Rat Cerebral Arteries Research Articles

Protective effect of chlorogenic acid on the focal cerebral ischemia reperfusion rat models

ObjectiveThe aim of the study was to investigate the protective characteristic of chlorogenic acid, a natural glucosyl xanthone found in Lonicera Japonica on the cerebral ischemia reperfusion injury and the underlying mechanism. MethodsFocal cerebral ischemia reperfusion model was built by blocking the left middle cerebral artery in rats by using the suture-occluded method. Before operation, the corresponding drugs were given for each group once a day for 7days. After 1h of final administration, the model was built, after operation, reperfusion was conducted for 22h, Before the reperfusion 10min tail vein injection of large, medium and small dose of chlorogenic acid and then mortality was calculated, and Neurological deficit score (NDS) was conducted, and serum was collected to measure the NSE level; a 2mm thick brain slice located at the intersection of optic nerves was collected for TTC staining, and the percentage of cerebral infarction area was calculated; brain homogenate was collected to measure the ICAM-1, VCAM-1, EPO and HIF-1α levels in brain tissue of cerebral ischemia reperfusion rat models; NGF was detected using immunohistochemical method; the morphological changes in brain tissue was observed with HE staining. ResultsAll focal cerebral ischemia reperfusion rat models were duplicated successfully. Every chlorogenic acid group with different dosage can significantly reduce the mortality, NDS and cerebral infarction area of rats, and significantly increase the EPO, HIF-1α and NGF levels in brain tissue; significantly improve the pathological lesions of hippocampus and cortex in brain tissue. ConclusionThe results showed that chlorogenic acid could protect the focal cerebral ischemia reperfusion injury rat models by adjusting the inflammatory factor, hypoxia factor and nerve growth factor.

Nifedipine Inhibition of High-Voltage Activated Calcium Channel Currents in Cerebral Artery Myocytes Is Influenced by Extracellular Divalent Cations.

Voltage-dependent calcium channels (VDCCs) play an essential role in regulating cerebral artery diameter and it is widely appreciated that the L-type VDCC, CaV1.2, encoded by the CACNA1C gene, is a principal Ca2+ entry pathway in vascular myocytes. However, electrophysiological studies using 10 mM extracellular barium ([Ba2+]o) as a charge carrier have shown that ~20% of VDCC currents in cerebral artery myocytes are insensitive to 1,4-dihydropyridine (1,4-DHP) L-type VDDC inhibitors such as nifedipine. Here, we investigated the hypothesis that the concentration of extracellular divalent cations can influence nifedipine inhibition of VDCC currents. Whole-cell VDCC membrane currents were obtained from freshly isolated rat cerebral artery myocytes in extracellular solutions containing Ba2+ and/or Ca2+. In the absence of [Ca2+]o, both nifedipine-sensitive and -insensitive calcium currents were observed in 10 mM [Ba2+]o. However, VDCC currents were abolished by nifedipine when using a combination of 10 mM [Ba2+]o and 100 μM [Ca2+]o. VDCC currents were also completely inhibited by nifedipine in either 2 mM [Ba2+]o or 2 mM [Ca2+]o. The biophysical characteristics of all recorded VDCC currents were consistent with properties of a high-voltage activated VDCC, such as CaV1.2. Further, VDCC currents recorded in 10 mM [Ba2+]o ± 100 μM [Ca2+]o or 2 mM [Ba2+]o exhibited similar sensitivity to the benzothiazepine L-type VDCC blocker, diltiazem, with complete current inhibition at 100 μM. These data suggest that nifedipine inhibition is influenced by both Ca2+ binding to an external site(s) on these channels and surface charge effects related to extracellular divalent cations. In sum, this work demonstrates that the extracellular environment can profoundly impact VDCC current measurements.

