Cerebral microcirculatory responses of insulin-resistant rats are preserved to physiological and pharmacological stimuli.

OBJECTIVE: Previously, we have shown that IR impairs the vascular reactivity of the major cerebral arteries of ZO rats prior to the occurrence of Type-II diabetes mellitus. However, the functional state of the microcirculation in the cerebral cortex is still being explored. METHODS: We tested the local CoBF responses of 11-13-week-old ZO (n = 31) and control ZL (n = 32) rats to several stimuli measured by LDF using a closed cranial window setup. RESULTS: The topical application of 1-100 µm bradykinin elicited the same degree of CoBF elevation in both ZL and ZO groups. There was no significant difference in the incidence, latency, and amplitude of the NMDA-induced CSD-related hyperemia between the ZO and ZL groups. Hypercapnic CoBF response to 5% carbon-dioxide ventilation did not significantly change in the ZO compared with the ZL. Topical bicuculline-induced cortical seizure was accompanied by the same increase of CoBF in both the ZO and ZL at all bicuculline doses. CONCLUSIONS: CoBF responses of the microcirculation are preserved in the early period of the metabolic syndrome, which creates an opportunity for intervention to prevent and restore the function of the major cerebral vascular beds.

N-methyl-D-aspartate induces cortical hyperemia through cortical spreading depression-dependent and -independent mechanisms in rats.

OBJECTIVE: N-methyl-d-aspartate (NMDA) is a powerful cerebrovascular dilator in vivo. Cortical spreading depression (CSD) has recently been shown to contribute to the pial arteriolar dilation in mice. Our main aim was to examine the participation of CSD in the overall cerebrovascular response to NMDA in the rat. METHODS: Anesthetized Wistar rats (eight weeks old) were equipped with a closed cranial window to allow topical application of NMDA (10(-5)-10(-3) M) to the parietal cortex. Cortical blood flow (CoBF) under and outside the cranial window was simultaneously monitored by using a two-channel laser-Doppler flowmeter. CSDs were detected by recording the changes in the cortical DC potential. RESULTS: Concentrations of 10(-4) and 10(-3) M of NMDA evoked single CSDs associated with rapid, transient hyperemia, followed by a sustained, but reduced, increase in CoBF. The latency and magnitude of the CoBF responses were dose dependent. The higher dose resulted in shorter latency (100+/-5* vs. 146+/-11 seconds, *P<0.05; mean+/-standard error of the mean) and larger overall flow response (77+/-12* vs. 28+/-3% from baseline) under, but not outside, the cranial window. CONCLUSIONS: NMDA elicits dose-dependent increases in CoBF that are composed of CSD-dependent and -independent components in rats.

Secretory phospholipase A2 inhibitor PX-18 preserves microvascular reactivity after cerebral ischemia in piglets.

Cerebral ischemia/reperfusion (I/R) results in cellular energy failure and dysfunction of the neurovascular unit that contribute to subsequent neuronal cell death in the neonate. PX-18 is a putative neuroprotective inhibitor of secretory phospholipase A(2) (sPLA(2)) but its in vivo testing has been limited by its poor solubility. Our purpose was to assess whether PX-18 preserved neuronal-vascular reactivity to I/R-sensitive endothelium-dependent (hypercapnia, bradykinin) and/or neuron-dependent (N-methyl-D-aspartate; NMDA) stimuli. To make the drug available for in vivo studies, PX-18 was formulated as a 3% nanosuspension applying high pressure homogenization. Newborn piglets (1-day old, n=40) were anesthetized and ventilated, and cerebrovascular reactivity to the above stimuli was determined by measuring changes in pial arteriolar diameters using the closed cranial window/intravital videomicroscopy technique. Intravenous infusion of PX-18 nanosuspension (6 mg/kg, 20 min) did not affect baseline arteriolar diameters, or hypercapnia-, bradykinin-, or NMDA-induced pial arteriolar vasodilation under normoxic conditions. Global cerebral ischemia (10 min) followed by 1 h of reperfusion significantly attenuated hypercapnia-, bradykinin-, and NMDA-induced vasodilation in untreated or vehicle-treated controls. However, PX-18 resulted in nearly full preservation of cerebrovascular reactivity to all these stimuli. In conclusion, inhibition of sPLA(2) by PX-18 improves neurovascular function both at the neuronal and the microvascular level following I/R. This effect of PX-18 likely contributes to its neuroprotective effect.

