor (ER) α agonist], and G1 [G protein-coupled ER (GPER) agonist] did it (4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol > bazedoxifene = G1 > 17-ß-estradiol). 2,3-Bis(4-hydroxyphenyl)-propionitrile (ERß agonist) had no effect. Expression profile of genes encoding for ERα (ESR1), ERß (ESR2), and GPER was GPER > ESR1 > ESR2. As to the endothelial mechanisms, endothelium removal, N-nitro-L-arginine methyl ester, and indomethacin, did not modify the relaxant responses to bazedoxifene. As to the K channels, both a high-K medium and the Kv blocker, 4-aminopyridine, inhibited the bazedoxifene-induced relaxations, whereas tetraethylammonium (nonselective K channel blocker), glibenclamide (selective KATP blocker) or iberiotoxin (selective KCa blocker) were without effect. Bazedoxifene also inhibited both Ca- and Bay K8644-elicited contractions. Therefore, bazedoxifene induces endothelium-independent relaxations of cerebral arteries through (1) activation of GPER and ERα receptors; (2) increase of K conductance through Kv channels; and (3) inhibition of Ca entry through L-type Ca channels. Such a profile is compatible with the beneficial effects of estrogenic compounds (eg, SERMs) on vascular function and, specifically, that concerning the brain. Therefore, bazedoxifene could be useful in the treatment of cerebral disorders in which the cerebrovascular function is compromised (eg, stroke).

The selective estrogen receptor modulator, bazedoxifene, reduces ischemic brain damage in male rat.

While the estrogen treatment of stroke is under debate, selective estrogen receptor modulators (SERMs) arise as a promising alternative. We hypothesize that bazedoxifene (acetate, BZA), a third generation SERM approved for the treatment of postmenopausal osteoporosis, reduces ischemic brain damage in a rat model of transient focal cerebral ischemia. For comparative purposes, the neuroprotective effect of 17ß-estradiol (E2) has also been assessed. Male Wistar rats underwent 60min middle cerebral artery occlusion (intraluminal thread technique), and grouped according to treatment: vehicle-, E2- and BZA-treated rats. Optimal plasma concentrations of E2 (45.6±7.8pg/ml) and BZA (20.7±2.1ng/ml) were achieved 4h after onset of ischemia, and maintained until the end of the procedure (24h). Neurofunctional score and volume of the damaged brain regions were the main end points. At 24h after ischemia-reperfusion, neurofunctional examination of the animals did not show significant differences among the three experimental groups. By contrast, both E2- and BZA-treated groups showed significantly lower total infarct volumes, BZA acting mainly in the cortical region and E2 acting mainly at the subcortical level. Our results demonstrate that: (1) E2 at physiological plasma levels in female rats is neuroprotective in male rats when given at the acute stage of the ischemic challenge and (2) BZA at clinically relevant plasma levels mimics the neuroprotective action of E2 and could be, therefore, a candidate in stroke treatment.

Novel antihypertensive hexa- and heptapeptides with ACE-inhibiting properties: from the in vitro ACE assay to the spontaneously hypertensive rat.

Bioactive ACE inhibiting peptides are gaining interest in hypertension treatment. We have designed and screened six synthetic heptapeptides (PACEI48 to PACEI53) based on two hexapeptide leads (PACEI32 and PACEI34) to improve ACE inhibitory properties and assess their antihypertensive effects. ACE activity was assayed in vitro and ex vivo. Selected peptides were administered to spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. In vitro cytotoxicity was assessed with the MTT reduction test. The six heptapeptides at low micromolar concentration produced different degrees of in vitro inhibition of ACE activity using the synthetic substrate HHL or the natural substrate angiotensin I; and ex vivo inhibition of ACE-dependent, angiotensin I-induced vasoconstriction, but not angiotensin II-induced vasoconstriction. Oral administration of the hexapeptide PACEI32L, and the heptapeptides PACEI50L and PACEI52L, induced reductions in systolic blood pressure lasting up to 3h in SHRs but not in WKY rats. Intravenous injection of PACEI32L and PACEI50L, but not PACEI52L, induced acute transient reductions in mean blood pressure of SHRs. d-Amino acid peptides showed five-fold less ACE inhibitory potency, no inhibitory effect on angiotensin I-induced vasoconstriction, and antihypertensive effect in SHRs after i.v. injection, but not after oral administration. The toxicity of peptides to reduce the viability of cultured cells was in the millimolar range. In conclusion, we have obtained novel rationally designed heptapeptides with improved ACE inhibitory properties when compared to lead hexapeptides. One selected hexapeptide and two heptapeptides show oral antihypertensive effects in SHRs and appear safe in cytotoxicity assays.