Protective effect and mechanism of scutellarin ethyl ester on focal cerebral ischemia induced by ligation of middle cerebral artery in rats

To observe the protective effect of scutellarin ethyl ester on focal cerebral ischemia injury induced by middle cerebral artery occlusion in rats(MCAOR), and explore its mechanism. Totally 84 male SD rats were randomly divided into seven groups： sham-operated group, model group,positive drug group(niomdipine,12 mg•kg⁻¹), Brevisapin tablets group(48 mg•kg⁻¹), and high, middle and low-dose scutellarin ethyl ester groups(100, 50, 25 mg•kg⁻¹). The MCAOR model was prepared by using thread embolism method to observe the neurological function of rats, the area of cerebral infarction was measured with TTC, and the levels of MDA, SOD and NO in serum were detected with semiautomatic biochemistry analyzer.Ox-LDL and TNF-α cell injury models was established by treating HUVECs with 200 mg•L⁻¹ ox-LDL and 100 μg•L⁻¹ TNF-α,and the levels of MDA, SOD, NO, ET, 6-keto-PGF1α,TXB2, IL-1, IL-6, IL-8, ICAM-1 and PECAM-1 in the cell supernatant were determined. The results showed that scutellarin ethyl ester could effectively improve the neurological function of MCAOR rats, and significantly reduce the area of cerebral infarction. Compared with the model group, activities of SOD and NO in serum increased, while content of MDA decreased. In the cell supernatant, activities of SOD, 6-keto-PGF1α and NO increased, content of IL-1, IL-6, IL-8, ICAM-1, PECAM-1, TXB2, ET and MDA decreased, which indicated that scutellarin ethyl ester has a certain protective effect on focal cerebral ischemia injury induced by middle cerebral artery occlusion in rats, and its mechanism may be related to antioxidative stress, improvement of endothelial function and reduction in inflammatory reaction.

PIP2‐Regulation of Endothelial KIR2.1 Channels in Cerebral Arteries

In the cerebral circulation, inwardly rectifying potassium (KIR) channels are robustly expressed in both endothelial and smooth muscle cells. This pattern of expression is unusual for vascular cells that are electrically coupled and it suggests that each pool is differentially regulated driving a distinct subset of physiological responses. In this context, this study characterized the electrical/molecular properties of KIR2.x channels in cerebral arteries and ascertained the importance of phosphatidylinositol‐bis‐phosphate (PIP2), a regulatory phospholipid, in modulating each channel pool. Endothelial and smooth muscle cells were freshly isolated from rat cerebral arteries; patch clamp electrophysiology and Q‐PCR were used to ascertain KIR activity and expression, respectively. Our electrophysiological results highlight that a Ba2+‐sensitive KIR current is present and stable in both cell types. Endothelial current density was ~8 fold greater than smooth muscle (−20 vs −2.5 pA/pF at −100mV) and Q‐PCR revealed that KIR2.x mRNA subunits were present in both cell types, albeit it at variable levels. Intriguingly, KIR channels in endothelial but not smooth muscle cells were PIP2 sensitive; neomycin impaired, and exogenous PIP2 elevated channel activity. In summary, our observations indicate that while endothelial and smooth muscle both expressed KIR channels, they are uniquely regulated by PIP2. A complete picture of the expression/regulation of KIR channels within the vascular wall will provide further advances in our understanding of their role in cerebral blood flow control in health and disease.Support or Funding InformationCanadian Institute of Health Research (CIHR)

Tyrphostin AG556 increases the activity of large conductance Ca2+ -activated K+ channels by inhibiting epidermal growth factor receptor tyrosine kinase.

The present study was designed to investigate whether large conductance Ca2+‐activated K+ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK‐HEK 293 cells expressing both functional α‐subunits and the auxiliary β1‐subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK‐HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α‐ and β1‐subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre‐contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.