Pituitary adenylate cyclase-activating polypeptide induces pial arteriolar vasodilation through cyclooxygenase-dependent and independent mechanisms in newborn pigs.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a cerebrovascular dilator and was found neuroprotective in numerous in vitro and in vivo models of cerebral ischemia. However, the mechanism of its cerebrovascular action is poorly known, especially in newborns. Therefore, we tested pial arteriolar responses to the two naturally occurring forms PACAP27 and 38 as well as to shorter sequences (PACAP6-27, 6-38, 1-15, 6-15, 20-31). We also investigated the involvement of nitric oxide synthase (NOS), cyclooxygenase-1 and -2 (COX-1 and -2) activity in PACAP-induced pial arteriolar responses using the NOS inhibitor N-omega-nitro-l-arginine methyl ester (L-NAME 15 mg/kg iv), the non-selective COX inhibitor indomethacin (5 mg/kg iv), and the selective COX-1 and COX-2 inhibitors SC-560 (1 mg/kg iv) and NS-398 (1 mg/kg iv), respectively. Anesthetized, ventilated piglets (n=127) were equipped with closed cranial windows, and pial arteriolar diameters were determined via intravital microscopy. Topical application of both natural PACAPs, but none of the PACAP segments, resulted in prominent, repeatable, dose-dependent vasodilation. Percentage changes ranged 5+/-1-29+/-6 (n=7) and 4+/-1-36+/-7 (n=9) to 10(-)(8) to 10(-)(6) M PACAP27 and 38 (mean+/-SEM), respectively. Vasodilation to both natural PACAPs was significantly reduced by co-application with PACAP6-27 or 6-38, but not by L-NAME. Indomethacin abolished PACAP38 but not PACAP27-induced vasodilation. Arteriolar responses to PACAP38 were also sensitive to SC-560 but not to NS-398 suggesting the unique involvement of COX-1 activity in this response. In summary, PACAP27 and 38 are potent vasodilators in the neonatal cerebral circulation with at least two distinct mechanisms of action: a COX-dependent and a COX-independent pathway.

Living standard is related to microregional differences in stroke characteristics in Central Europe: the Budapest Districts 8-12 Project.

OBJECTIVES: To test whether str oke features relate to living standard within one city by comparing 2 districts. METHODS: District-8 (D-8) ranks the last, whereas District-12 (D-12) is the second regarding personal monthly income of the 23 districts of Budapest, Hungary. Stroke cases hospitalized in 2007 were identified by the database of the National Health Insurance Fund and postal codes for living address. Case certification was performed by personal visits to the general practitioners. Demographic data, risk factors and survival status in 2010 were analyzed using the anonymized database. RESULTS: Three-year case fatality was 36.6 % in D-8 and 31.5 % in D-12 (p = 0.24). Of the fatal cases, men were more than 12 years younger in D-8 than in D-12 (69.2 ± 13.3 vs. 82.4 ± 9.2 years, p < 0.001). Men died younger than women in D-8 (69.2 ± 13.3 vs. 75.2 ± 12.4; p = 0.036), but not in D-12 (82.4 ± 9.2 vs. 81.9 ± 7.3, p = 0.8). Non-treated hypertension, alcohol dependence, and smoking were significantly more prevalent in the poor district (p < 0.01 for all). CONCLUSION: In national stroke programs of former Eastern Block countries, primary prevention should focus especially on male populations of less wealthy regions.

