María C. Burguete

Cannabinoid Type 2 Receptor Activation Downregulates Stroke-Induced Classic and Alternative Brain Macrophage/Microglial Activation Concomitant to Neuroprotection

Background and Purpose— Ischemic stroke continues to be one of the main causes of death worldwide. Inflammation accounts for a large part of damage in this pathology. The cannabinoid type 2 receptor (CB2R) has been proposed to have neuroprotective properties in neurological diseases. Therefore, our aim was to determine the effects of the activation of CB2R on infarct outcome and on ischemia-induced brain expression of classic and alternative markers of macrophage/microglial activation. Methods— Swiss wild-type and CB2R knockout male mice were subjected to a permanent middle cerebral artery occlusion. Mice were treated with either a CB2R agonist (JWH-133), with or without a CB2R antagonist (SR144528) or vehicle. Infarct outcome was determined by measuring infarct volume and neurological outcome. An additional group of animals was used to assess mRNA and protein expression of CB2R, interleukin (IL)-1&bgr;, IL-6, tumor necrosis factor &agr; (TNF-&agr;), monocyte chemoattractant protein–1 (MCP-1), macrophage inflammatory peptide (MIP) –1&agr;, RANTES, inducible nitric oxide synthase (iNOS), cyclooxygenase-2, IL-4, IL-10, transforming growth factor &bgr; (TGF-&bgr;), arginase I, and Ym1. Results— Administration of JWH-133 significantly improved infarct outcome, as shown by a reduction in brain infarction and neurological impairment. This effect was reversed by the CB2R antagonist and was absent in CB2R knockout mice. Concomitantly, administration of JWH-133 led to a lower intensity of Iba1+ microglia/macrophages and a decrease in middle cerebral artery occlusion–induced gene expression of both classic (IL-6, TNF-&agr;, MCP-1, MIP-1&agr;, RANTES, and iNOS) and alternative mediators/markers (IL-10, TGF-&bgr;, and Ym1) of microglial/macrophage activation after permanent middle cerebral artery occlusion. Conclusions— The inhibitory effect of CB2R on the activation of different subpopulations of microglia/macrophages may account for the protective effect of the selective CB2R agonist JWH-133 after stroke.

Relaxant Effects of 17-β-Estradiol in Cerebral Arteries through Ca2+ Entry Inhibition

Estrogens account for gender differences in the incidence and outcome of stroke, but it remains unclear to what extent neuroprotective effects of estrogens are because of parenchymal or vascular actions. Because reproductive steroids have vasoactive properties, the authors assessed the effects and mechanisms of action of 17-β-estradiol in rabbit isolated basilar artery. Cumulative doses of 17-β-estradiol (0.3 μmol/L to 0.1 mmol/L) induced concentration-dependent relaxation that was larger in basilar than carotid artery, in male than female basilar artery, and in KCl-precontracted than UTP-precontracted male basilar artery. Endothelium removal did not modify relaxation induced by 17-β-estradiol in basilar artery, whereas relaxation induced by acetylcholine (1 nmol/L to 0.1 mmol/L) was almost abolished. Neither the estrogen receptor antagonist ICI 182,780 (1 μmol/L), nor the protein synthesis inhibitor cycloheximide (1 μmol/L) affected 17-β-estradiol–induced relaxations. Relaxations induced by the K+ channel openers NS1619 and pinacidil in the same concentration range were greater and lower, respectively, when compared with relaxation to 17-β-estradiol, which was not significantly modified by incubation with the K+ channel blockers charybdotoxin (1 nmol/L and 0.1 μmol/L) or glibenclamide (10 nmol/L and 1 μmol/L). Preincubation with 17-β-estradiol (3 to 100 μmol/L) produced concentration-dependent inhibition of CaCl2-induced contraction, with less potency than the Ca2+ entry blocker nicardipine (0.01 to 10 nmol/L). The authors conclude that 17-β-estradiol induces endothelium-independent relaxation of cerebral arteries with tissue and gender selectivity. The relaxant effect is because of inhibition of extracellular Ca2+ influx to vascular smooth muscle, but activation of estrogen receptors, protein synthesis, or K+ efflux are not involved. Relatively high pharmacologic concentrations of 17-β-estradiol causing relaxation preclude acute vascular effects of physiologic circulating levels on the cerebral circulation.

