James Javellaud

Synergistic protective effects of erythropoietin and olmesartan on ischemic stroke survival and post-stroke memory dysfunctions in the gerbil.

Objectives Treatment with erythropoietin and AT1 blockers is protective in experimental acute cerebral ischemia, with promising results in pilot clinical studies in human stroke. This paper examines the effects of using both agents as combination therapy in acute ischemic stroke. Methods We used the single carotid ligation stroke model in the gerbil. Six groups of 50 gerbils were treated either with placebo, erythropoietin (intraperitoneally, 5000 IU/kg, 2 and 48 h after stroke), olmesartan (10 mg/kg per day in drinking water started 36 h after stroke), ramipril (2.5 mg/kg per day in drinking water started 36 h after stroke), erythropoietin + olmesartan, or erythropoietin + ramipril. Long-term (1 month) Kaplan–Meyer survival curves were obtained, and survivors were submitted at day 30 to immediate (object recognition test) and spatial (Morris water maze) memory function tests. Results Erythropoietin alone and olmesartan alone, but not ramipril, significantly increased survival at day 30 compared with untreated controls (38, 30 and 6% versus 12%, respectively). Combined treatment with erythropoietin and olmesartan further increased the survival rate to 56%, whereas combined therapy with erythropoietin and ramipril decreased 30-day survival to 24% (P < 0.0001, erythropoietin + olmesartan versus erythropoietin + ramipril). Untreated stroke survivors had markedly altered performances in both the object recognition test (P = 0.0007) and the Morris water maze (P < 0.0001) tests at day 30 compared with normal gerbils. In erythropoietin-treated animals, ramipril therapy had no beneficial effect whereas olmesartan fully restored normal response to the memory tests. Conclusion Post-infarct treatment with olmesartan combined with early erythropoietin therapy has a protective effect on survival, and markedly improves long-term memory dysfunction in this experimental model.

Vasoconstrictive effect of angiotensin IV in isolated rat basilar artery independent of AT1 and AT2 receptors.

The effect of angiotensin IV (AngIV) was studied in freshly isolated rat basilar arteries (BAs) perfused at a constant rate. AngIV had no effect on basal BA perfusion pressure, but induced a marked concentration-dependent contraction in vessels precontracted by a 50-mM KCl solution (EC₅₀ = 44.5 ± 16 nM). This contraction was unaffected by the angiotensin AT₁ receptor antagonist candesartan or the angiotensin AT₂ receptor blocker PD123319, but was markedly inhibited by two different specific AT₄ receptor antagonists, Nle¹-Leu³¥(CH₂-NH₂)^3–4-AngIV and divalinal-AngIV. Removal of the endothelium abolished the contractile response to AngIV, and pretreatment of endothelium-intact arteries with the endothelin ET_A/ET_B receptors inhibitor PD142893 blocked the AngIV-induced contraction to the same extent. In BA pretreated with endothelin-1 (ET-1; 0.01 µM), AngIV-induced a concentration-dependent contraction, shifted to the left, compared with that observed with KCl precontraction, unaffected by candesartan but completely abolished by Nle¹-Leu³¥(CH₂-NH₂)^3–4-AngIV. The contractile effect was not affected by endothelium removal in the presence of exogenous ET-1, in contrast to KCl pretreated BA, suggesting that endothelium was mandatory to unmask the effect of AngIV as a source of endogenous ET-1 release. Taken together, these results indicate that low (nanomolar) concentrations of AngIV exert a constrictive effect mediated by its specific binding site AT₄ in the rat BA, and that this vasoactive effect is indirect and involves endogenous endothelin(s).

Involvement of potassium channels in the protective effect of 17β-estradiol on hypercholesterolemic rabbit carotid artery

The involvement of endothelium-derived hyperpolarizing factor (EDHF) in the protective effect of 17beta-estradiol was investigated on the phenylephrine-precontracted carotid artery from cholesterol fed rabbits. Animals were fed for 8 weeks as follows: control group, standard chow; (control+estradiol) group, standard chow+17beta-estradiol; standard chow+1% cholesterol, cholesterol group; or (cholesterol+estradiol) group, 1% cholesterol chow+17beta-estradiol. Relaxations to acetylcholine (ACh) (3 nM-30 microM) were performed with N(omega) nitro-L-arginine methyl ester (300 microM) and indomethacin (10 microM). Charybdotoxin (50 nM)+apamin (50 nM), glibenclamide (10 microM) or 4-aminopyridine (1 mM) were used to block, respectively, calcium-activated-K(+), adenosine triphosphate (ATP)-sensitive-K(+) and voltage-dependent K(+) channels. In the control group, ACh induced a residual concentration-dependent relaxation. This response was impaired by hypercholesterolemia and restored by 17beta-estradiol. In control and cholesterol groups, 4-aminopyridine or glibenclamide did not affect this relaxation, but in (control+estradiol) and (cholesterol+estradiol) groups, glibenclamide suppressed it. In all groups, this persisting relaxation was completely abolished by charybdotoxin alone or with apamin, by hemoglobin (10 microM), a nitric oxide scavenger, or by LY83183 (10 microM), a guanylate cyclase inhibitor. Thus, in the rabbit carotid artery, the protective effect of 17beta-estradiol against hypercholesterolemia is probably mediated by a nitric oxide/cyclic GMP pathway which activates calcium-targeted and ATP-dependent K(+) channels.