Stroke care in Central Eastern Europe: current problems and call for action.

Stroke is a major medical problem and one of the leading causes of mortality and disability all over in Europe. However, there are significant East-West differences in stroke care as well as in stroke mortality and morbidity rates. Central and Eastern European countries that formerly had centralized and socialist health care systems have serious and similar problems in organizing health and stroke care 20 years after the political transition. In Central and Eastern Europe, stroke is more frequent, the mortality rate is higher, and the victims are younger than in Western Europe. High-risk patients live in worse environmental conditions, and the socioeconomic consequences of stroke further weaken the economic development of these countries. To address these issues, a round table conference was organized. The main aim of this conference was to discuss problems to be solved related to acute and chronic stroke care in Central and Eastern European countries, and also, to exchange ideas on possible solutions. In this article, the discussed problems and possible solutions will be summarized, and introduce 'The Budapest Statement of Stroke Experts of Central and Eastern European countries'.

Neuroprotective effect of adenoviral catalase gene transfer in cortical neuronal cultures.

Reduced availability of reactive oxygen species is a key component of neuroprotection against various toxic stimuli. Recently we showed that the hydrogen peroxide scavenger catalase plays a central role in delayed preconditioning induced by the mitochondrial ATP-sensitive potassium channel opener BMS-191095. The purpose of the experiments discussed here was to investigate the neuroprotective effect of catalase in vitro using a recombinant adenoviral catalase gene transfer protocol. To induce catalase overexpression, cultured rat cortical neurons were infected with the adenoviral vector Ad5CMVcatalase and control cells were incubated with Ad5CMVntLacZ for 24 h. Gene transfer effectively increased catalase protein levels and activity, but did not influence other antioxidants tested. Ad5CMVcatalase, with up to 10 plaque forming units (pfu) per neuron, did not affect cell viability under control conditions and did not protect against glutamate excitotoxicity or oxygen-glucose deprivation. In contrast, catalase overexpression conferred a dose-dependent protection against exposure to hydrogen peroxide (viability: control, 33.02+/-1.09%; LacZ 10 pfu/cell, 32.85+/-1.51%; catalase 1 pfu/cell, 62.09+/-4.17%*; catalase 2 pfu/cell, 98.71+/-3.35%*; catalase 10 pfu/cell, 99.68+/-1.99%*; *p<0.05 vs. control; mean+/-SEM). Finally, the protection could be antagonized using the catalase inhibitor 3-aminotriazole. Our results support the view that enhancing cellular antioxidant capacity may play a crucial role in neuroprotective strategies.

Immediate neuronal preconditioning by NS1619.

The objectives of our present experiments were to determine whether the BK(Ca) channel agonist NS1619 is able to induce immediate preconditioning in cultured rat cortical neurons and to elucidate the role of BK(Ca) channels in the initiation of immediate preconditioning. NS1619 depolarized mitochondria and increased reactive oxygen species (ROS) generation, but neither of these effects was inhibited by BK(Ca) channel antagonists. NS1619 also activated the extracellular signal-regulated kinase signaling pathways. One-hour treatment with NS1619 induced immediate protection against glutamate excitotoxicity (viability 24 h after glutamate exposure: control, 58.45+/-0.95%; NS1619 50 microM, 78.99+/-0.90%; NS1619 100 microM, 86.89+/-1.20%; NS1619 150 microM, 93.23+/-1.23%; mean+/-SEM; p<0.05 vs. control; n=16-32). Eliminating ROS during the preconditioning phase effectively blocked the development of cytoprotection. In contrast, the BK(Ca) channel blockers iberiotoxin and paxilline, the phosphoinositide 3-kinase inhibitor wortmannin, the protein kinase C blocker chelerythrine, and the mitogen activated protein kinase antagonist PD98059 were unable to antagonize the immediate neuroprotective effect. Finally, preconditioning with NS1619 reduced the calcium load and ROS surge upon glutamate exposure and increased superoxide dismutase activity. Our results indicate that NS1619 is an effective inducer of immediate neuronal preconditioning, but the neuroprotective effect is independent of the activation of BK(Ca) channels.