Lactoferricin B-derived peptides with inhibitory effects on ECE-dependent vasoconstriction.

Endothelin-converting enzyme (ECE), a key peptidase in the endothelin (ET) system, cleaves inactive big ET-1 to produce active ET-1, which binds to ET(A) receptors to exert its vasoconstrictor and pressor effects. ECE inhibition could be beneficial in the treatment of hypertension. In this study, a set of eight lactoferricin B (LfcinB)-derived peptides, previously characterized in our laboratory as angiotensin-converting enzyme (ACE) inhibitory peptides, was examined for their inhibitory effects on ECE. In vitro inhibitory effects on ECE activity were assessed using both the synthetic fluorogenic peptide substrate V (FPS V) and the natural substrate big ET-1. To study vasoactive effects, an ex vivo functional assay was developed using isolated rabbit carotid artery segments. With FPS V, only four LfcinB-derived peptides induced inhibition of ECE activity, whereas the eight peptides showed ECE inhibitory effects with big ET-1 as substrate. Regarding the ex vivo assays, six LfcinB-derived peptides showed inhibition of big ET-1-induced, ECE-dependent vasoconstriction. A positive correlation between the inhibitory effects of LfcinB-derived peptides on ECE activity when using big ET-1 and the inhibitory effects on ECE-dependent vasoconstriction was shown. ECE-independent vasoconstriction induced by ET-1 was not affected, thus discarding effects of LfcinB-derived peptides on ET(A) receptors or intracellular signal transduction mechanisms. In conclusion, a combined in vitro and ex vivo method to assess the effects of potentially antihypertensive peptides on the ET system has been developed and applied to show the inhibitory effects on ECE-dependent vasoconstriction of six LfcinB-derived peptides, five of which were dual vasopeptidase (ACE/ECE) inhibitors.

Antihypertensive properties of lactoferricin B-derived peptides.

A set of eight lactoferricin B (LfcinB)-derived peptides was examined for inhibitory effects on angiotensin I-converting enzyme (ACE) activity and ACE-dependent vasoconstriction, and their hypotensive effect in spontaneously hypertensive rats (SHR). Peptides were derived from different elongations both at the C-terminal and N-terminal ends of the representative peptide LfcinB(20-25), which is known as the LfcinB antimicrobial core. All of the eight LfcinB-derived peptides showed in vitro inhibitory effects on ACE activity with different IC(50) values. Moreover, seven of them showed ex vivo inhibitory effects on ACE-dependent vasoconstriction. No clear correlation between in vitro and ex vivo inhibitory effects was found. Only LfcinB(20-25) and one of its fragments, F1, generated after a simulated gastrointestinal digestion, showed significant antihypertensive effects in SHR after oral administration. Remarkably, F1 did not show any effect on ACE-dependent vasoconstriction in contrast to the inhibitory effect showed by LfcinB(20-25). In conclusion, two LfcinB-derived peptides lower blood pressure and exhibit potential as orally effective antihypertensive compounds, yet a complete elucidation of the mechanism(s) involved deserves further ongoing research.

Mechanisms involved in the relaxant action of testosterone in the renal artery from male normoglycemic and diabetic rabbits.

Kidney disease is a frequent complication in diabetes, and significant differences have been reported between male and female patients. Our working hypothesis was that diabetes might modify the vascular actions of testosterone in isolated rabbit renal arteries and the mechanisms involved in these actions. Testosterone (10(-8) to 10(-4)M) induced relaxation of precontracted arteries, without significant differences between control and diabetic rabbits. Both in control and diabetic rabbits endothelium removal inhibited testosterone relaxant action. In arteries with endothelium, incubation with indomethacin (10(-5)M), N(G)-nitro-l-arginine (10(-5)M) or tetraethylammonium (10(-5)M) did not modify relaxations to testosterone neither in control nor in diabetic rabbits. In endothelium-denuded arteries indomethacin enhanced the relaxant action of testosterone, both in control and diabetic rabbits. In arteries from diabetic rabbits, eNOS, iNOS and COX-1 expression and testosterone-induced release of thromboxane A(2) and prostacyclin were not significantly different from those observed in control rabbits. However, COX-2 expression was significantly lower in diabetic rabbits that in control rabbits. In nominally Ca(2+)-free medium, cumulative addition of CaCl2 (10(-5) to 3x10(-2)M) contracted previously depolarized arteries. Testosterone (10(-4)M) inhibited CaCl2 contractions of the renal artery both in control and diabetic rabbits. These results show that testosterone relaxes the renal artery both in control and diabetic rabbits. This relaxation is modulated by muscular thromboxane A(2), it is partially mediated by endothelial prostacyclin, and it involves the blocking of extracellular Ca2+ entry. Diabetes does not modify the mechanisms involved in the relaxant action of testosterone in the rabbit renal artery.