The augmentation of BK channel activity by nitric oxide signaling in rat cerebral arteries involves co-localized regulatory elements

Large conductance, Ca2+-activated K+ (BK) channels control cerebrovascular tone; however, the regulatory processes influencing these channels remain poorly understood. Here, we investigate the cellular mechanisms underlying the enhancement of BK current in rat cerebral arteries by nitric oxide (NO) signaling. In isolated cerebral myocytes, BK current magnitude was reversibly increased by sodium nitroprusside (SNP, 100 μM) and sensitive to the BK channel inhibitor, penitrem-A (100 nM). Fostriecin (30 nM), a protein phosphatase type 2A (PP2A) inhibitor, significantly prolonged the SNP-induced augmentation of BK current and a similar effect was produced by sildenafil (30 nM), a phosphodiesterase 5 (PDE5) inhibitor. Using proximity ligation assay (PLA)-based co-immunostaining, BK channels were observed to co-localize with PP2A, PDE5, and cGMP-dependent protein kinase (cGKI) (spatial restriction < 40 nm); cGKI co-localization increased following SNP exposure. SNP (10 μM) reversibly inhibited myogenic tone in cannulated cerebral arteries, which was augmented by either fostriecin or sildenafil and inhibited by penitrem-A. Collectively, these data suggest that (1) cGKI, PDE5, and PP2A are compartmentalized with cerebrovascular BK channels and determine the extent of BK current augmentation by NO/cGMP signaling, and (2) the dynamic regulation of BK activity by co-localized signaling enzymes modulates NO-evoked dilation of cerebral resistance arteries.

Differential Expression of BK Channel Beta1 Subunits in Rat Mesenteric, Coronary and Cerebral Arteries

Large conductance, Ca2+i/voltage-gated K+ (BK) channels regulate several physiological processes (Yang et et al., 2015). In smooth muscle, BK complexes include channel-forming α and auxiliary β1 subunits (Brenner et al., 2000). BK β1 increases the channel's apparent Ca2+ sensitivity (Brenner et al.,2000) and is required for channel activation by lithocholate (LCA)(Bukiya et al., 2009). Here, we studied the expression of BK α and β1 subunits in mesenteric, coronary and cerebral (anterior, middle, posterior and basilar) arteries, and the contribution of such differential expression to LCA-induced artery dilation. Thus, we conducted quantitative real-time PCR, fluorescence confocal imaging and patch-clamp electrophysiology on freshly isolated cerebral artery myocytes, and diameter determination of arteries pressurized in vitro. Data show that: 1) BK α mRNA is higher in cerebral anterior and basilar arteries. In turn, β1 mRNA is higher in basilar and coronary arteries; 2) BK α protein levels at the myocyte membrane surface are similar across the different arteries whereas β1 levels are higher in basilar and coronary arteries; 3) BK channel basal activity is also higher in basilar and coronary artery myocytes; 4) Likewise, BK channels from basilar and coronary artery myocytes are more responsive to LCA; 5) Finally, LCA-induced dilation of basilar and coronary arteries is higher than dilation of other arteries. Our results demonstrate a differential protein expression and functional impact on both basal channel activity and its response to LCA, of BK β1 subunits across arteries from cerebral and systemic vasculatures. In particular, basilar and coronary artery myocytes show an increased biochemical and functional presence of BK β1 subunits, suggesting that these vascular territories are more susceptible to modulation via BK β1. Support: HL104631, R37AA11560 (AMD); AHA Pre-doctoral Fellowship (GK).

Role of the slo1 CRAC4 Motif in BK Channel's Ethanol Sensitivity

Cholesterol (CLR) has been recognized for its ability to interact with ion channels. Among ion channels that sense CLR presence are large conductance, voltage/Ca2+-gated K+ (BK) channels. Functional BK channels result from tetrameric association of pore-forming slo1 subunits. CLR inhibition of slo1 current may result from CLR-sensing by the slo1 cytosolic tail domain (CTD). CTD contains seven CLR-recognition amino acid consensus (CRAC) motifs that contribute to the overall CLR sensitivity of the channel, with the contribution by CRAC4 being studied (Singh et al., 2012). Alcohol (ethanol) also modifies BK channel function, this effect resulting from an orchestration of several factors that include [Ca2+]i. In native membranes, ethanol increases slo1 channel open probability (Po) at [Ca2+]i<20-30 µM whereas reduces Po at [Ca2+]i<30 µM. Both CLR-enrichment and CLR-depletion of native membranes modify ethanol action on BK channels. Thus, we set to determine the role of CLR-sensing by the CRAC4 motif in the slo1 channel's ethanol sensitivity. Slo1 cloned from rat cerebral artery myocytes (cbv1) and the cbv1CRAC4T450F were incorporated into POPE:POPS (3:1 w/w) bilayers in 300/30 mM K+i/K+o gradient. At 0.3 μM Ca2+i, ethanol increased cbv1 Po by ≈3-fold. At 300 μM Ca2+i, ethanol failed to modify cbv1 Po in CLR-free bilayers but increased cbv1 Po in CLR-containing (33 mol%) bilayers. The discrepancy in ethanol effect between native membranes and planar bilayers may likely arise from their difference in proteo-lipid, including CLR, composition. Cbv1CRAC4T450F did not respond to ethanol in CLR-free bilayers, but was activated by ethanol in presence of 33 mol% CLR. Collectively, the similarity of ethanol modulation of BK channel activity studied with cbv1 vs. cbv1CRAC4T450F suggests that CRAC4 does not contribute to CLR modulation of ethanol action on slo1 at high [Ca2+]i. Support: R01AA023764 (AB), R37AA11560 (AD).