Cerebrovascular responses to insulin in rats.

Effects of insulin on cerebral arteries have never been examined. Therefore, we determined cerebrovascular actions of insulin in rats. Both PCR and immunoblot studies identified insulin receptor expression in cerebral arteries and in cultured cerebral microvascular endothelial cells (CMVECs). Diameter measurements (% change) of isolated rat cerebral arteries showed a biphasic dose response to insulin with an initial vasoconstriction at 0.1 ng/mL (-9.7%+/-1.6%), followed by vasodilation at 1 to 100 ng/mL (31.9%+/-1.4%). Insulin also increased cortical blood flow in vivo (30%+/-8% at 120 ng/mL) when applied topically. Removal of reactive oxygen species (ROS) abolished the vasoconstriction to insulin. Endothelial denudation, inhibition of K(+) channels, and nitric oxide (NO) synthase, all diminished insulin-induced vasodilation. Inhibition of cytochrome P450 enhanced vasodilation in endothelium-intact arteries, but promoted vasoconstriction after endothelial denudation. Inhibition of cyclooxygenase abolished vasoconstriction and enhanced vasodilation to insulin in all arteries. Inhibition of endothelin type A receptors enhanced vasodilation, whereas endothelin type B receptor blockade diminished vasodilation. Insulin treatment in vitro increased Akt phosphorylation in cerebral arteries and CMVECs. Fluorescence studies of CMVECs showed that insulin increased intracellular calcium and enhanced the generation of NO and ROS. Thus, cerebrovascular responses to insulin were mediated by complex mechanisms originating in both the endothelium and smooth muscle.

PACAP and VIP differentially preserve neurovascular reactivity after global cerebral ischemia in newborn pigs.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are neuroprotective in numerous models. Impairment of cerebrovascular reactivity (CR) contributes to ischemia/reperfusion (I/R)-induced neuronal damage. We tested whether PACAP and/or VIP preserve CR to I/R-sensitive dilator responses dependent on endothelial and/or neuronal function. Accordingly, changes in pial arteriolar diameters in response to hypercapnia (5-10% CO(2) ventilation) or topical N-methyl-d-aspartate (NMDA, 10(-4) M) were determined before and after I/R via intravital microscopy in anesthetized/ventilated piglets. Local pretreatment with non-vasoactive doses of PACAP (10(-8) M) and VIP (10(-9) M) prevented the attenuation of postischemic CR to hypercapnia; to 10% CO(2), the CR values were 27+/-8% vs 92+/-5% vs 88+/-13% (vehicle vs PACAP38 vs VIP, CR expressed as a percentage of the response before I/R, mean+/-SEM, n=8-8, p<0.05). PACAP, but not VIP, preserved CR to NMDA after I/R, with CR values of 31+/-10% vs 87+/-8% vs 35+/-12% (vehicle vs PACAP38 vs VIP, n=6-6). Unlike PACAP, VIP-induced vasodilation has not yet been investigated in the piglet. We tested whether VIP-induced arteriolar dilation was sensitive to inhibitors of cyclooxygenase (COX)-1 (SC-560, 1 mg/kg), COX-2 (NS-398, 1 mg/kg), indomethacin (5 mg/kg), and nitric oxide synthase (L-NAME, 15 mg/kg). VIP (10(-8)-10(-7)-10(-6) M, n=8) induced reproducible, dose-dependent vasodilation of 16+/-3%, 33+/-6%, and 70+/-8%. The response was unaffected by all drugs, except that the vasodilation to 10(-8) M VIP was abolished by SC-560 and indomethacin. In conclusion, PACAP and VIP differentially preserve postischemic CR; independent of their vasodilatory effect.