Role of NO-synthases and cyclooxygenases in the hyperreactivity of male rabbit carotid artery to testosterone under experimental diabetes.

Cardiovascular disease is the major cause of morbidity and mortality in diabetic patients, which in turn is also associated with low levels of serum testosterone. The working hypothesis was that diabetes might modify the mechanisms involved in the vascular actions of testosterone in isolated rabbit carotid arteries. Testosterone (10(-8)-3x10(-4)M) induced a concentration-dependent relaxation of precontracted carotid arteries, which was higher in diabetic than in control rabbits. In control rabbits neither endothelium removal nor the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine (l-NOArg, 10(-5)M) modified the relaxant action of testosterone, and the cyclooxygenase (COX) inhibitor indomethacin (10(-5)M) enhanced this relaxation. In contrast, in diabetic rabbits endothelium removal, l-NOArg (10(-5)M) or indomethacin (10(-5)M) inhibited the testosterone induced relaxation. In arteries from diabetic rabbits, eNOS, iNOS and COX-2 expression and testosterone induced release of prostacyclin resulted enhanced in comparison with arteries from control rabbits. Testosterone (10(-4)M) strongly inhibited CaCl(2) (10(-5)-3x10(-2)M) concentration-related contractions of the carotid artery both in control and diabetic rabbits. These results suggest that testosterone relaxes the rabbit carotid artery by blocking the extracellular calcium entry. Diabetes enhances the vasodilator response of the rabbit carotid artery to testosterone by a mechanism that at least includes an increased modulatory activity of the endothelial nitric oxide and an augmented release of COX-2 vasodilator, prostacyclin rather than the absence of COX-1 vasoconstrictor, thromboxane A(2). The hypotestosteronemia observed in diabetic rabbits could be a consequence of the increased expression of iNOS and could contribute to the hyperreactivity of the rabbit carotid artery to testosterone.

Lactoferricin-related peptides with inhibitory effects on ACE-dependent vasoconstriction.

A selection of lactoferricin B (LfcinB)-related peptides with an angiotensin I-converting enzyme (ACE) inhibitory effect have been examined using in vitro and ex vivo functional assays. Peptides that were analyzed included a set of sequence-related antimicrobial hexapeptides previously reported and two representative LfcinB-derived peptides. In vitro assays using hippuryl-L-histidyl-L-leucine (HHL) and angiotensin I as substrates allowed us to select two hexapeptides, PACEI32 (Ac-RKWHFW-NH2) and PACEI34 (Ac-RKWLFW-NH2), and also a LfcinB-derived peptide, LfcinB17-31 (Ac-FKCRRWQWRMKKLGA-NH2). Ex vivo functional assays using rabbit carotid arterial segments showed PACEI32 (both D- and L-enantiomers) and LfcinB17-31 have inhibitory effects on ACE-dependent angiotensin I-induced contraction. None of the peptides exhibited in vitro ACE inhibitory activity using bradykinin as the substrate. In conclusion, three bioactive lactoferricin-related peptides exhibit inhibitory effects on both ACE activity and ACE-dependent vasoconstriction with potential to modulate hypertension that deserves further investigation.

Inhibition of iNOS activity by 1400W decreases glutamate release and ameliorates stroke outcome after experimental ischemia.