The neuroprotective effects of Tao-Ren-Cheng-Qi Tang against embolic stroke in rats

BackgroundCombinations of the traditional Chinese and Western medicines have been used to treat numerous diseases throughout the world, and there is a growing body of evidence showing that some of the herbs used in traditional Chinese medicine elicit significant pharmacological effects. The aim of this study was to demonstrate the neuroprotective effects of Tao-Ren-Cheng-Qi Tang (TRCQT) in combination with aspirin following middle cerebral artery occlusion (MCAO)—induced embolic stroke in rats.MethodsA blood clot was embolized into the middle cerebral artery of rats to induce focal ischemic brain injury. After 24 h of MCAO occlusion, the rats were arbitrarily separated into five groups and subjected to different oral treatment processes with TRCQT and aspirin for 30 days before being evaluated in terms of their neurological behavior using a four-point system. The rats were sacrificed at 30 days after drug treatment and the infarct volumes were measured using a 2,3,5-triphenyltetrazolium chloride staining method. Tumor necrosis factor-α (TNF-α), c-Jun N-terminal kinases (JNK), activated caspase-3 and Bax were detected by western blot analysis. The apoptotic cells were identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. ROS generation was also measured by electron spin resonance spectrometry.ResultsRats treated with TRCQT alone or in combination with aspirin showed a significantly reduced infarct volume (P < 0.001) and improved neurological outcome compared with those treated with distilled water. Rats treated with TRCQT alone (P = 0.021) or in combination with aspirin (P = 0.02) also showed significantly reduced MCAO-induced expression levels of TNF-α and pJNK (P < 0.001) in their ischemic regions. Rats treated with TRCQT alone or in combination with aspirin showed decreased apoptosis by a reduction in the number of TUNEL positive cells, which inhibited the expression of activated caspase-3 (P = 0.038) and Bax (P = 0.004; P = 0.003). TRCQT also led to a significant concentration-dependent reduction in the formation of hydroxyl radicals (P < 0.001).ConclusionsTRCQT reduced brain infarct volume and improved neurological outcomes by reducing apoptosis, attenuating the expression of TNF-α and p-JNK, and reducing the formation of hydroxyl radicals in MCAO-induced embolic stroke of rats.

The experimental strategies in the study of ischemic stroke

Literature data and own experience in the studies of experimental stroke methodology are reviewed. Advantages and disadvantages of the common models of focal ischemia used in the laboratory practice are discussed in details. The advantages of the filament occlusion of the middle cerebral artery in rats as the most adequate model of human stroke are substantiated. The authors suggest a modification of this variant using an additional coagulation of the pterygopalatine artery that allows the exclusion of the retrograde and collateral blood flow into the inner carotid artery after ligation of the common and external carotid arteries.

Interplay among distinct Ca2+ conductances drives Ca2+ sparks/spontaneous transient outward currents in rat cerebral arteries.