Effects of treatment with 17β-estradiol on the hypercholesterolemic rabbit middle cerebral artery

Objective: The effects of acute and long-term treatment with 17β-estradiol on the vasomotor responses of rabbit middle cerebral artery (RMCA) were investigated. Methods: For 8 weeks, male rabbits consumed standard chow (control group), standard chow+1% cholesterol (cholesterol group) or 1% cholesterol chow+17β-estradiol (i.m. injection 700 μg per week) (estradiol group). The RMCA was precontracted with high K+ solution and exposed to agonists. Results: Acute exposure to 17β-estradiol strongly induced relaxation of the RMCA isolated from either control or cholesterol groups. This effect was endothelium independent. Incubation with 17β-estradiol shifted the calcium contraction curve to the right. High cholesterol diet impaired the relaxation induced by acetylcholine and did not alter relaxation to sodium nitroprusside or to papaverine. Chronic treatment with 17β-estradiol restored this impaired relaxation to acetylcholine. This protective effect of estradiol was significantly reduced in the presence of Nω nitro-l-arginine methyl ester, a constitutive nitric oxide-synthase inhibitor and was not modified in the presence of aminoguanidine, an inducible nitric oxide-synthase inhibitor. Neither tetrabutylammonium, a blocker of calcium-activated K+ channels, nor glibenclamide, a blocker of ATP-sensitive K+ channels, affected concentration–response to acetylcholine in the RMCA of the estradiol group, whereas 4-aminopyridine, a blocker of voltage-dependent K+ channels strongly inhibited this relaxation. Conclusions: These results suggest that acute effects of 17β-estradiol in the RMCA is mediated through blockade of calcium entry into vascular smooth muscle cells, while chronic treatment with this hormone seems to be mediated by release of nitric oxide which activates voltage-dependent potassium channels.

Erythropoietin restores C-fiber function and prevents pressure ulcer formation in diabetic mice.

Pressure-induced vasodilatation (PIV), a cutaneous physiological neurovascular (C-fiber/endothelium) mechanism, is altered in diabetes and could possibly contribute to pressure ulcer development. We wanted to determine whether recombinant human erythropoietin (rhEPO), which has protective neurovascular effects, could prevent PIV alteration and pressure ulcer formation. We developed a skin pressure ulcer model in mice by applying two magnetic plates to the dorsal skin. This induced significant stage 2 ulcers (assessed visually and histologically) in streptozotocin-treated mice with 8 weeks of diabetes compared with very few in controls. Control and streptozotocin mice received either no treatment or systematic rhEPO (3,000 UI kg(-1) intraperitoneally, twice a week) during the last 2 weeks of diabetes. After 8 weeks of diabetes, we assessed ulcer development, PIV, endothelium-dependent vasodilation, C-fiber-mediated nociception threshold, and skin innervation density. Pretreatment with rhEPO fully prevented ulcer development in streptozotocin mice and also fully restored C-fiber nociception, skin innervation density, and significantly improved PIV, but had no effect on endothelium-dependent vasodilation. Our finding that rhEPO treatment protects the skin against pressure-induced ulcers in diabetic mice encourages evaluation of the therapeutic potential for non-hematopoietic analogs of EPO in preventing neuropathic diabetic ulcers.

PO34 Un traitement préventif à l’érythropoïétine améliore la vasodilatation induite par la pression et prévient la formation d’un ulcère de pression chez la souris diabétique exprimant des neuropathies

Introduction Une pression excessive aux zones d’appui du pied associee aux complications neurovasculaires lors d’un diabete favorise la formation d’un ulcere de pression. La vasodilatation induite par la pression (PIV), mecanisme physiologique (fibre C – endothelium) permettant une augmentation du flux sanguin cutane suite a l’application d’une pression non nociceptive, est tres alteree lors d’un diabete de 8 semaines chez la souris. Cette alteration peut refleter un important facteur de risque au developpement du mal perforant plantaire. L’objectif de cette etude est de savoir si l’erythropoietine (rhEPO) qui a montre recemment des effets protecteurs neurovasculaires pourrait prevenir l’atteinte de la PIV et la formation d’un ulcere de pression lors d’un diabete de type 1 induit par la streptozotocine (STZ) chez la souris. Materiels et Methodes Les souris diabetiques de 8 semaines et temoins sont non traitees ou traitees par de l’rhEPO (3 000 UI en intraperitoneal 2 fois par semaine) les 2 dernieres semaines de diabete. Nous avons developpe un modele d’ulcere de pression cutane chez la souris diabetique. Une pression continue est appliquee avec des aimants pendant 3 heures sur les flancs de la souris et induit un ulcere chez les souris diabetiques et pas chez les souris temoins. La technique de fluxmetrie laser Doppler a ete utilisee pour evaluer la PIV et la vasodilatation endothelium-dependante apres une stimulation iontophoretique a l’acetylcholine. Nous avons mesure les fonctions nociceptives des fibres nerveuses sensitives de type C avec le test de Randall-Sellito. Resultats L’rhEPO restaure completement la fonction nociceptive des fibres C et ameliore significativement la PIV et la vasodilatation endothelium-dependante. Chez les STZ, l’ulcere de pression (ulcere de stade 3 : necrose et atteinte du tissu sous-cutane) apparait 24 h apres l’application de la pression, puis sa surface augmente avec une surface maximale au troisieme jour et ensuite diminue. Un pretraitement a l’rhEPO previent completement le developpement de l’ulcere. Conclusion Dans ce modele de souris diabetique de 8 semaines exprimant des complications neurovasculaires, un traitement preventif de deux semaines a l’rhEPO ameliore la PIV et protege efficacement la peau contre une pression induisant un ulcere.