Dietary phytoestrogens improve stroke outcome after transient focal cerebral ischemia in rats

As phytoestrogens are postulated as being neuroprotectants, we assessed the hypothesis that dietary isoflavone‐type phytoestrogens are neuroprotective against ischemic stroke. Transient focal cerebral ischemia (90 min) was induced by middle cerebral artery occlusion (MCAO) following the intraluminal thread technique, both in rats fed with soy‐based diet and in rats fed with isoflavone‐free diet. Cerebro‐cortical laser‐Doppler flow (cortical perfusion, CP), arterial blood pressure, core temperature, PaO2, PaCO2, pH and glycemia were measured before, during and after MCAO. Neurological examination and infarct volume measurements were carried out 3 days after the ischemic insult. Dietary isoflavones (both glycosides and aglycones) were measured by high‐performance liquide chromatography. Neither pre‐ischemic, intra‐ischemic nor post‐ischemic CP values were significantly different between the soy‐based diet and the isoflavone‐free diet groups. Animals fed with the soy‐based diet showed an infarct volume of 122 ± 20.2 mm3 (19 ± 3.3% of the whole ipsilateral hemisphere volume). In animals fed with the isoflavone‐free diet the mean infarct volume was significantly higher, 191 ± 26.7 mm3 (28 ± 4.1%, P < 0.05). Neurological examination revealed significantly higher impairment in the isoflavone‐free diet group compared with the soy‐based diet group (3.3 ± 0.5 vs. 1.9 ± 0.5, P < 0.05). These results demonstrate that dietary isoflavones improve stroke outcome after transient focal cerebral ischemia in such a way that a higher dietary isoflavone content results in a lower infarct volume and a better neurological status.

Acute relaxant effects of 17-β-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.

Administration of Transforming Growth Factor-α Reduces Infarct Volume after Transient Focal Cerebral Ischemia in the Rat

Growth factors promote cell growth and survival and protect the brain from developing injury after ischemia. In this article, the authors examined whether transforming growth factor-α (TGF-α) was protective in transient focal ischemia and whether alteration of cerebral circulation was involved. Rats received intraventricular TGF-α (50 ng, either split into 2 doses given 30 minutes before and 30 minutes after middle cerebral artery occlusion (MCAO), or 1 dose given 30 minutes after MCAO) or vehicle. Rats were subjected to 1-hour intraluminal MCAO and cerebral blood flow was recorded continuously by laser–Doppler flowmetry. Infarct volume was measured 1 and 4 days later. The effects of TGF-α on arterial tone were assessed in isolated rabbit basilar and common carotid arteries. Transforming growth factor-α before and after ischemia reduced infarct volume by 70% at 1 day and 50% at 4 days. Transforming growth factor-α given only after ischemia also did reduce infarct volume by 70% at 1 day and 80% at 4 days. The protective effect was more marked in cortex than in striatum. Transforming growth factor-α did not change cortical microvascular perfusion and did not modify arterial passive tone nor agonist-induced active tone. It can be concluded that TGF-α reduces infarct volume, even when the factor is exclusively administered at reperfusion, and that this effect is not mediated by changes in microvascular perfusion or cerebral arteries. It is therefore suggested that TGF-α has a protective effect against neuronal cell death after transient focal ischemia.

Administration of transforming growth factor-alpha reduces infarct volume after transient focal cerebral ischemia in the rat.

Growth factors promote cell growth and survival and protect the brain from developing injury after ischemia. In this article, the authors examined whether transforming growth factor-α (TGF-α) was protective in transient focal ischemia and whether alteration of cerebral circulation was involved. Rats received intraventricular TGF-α (50 ng, either split into 2 doses given 30 minutes before and 30 minutes after middle cerebral artery occlusion (MCAO), or 1 dose given 30 minutes after MCAO) or vehicle. Rats were subjected to 1-hour intraluminal MCAO and cerebral blood flow was recorded continuously by laser–Doppler flowmetry. Infarct volume was measured 1 and 4 days later. The effects of TGF-α on arterial tone were assessed in isolated rabbit basilar and common carotid arteries. Transforming growth factor-α before and after ischemia reduced infarct volume by 70% at 1 day and 50% at 4 days. Transforming growth factor-α given only after ischemia also did reduce infarct volume by 70% at 1 day and 80% at 4 days. The protective effect was more marked in cortex than in striatum. Transforming growth factor-α did not change cortical microvascular perfusion and did not modify arterial passive tone nor agonist-induced active tone. It can be concluded that TGF-α reduces infarct volume, even when the factor is exclusively administered at reperfusion, and that this effect is not mediated by changes in microvascular perfusion or cerebral arteries. It is therefore suggested that TGF-α has a protective effect against neuronal cell death after transient focal ischemia.