Distinct Ca2+ channels work in a coordinated manner to grade Ca2+ spark/spontaneous transient outward currents (STOCs) in rat cerebral arteries. The relative contribution of each Ca2+ channel to Ca2+ spark/STOC production depends upon their biophysical properties and the resting membrane potential of smooth muscle. Na+ /Ca2+ exchanger, but not TRP channels, can also facilitate STOC production. Ca2+ sparks are generated in a voltage-dependent manner to initiate spontaneous transient outward currents (STOCs), events that moderate arterial constriction. In this study, we defined the mechanisms by which membrane depolarization increases Ca2+ sparks and subsequent STOC production. Using perforated patch clamp electrophysiology and rat cerebral arterial myocytes, we monitored STOCs in the presence and absence of agents that modulate Ca2+ entry. Beginning with CaV 3.2 channel inhibition, Ni2+ was shown to decrease STOC frequency in cells held at hyperpolarized (-40mV) but not depolarized (-20mV) voltages. In contrast, nifedipine, a CaV 1.2 inhibitor, markedly suppressed STOC frequency at -20mV but not -40mV. These findings aligned with the voltage-dependent profiles of L- and T-type Ca2+ channels. Furthermore, computational and experimental observations illustrated that Ca2+ spark production is intimately tied to the activity of both conductances. Intriguingly, this study observed residual STOC production at depolarized voltages that was independent of CaV 1.2 and CaV 3.2. This residual component was insensitive to TRPV4 channel modulation and was abolished by Na+ /Ca2+ exchanger blockade. In summary, our work highlights that the voltage-dependent triggering of Ca2+ sparks/STOCs is not tied to a single conductance but rather reflects an interplay among multiple Ca2+ permeable pores with distinct electrophysiological properties. This integrated orchestration enables smooth muscle to grade Ca2+ spark/STOC production and thus precisely tune negative electrical feedback.

The expression of bitter taste receptors in mesenteric, cerebral and omental arteries

AimBitter taste is sensed by the bitter taste receptor (TAS2R), which is mainly expressed in the tongue as well as in extra-oral organs, such as the gastrointestinal tract, respiratory tract, brain, heart and testis. This study aimed to investigate whether TAS2R is expressed in the mesenteric, cerebral and omental arteries. Main methodsThe expression levels of TAS2R mRNA and protein were determined by reverse-transcription polymerase chain reaction and Western blotting, respectively. The location of TAS2R was determined by immunofluorescence imaging. TAS2R agonists were used in a sensitive myograph to study the function of TAS2R in arteries. Key findingsThe mRNA of rat TAS2Rs, including rTAS2R39, rTAS2R40, rTAS2R108, rTAS2R114, rTAS2R130, rTAS2R137, and rTAS2R140, was expressed in rat mesenteric and cerebral arteries, but rTAS2R114 was not expressed in the cerebral arteries. The mRNA of human TAS2Rs, including hTAS2R3, hTAS2R4, hTAS2R7, hTAS2R10, hTAS2R14, hTAS2R39 and hTAS2R40, was expressed in human omental arteries. The TAS2R7 protein was expressed in rat mesenteric and cerebral arteries, as well as in human omental arteries. Immunofluorescence imaging confirmed that TAS2R7 was located in vascular smooth muscle cells and endothelial cells. The TAS2R agonists, chloroquine and quinine relaxed rat mesenteric arteries and cerebral arteries and human omental arteries in a concentration-dependent manner. SignificanceTAS2R is expressed in the arteries of systemic circulation, including rat mesenteric and cerebral arteries and human omental arteries. This study provides evidence that TAS2R do exist in the arteries and may be involved in the mediation of vessel functions.

Alcohol modulation of BK channel gating depends on β subunit composition.