BACKGROUND AND PURPOSE: It has been shown that the reversed operation of glutamate transporters when ATP levels fall accounts for most glutamate release induced by severe cerebral ischemia. Nitric oxide (NO) is formed after ischemia and causes ATP depletion. Our purpose is to test if NO release from inducible NO synthase (iNOS) after stroke may cause a delayed glutamate release due to ATP depletion that might underlie progression of the ischemic infarct. We have studied the effect of the highly selective inhibitor of iNOS activity 1400W on brain ATP levels, extracellular glutamate, and stroke outcome after transient focal cerebral ischemia in rats. METHODS: To induce focal ischemia, the middle cerebral artery (MCA) was occluded by using the intraluminal thread technique (tMCAO). 1400W was administered, after tMCAO, by using an Alzet osmotic pump to yield a drug delivery rate of 2.5 mg/kg/h. Results. Postischemic treatment with 1400W induced a reduction in the neurofunctional impairment and in the total volume of brain infarct. Western blot analysis showed ischemia-induced expression of iNOS. Treatment with 1400W partially prevented delayed ATP reduction and produced inhibition of the subsequent delayed increase in glutamate levels caused by the ischemic insult. CONCLUSIONS: Our data indicate that 1400W improves stroke outcome, an effect concomitant to the inhibition of both ischemia-induced decrease in brain ATP levels and increase in glutamate release. These results provide evidence indicating that the expression of iNOS induced by ischemia may contribute to the progression of the ischemic infarct and have important therapeutic implications for the management of stroke.

Single-dose ebselen does not afford sustained neuroprotection to rats subjected to severe focal cerebral ischemia.

Oxygen free radicals have been involved in the pathophysiology of cerebral ischemia, especially after spontaneous or thrombolytic reperfusion. In this study with rats, we have combined a severe focal ischemic insult (2 h) and a prolonged reperfusion time (7 days) to assess the possible sustained neuroprotective effect of ebselen (10 or 100 mg/kg), a small, lipophilic organoselenium compound which mimics glutathione peroxidase. Parietal cortical perfusion was measured by laser-Doppler flowmetry, and focal cerebral ischemia was carried out by the intraluminal thread method. We have measured plasma selenium levels, brain reduced glutathione levels, as a marker of oxidative stress, and infarct volume associated with cerebral ischemia. Focal ischemia did not alter reduced glutathione levels, while 60 min reperfusion following ischemia induced a significant (P < 0.05) decrease in reduced glutathione levels of the ipsilateral hemisphere. Pretreatment with ebselen, which induced significant (P < 0.05) increase in plasma selenium levels, did not significantly alter the decrease in reduced glutathione levels. The ischemic insult induced 30% mortality on average, with deaths always occurring within 12-48 h. Surviving rats suffered up to 25% body weight loss 1 week after the ischemic insult. Infarct volumes were 26.8 +/- 4.7% of the hemisphere in placebo-treated rats, 26.6 +/- 3.6% in 10 mg/kg ebselen-treated rats, and 25.6 +/- 6.4% in 100 mg/kg ebselen-treated rats (not significantly different). Single-dose administration of ebselen does not reduce the size of brain infarct resulting from severe focal cerebral ischemia in rats. In contrast to previous studies with relatively earlier endpoints, we have delayed the measurement of infarct volume to 1 week after the ischemic insult.

Acute relaxant effects of 17-beta-estradiol through non-genomic mechanisms in rabbit carotid artery.

Estrogens could play a cardiovascular protective role not only by means of systemic effects but also by means of direct effects on vascular structure and function. We have studied the acute effects and mechanisms of action of 17-beta-estradiol on vascular tone of rabbit isolated carotid artery. 17-Beta-estradiol (10, 30, and 100 microM) elicited concentration-dependent relaxation of 50 mM KCl-induced active tone in male and female rabbit carotid artery. The stereoisomer 17-alpha-estradiol showed lesser relaxant effects in male rabbits. Endothelium removal did not modify relaxation induced by 17-beta-estradiol. The NO synthase inhibitor L-NAME (100 microM) only reduced significantly relaxation produced by 30 microM 17-beta-estradiol. Relaxation was not modified by the estrogen receptor antagonist ICI 182,780 (1 microM), the protein synthesis inhibitor cycloheximide (1 microM), and the selective K(+) channel blockers charybdotoxin (0.1 microM) and glibenclamide (1 microM). CaCl(2) (30 microM -10 mM) induced concentration-dependent contraction in rabbit carotid artery depolarized by 50 mM KCl in Ca(2+) free medium. Preincubation with 17-beta-estradiol (3, 10, 30, or 100 microM) or the L-type Ca(2+) channel blocker nicardipine (0.01, 0.1, 1, or 10 nM) produced concentration-dependent inhibition of CaCl(2)-induced contraction. In conclusion, 17-beta-estradiol induces endothelium-independent relaxation of rabbit carotid artery, which is not mediated by classic estrogen receptor and protein synthesis activation. The relaxant effect is due to inhibition of extracellular Ca(2+) influx to vascular smooth muscle, but activation of K(+) efflux is not involved. Relatively high pharmacological concentrations of estrogen causing relaxation preclude acute vasoactive effects of plasma levels in the carotid circulation.