Transient focal cerebral ischemia significantly alters not only EAATs but also VGLUTs expression in rats: relevance of changes in reactive astroglia.

J. Neurochem. (2010) 113, 1343–1355.

Soy-derived phytoestrogens as preventive and acute neuroprotectors in experimental ischemic stroke: Influence of rat strain

The ability of a soy-based high-phytoestrogen diet (nutritional intervention) or genistein (pharmacological intervention), to limit ischemic brain damage in Wistar, Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats, has been assessed. As to the nutritional intervention, two groups from each strain received either a phytoestrogen-free (PE-0) or a high-phytoestrogen (PE-600) diet from weaning to adulthood. As to the pharmacological intervention, all animals were fed the standard soy-free AIN-93G diet and subsequently separated into two groups from each strain to receive either pure genistein (aglycone form, 1mg/kg/day intraperitoneal) or vehicle at 30 min reperfusion. After an episode of 90 min ischemia (intraluminal thread procedure) followed by 3 days reperfusion, cerebral infarct volume was measured. Arterial blood pressure (ABP) was significantly higher at the basal stage (just before ischemia) in SHR (140 ± 7 mmHg, n=17, p<0.05) than in Wistar (113 ± 4mmHg, n=23) and WKY (111 ± 6mmHg, n=14) rats. No significant differences were shown among the three stages (basal, ischemia, reperfusion) within each rat strain for both PE-0 and PE-600 diets. Wistar, but not WKY or SHR, rats fed the PE-600 diet showed significantly lower infarct volumes than their counterparts fed the PE-0 diet (30 ± 3% vs. 17 ± 3%, p<0.01). Genistein-treated Wistar, but not WKY or SHR, rats showed significantly lower infarct volumes than their vehicle-treated controls (27 ± 2% vs. 15 ± 2%, p<0.01). Our results demonstrate that: (1) the neuroprotective action of either chronic or acute exposure to soy isoflavones is strain-dependent, since it was shown in Wistar but not WKY or SHR rats; and (2) the soy-based diet does not prevent development of hypertension in SHR rats.

Improvement of the circulatory function partially accounts for the neuroprotective action of the phytoestrogen genistein in experimental ischemic stroke

We tested the hypothesis that the phytoestrogen genistein protects the brain against ischemic stroke by improving the circulatory function in terms of reduced production of thromboxane A2 and leukocyte-platelet aggregates, and of preserved vascular reactivity. Ischemia-reperfusion (90 min-3 days, intraluminal filament) was induced in male Wistar rats, and functional score and cerebral infarct volume were the end points examined. Genistein (10mg/kg/day) or vehicle (β-cyclodextrin) was administered at 30 min after ischemia or sham-operation. Production of thromboxane A2 and leukocyte-platelet aggregates, as well as reactivity of carotid artery to U-46619 (thromboxane A2 analogue) and to platelet releasate was measured. At 3 days post-ischemia, both improvement in the functional examination and reduction in the total infarct volume were shown in the ischemic genistein-treated group. Genistein significantly reverted both the increased thromboxane A2 concentration and the increased leukocyte-platelet aggregates production found in samples from the ischemic vehicle-treated group. Both U-46619 and platelet releasate elicited contractions of the carotid artery, which were significantly lower in the ischemic vehicle-treated group. Genistein significantly restored both the decreased U-46619- and the decreased platelet releasate-elicited contractile responses. In conclusion, genistein protects the brain against an ischemia-reperfusion challenge, at least in part, by its beneficial effects on the circulatory function.

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 17β-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.