In most mammalian tissues, Ca2+i/voltage-gated, large conductance K+ (BK) channels consist of channel-forming slo1 and auxiliary (β1-β4) subunits. When Ca2+i (3-20 µM) reaches the vicinity of BK channels and increases their activity at physiological voltages, β1- and β4-containing BK channels are, respectively, inhibited and potentiated by intoxicating levels of ethanol (50 mM). Previous studies using different slo1s, lipid environments, and Ca2+i concentrations-all determinants of the BK response to ethanol-made it impossible to determine the specific contribution of β subunits to ethanol action on BK activity. Furthermore, these studies measured ethanol action on ionic current under a limited range of stimuli, rendering no information on the gating processes targeted by alcohol and their regulation by βs. Here, we used identical experimental conditions to obtain single-channel and macroscopic currents of the same slo1 channel ("cbv1" from rat cerebral artery myocytes) in the presence and absence of 50 mM ethanol. First, we assessed the role five different β subunits (1,2,2-IR, 3-variant d, and 4) in ethanol action on channel function. Thus, two phenotypes were identified: (1) ethanol potentiated cbv1-, cbv1+β3-, and cbv1+β4-mediated currents at low Ca2+i while inhibiting current at high Ca2+i, the potentiation-inhibition crossover occurring at 20 µM Ca2+i; (2) for cbv1+β1, cbv1+wt β2, and cbv1+β2-IR, this crossover was shifted to ∼3 µM Ca2+i Second, applying Horrigan-Aldrich gating analysis on both phenotypes, we show that ethanol fails to modify intrinsic gating and the voltage-dependent parameters under examination. For cbv1, however, ethanol (a) drastically increases the channel's apparent Ca2+ affinity (nine-times decrease in Kd) and (b) very mildly decreases allosteric coupling between Ca2+ binding and channel opening (C). The decreased Kd leads to increased channel activity. For cbv1+β1, ethanol (a) also decreases Kd, yet this decrease (two times) is much smaller than that of cbv1; (b) reduces C; and (c) decreases coupling between Ca2+ binding and voltage sensing (parameter E). Decreased allosteric coupling leads to diminished BK activity. Thus, we have identified critical gating modifications that lead to the differential actions of ethanol on slo1 with and without different β subunits.

Peroxynitrite formed during a transient episode of brain ischaemia increases endothelium‐derived hyperpolarization‐type dilations in thromboxane/prostaglandin receptor‐stimulated rat cerebral arteries

Increased thromboxane A2 and peroxynitrite are hallmarks of cerebral ischaemia/reperfusion (I/R). Stimulation of thromboxane/prostaglandin receptors (TP) attenuates endothelium-derived hyperpolarization (EDH). We investigated whether EDH-type middle cerebral artery (MCA) relaxations following TP stimulation are altered after I/R and the influence of peroxynitrite. Vascular function was determined by wire myography after TP stimulation with the thromboxane A2 mimetic 9,11-dideoxy-9α, 11α -methano-epoxy prostaglandin F2α (U46619) in MCA of Sprague Dawley rats subjected to MCA occlusion (90min)/reperfusion (24h) or sham operation, and in non-operated (control) rats. Some rats were treated with saline or the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) (20mgkg-1 ). Protein expression was evaluated in MCA and in human microvascular endothelial cells submitted to hypoxia (overnight)/reoxygenation (24h) (H/R) using immunofluorescence and immunoblotting. In U46619-pre-constricted MCA, EDH-type relaxation by the proteinase-activated receptor 2 agonist serine-leucine-isoleucine-glycine-arginine-leucine-NH2 (SLIGRL) was greater in I/R than sham rats due to an increased contribution of small-conductance calcium-activated potassium channels (SKCa ), which was confirmed by the enlarged relaxation to the SKCa activator N-cyclohexyl-N-2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine. I/R and H/R induced endothelial protein tyrosine nitration and filamentous-actin disruption. In control MCA, either cytochalasin D or peroxynitrite disrupted endothelial filamentous-actin and augmented EDH-type relaxation. Furthermore, peroxynitrite decomposition during I/R prevented the increase in EDH-type responses. Following TP stimulation in MCA, EDH-type relaxation to SLIGRL is greater after I/R due to endothelial filamentous-actin disruption by peroxynitrite, which prevents TP-induced block of SKCa input to EDH. These results reveal a novel mechanism whereby peroxynitrite could promote post-ischaemic brain injury.

Involvement of Hydrogen Sulfide in Endothelium-Derived Relaxing Factor-Mediated Responses in Rat Cerebral Arteries

Background/Aim: H₂S is a novel vasoactivator. To verify the hypothesis that H₂S may act as an endothelium-derived hyperpolarizing factor (EDHF) in the rat cerebrovasculature, the role of H₂S in endothelium-derived relaxing factor (EDRF)-mediated responses was investigated. Methods: Cystathionine-γ-lyase (CSE) was knocked down with an siRNA technique. Artery diameter, hyperpolarization and Ca²⁺-activated K⁺ (K_Ca) current were measured. Results: CSE knockdown was indicated by a decrease in protein and mRNA expression in the rat middle cerebral artery (MCA) and cerebral basilar artery (CBA). Acetylcholine (ACh) induced significant hyperpolarization and vasodilation in endothelium-intact MCA and CBA. Removal of the endothelium abolished these responses. The nitric oxide (NO) synthase inhibitor <smlcap>L</smlcap>-NAME, but not the PGI₂ production inhibitor indomethacin, significantly inhibited ACh-induced hyperpolarization and vasodilation in the CBA. In the presence of <smlcap>L</smlcap>-NAME and indomethacin, ACh-induced hyperpolarization and vasodilation in the MCA and CBA were attenuated. The non-NO/PGI₂-mediated responses were abolished by the K_Ca channel blockers charybdotoxin and apamin. In the cerebral arteries from the CSE knockdown rat, non-NO/PGI₂-mediated responses were significantly attenuated, and the remaining responses were abolished by charybdotoxin and apamin or the CSE inhibitor propargylglycine. CSE knockdown did not affect <smlcap>L</smlcap>-NAME-sensitive responses in the CBA. Sodium hydrosulfide (NaHS) augmented the K_Ca current in CBA vascular smooth muscle cells. Conclusion: EDHF-mediated responses in rat cerebral arteries were due to H₂S activating the K_Ca channel.

Diving Response in Rats: Role of the Subthalamic Vasodilator Area.

Diving response (DR) is a powerful integrative response targeted toward survival of the hypoxic/anoxic conditions. Being present in all animals and humans, it allows to survive adverse conditions like diving. Earlier, we discovered that forehead stimulation affords neuroprotective effect, decreasing infarction volume triggered by permanent occlusion of the middle cerebral artery in rats. We hypothesized that cold stimulation of the forehead induces DR in rats, which, in turn, exerts neuroprotection. We compared autonomic [AP, heart rate (HR), cerebral blood flow (CBF)] and EEG responses to the known DR-triggering stimulus, ammonia stimulation of the nasal mucosa, cold stimulation of the forehead, and cold stimulation of the glabrous skin of the tail base in anesthetized rats. Responses in AP, HR, CBF, and EEG to cold stimulation of the forehead and ammonia vapors instillation into the nasal cavity were comparable and differed significantly from responses to the cold stimulation of the tail base. Excitotoxic lesion of the subthalamic vasodilator area (SVA), which is known to participate in CBF regulation and to afford neuroprotection upon excitation, failed to affect autonomic components of the DR evoked by forehead cold stimulation or nasal mucosa ammonia stimulation. We conclude that cold stimulation of the forehead triggers physiological response comparable to the response evoked by ammonia vapor instillation into nasal cavity, which is considered as stimulus triggering protective DR. These observations may explain the neuroprotective effect of the forehead stimulation. Data demonstrate that SVA does not directly participate in the autonomic adjustments accompanying DR; however, it is involved in diving-evoked modulation of EEG. We suggest that forehead stimulation can be employed as a stimulus capable of triggering oxygen-conserving DR and can be used for neuroprotective therapy.

PPARγ activation attenuates oxidative stress and improves cerebral arteries endothelial dysfunction in aging rat through up-regulating UCP2

Objectives: Increasing amounts of evidence implicate oxidative stress as having a pivotal role in age-related cerebrovascular dysfunction, which is an important risk factor for the development of cerebrovascular disease. Previous studies have shown that the activation of the expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) in vascular endothelial cells results in an improvement of vascular function. Pioglitazone, a well-known PPAR-γ agonist, protects against oxidative stress in the rostral ventrolateral medulla by the upregulation of mitochondrial uncoupling protein 2 (UCP2).In this study, we sought to explore the effects and the underlying mechanisms of pioglitazone on age-related oxidative stress elevation and cerebrovascular dysfunction in aging rat cerebral arteries.

The influence of oxygen tension on the local regulation of blood flow by shear stress

Introduction and purpose. Vascular response to mechanical stimuli, namely transmural pressure (Bayliss effect) and wall shear stress (response to blood flow), play an important role in regulation of vascular tone. The purpose of the work was to study an influence of hypoxia on the vessel radius and blood flow control by response to shear stress. Methodology/approach. Mathematical simulation was used. The model is based on published data of experiments on small cerebral arteries of rats. The main assumptions of the model are: 1) the vessel is a thin wall cylinder; 2) the radius is controlled by two parameters: concentration of free calcium ions in the cytoplasm of the smooth muscle cells and concentration of nitric oxide (NO) in the smooth muscle layer; 3) the rate of NO production by endothelium is proportional to modulus of shear stress on the vessel wall. The apparent blood viscosity is calculated using the solution of the problem of two-layer flow. The numerical experiments were performed in Turbo Pascal. The main results and discussion. The dependence of vessel tone regulation by response to altered shear stress on oxygen tension is caused by dependence of NO synthesis in endothelium and NO consumption on oxygen concentration. As it follows from mathematical simulation, hypoxia reduces the role of mechanogenic regulation, and the increase of the wall sensitivity to NO makes this effect more appreciable. Calculations performed for typical value of cerebral vessel response to shear stress, show that the fall in oxygen tension from 100 to 30 per cent leads to decrease in diameter by 6 %, in blood flow rate by 11 %. The rheological factors prevent flow rate diminution, but their contribution is very small: less than 3 %. The fall in oxygen tension reduces NO production rate by endothelial cells and NO concentration in the vessel wall. At strong hypoxia (reduction in oxygen tension from 100 to 30 % and less) NO concentration in smooth muscle layer drops by more than 15 %. Conclusions. Hypoxia decreases NO-dependent vessel response to altered shear rate. This effect increases with the value of vessel response to shear stress. The rheological factors impede the decrease of this response.

The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries

Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ETA receptor mRNA and protein levels as well as contractile responses mediated by ETA receptors. The immunoreactivity of increased ETA receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ETB receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ETA receptor upregulation in rat cerebral arteries.

Epidermal growth factor-like repeats of tenascin-C-induced constriction of cerebral arteries via activation of epidermal growth factor receptors in rats

Tenascin-C (TNC), one of matricellular proteins, has been suggested to be involved in cerebral vasospasm after aneurysmal subarachnoid hemorrhage. However, the mechanisms of how TNC constricts cerebral arteries remain unclear. The aim of this study was to examine if epidermal growth factor (EGF)-like repeats of TNC is involved in TNC-induced constriction of cerebral arteries in rats via EGF receptor (EGFR) activation. Two dosages of recombinant TNC (r-TNC) consisting of the EGF-like repeats was administered intracisternally to healthy rats, and its vasoconstrictor effects were evaluated by neurobehavioral tests and India-ink angiography at 24, 48, and 72 hours after the administration. Western blotting and immunohistochemistry were performed to explore the underlying mechanisms on constricted cerebral arteries after 24 hours. The effects of a selective EGFR tyrosine kinase inhibitor (AG1478) on r-TNC-induced vasoconstriction were evaluated by neurobehavioral tests, India-ink angiography and immunohistochemistry at 24 hours after the administration. A higher dosage of r-TNC induced cerebral arterial constriction more severely, which continued for 48 hours. The effects were associated with the activation of EGFR and extracellular signal-regulated kinase (ERK)1/2 in the smooth muscle cell layer of the constricted cerebral artery, while c-Jun N-terminal kinase and p38 were not activated. AG1478 blocked r-TNC-induced vasoconstrictive effects, as well as activation of EGFR and ERK1/2. These findings demonstrate that TNC induces constriction of cerebral arteries via activation of EGFR and ERK1/2.