Canine Basilar Artery Research Articles

Von Willebrand factor targeted thrombolysis in canine basilar artery occlusion.

Posterior circulation strokes, accounting for 20% of acute ischemic strokes, significantly contribute to morbidity and mortality. Fibrinolysis by rtPA improves outcomes in stroke but the risk of intracranial hemorrhage limits benefit. Arterial recanalization of basilar artery occlusion by thrombolysis or endovascular thrombectomy improves outcomes in posterior circulation strokes. This study investigates a VWF-targeting RNA aptamer as a safer and more effective alternative to rtPA in a canine model. Autologous clots were placed into the basilar artery to induce stroke in 24 beagles. To compare reperfusion, 0.9 mg/kg rtPA, 0.5 mg/kg BB-031, or vehicle were administered 60 min after the initiation of occlusion. Digital subtraction angiography, laser speckle imaging and magnetic resonance imaging were used to assess recanalization, reperfusion and infarct volume, respectively. Treatment with BB-031 resulted in recanalization of the posterior circulation on digital subtraction angiography with no evidence of microembolism assessed at sacrifice. 66.5% of animals treated with BB-031 resulted in reperfusion with a TICI score of ≥1 whereas vehicle remained at TICI score 0 as did all but one rtPA animal at sacrifice. Improved perfusion was seen in the basilar artery and surrounding blood vessels visualized through the cranial window with laser speckle imaging to ~47% of its original baseline in BB-031 group compared to rtPA at 37% and vehicle at 22%. Finally, BB-031-treatment resulted in an approximate 32% mean infarct volume, significantly smaller on magnetic resonance imaging compared to 56% in vehicle treated and 48% with rtPA treatment. Targeted inhibition of VWF by BB-031 increased recanalization and reperfusion, and reduced infarct volume in a canine model of BAO stroke. It represents a promising target based on preliminary results for treating acute ischemic stroke.

Comment on, “Gene expression in a canine basilar artery vasospasm model: a genome-wide network-based analysis”

Comment on, “Gene expression in a canine basilar artery vasospasm model: a genome-wide network-based analysis”

Comprehensive Assessment of Drug Kinetics, Neurotoxicity, and Safety of Sirolimus-Eluting Intracranial Stents in Canine Basilar Artery.

Sirolimus-eluting stents (SESs) have shown promise in treating intracranial atherosclerosis but concerns about potential neurotoxicity due to prolonged drug release exist. The aim of this study was to comprehensively assess the safety of SES, with a focus on neurotoxicity. Stents (1.50 × 7 or 12 mm) were implanted into the basilar arteries of 154 Labrador Retrievers (weighing >25 kg and aged older than 1 year) divided into 4 groups: baer-metal stent, polymer-coated stent, standard-dose SES (sirolimus dose: 71 μg), and high-dose SES group (sirolimus dose: 284 μg). Pharmacokinetic analysis was conducted using liquid chromatography-mass spectrometry on blood and tissue samples, and analysis of brain tissue was performed with 5 different special stains and immunohistochemistry protocols to assess axonal degeneration, vacuolization, astrocyte proliferation, microglial activation, or widespread neurodegeneration. In the standard-dose SES group, the stent released 10.56% of the drug on day 1 and 95.41% on day 28 postimplantation. In the high-dose SES group, corresponding figures were 40.20% on day 1 and 98.08% on day 28. Systemic drug concentration consistently remained below 1.5 ng/mL throughout the study. Arterial tissue concentration reached its peak at day 28 days in the standard-dose group and at 7 days in the high-dose group. Importantly, the brain and related tissue concentrations remained below 0.4 µg/g in both standard-dose and high-dose SES groups, peaking on day 21 in the standard-dose group and day 1 in the high-dose group. The detailed 180-day safety assessment revealed no adverse effects on the brain, even at high sirolimus doses in the SES group. This study provides robust evidence supporting the long-term pharmacokinetic safety of SESs in the context of intracranial interventions for high-grade intracranial atherosclerosis. The results adequately alleviate concerns related to neurotoxicity and substantiate the feasibility of using these stents as a therapeutic choice in neurosurgery.

Pharmacological characterization of the mechanisms involved in the vasorelaxation induced by progesterone and 17β‐estradiol on isolated canine basilar and carotid arteries

Pharmacological characterization of the mechanisms involved in the vasorelaxation induced by progesterone and 17β‐estradiol on isolated canine basilar and carotid arteries

Pharmacological characterization of the mechanisms involved in the vasorelaxation induced by progesterone and 17β-estradiol on isolated canine basilar and internal carotid arteries

Progesterone and 17β-estradiol induce vasorelaxation through non-genomic mechanisms in several isolated blood vessels; however, no study has systematically evaluated the mechanisms involved in the relaxation induced by 17β-estradiol and progesterone in the canine basilar and internal carotid arteries that play a key role in cerebral circulation. Thus, relaxant effects of progesterone and 17β-estradiol on KCl- and/or PGF2α-pre-contracted arterial rings were investigated in absence or presence of several antagonists/inhibitors/blockers; the effect on the contractile responses to CaCl2 was also determined. In both arteries progesterone (5.6–180μM) and 17β-estradiol (1.8–180μM): (1) produced concentration-dependent relaxations of KCl- or PGF2α-pre-contracted arterial rings; (2) the relaxations were unaffected by actinomycin D (10μM), cycloheximide (10μM), SQ 22,536 (100μM) or ODQ (30μM), potassium channel blockers and ICI 182,780 (only for 17β-estradiol). In the basilar artery the vasorelaxation induced by 17β-estradiol was slightly blocked by tetraethylammonium (10mM) and glibenclamide (KATP; 10μM). In both arteries, progesterone (10–100μM), 17β-estradiol (3.1–31μM) and nifedipine (0.01–1μM) produced a concentration-dependent blockade of the contraction to CaCl2 (10μM–10mM). These results suggest that progesterone and 17β-estradiol produced relaxation in the basilar and internal carotid arteries by blockade of L-type voltage dependent Ca2+ channel but not by genomic mechanisms or production of cAMP/cGMP. Potassium channels did not play a role in the relaxation to progesterone in both arteries or in the effect of 17β-estradiol in the internal carotid artery; meanwhile KATP channels play a minor role on the effect of 17β-estradiol in the basilar artery.

Abstract 139: Preclinical In-vivo Safety Evaluation Of Thrombectomy Devices In The Canine Cerebrovasculature

Introduction: Thrombectomy devices have become an integral tool in the armamentarium of neuro-interventionalists to treat acute ischemic stroke (AIS). Previous preclinical evaluation of these devices has not assessed their safety in the native cerebrovasculature. We assessed the operation of stent-trievers in the canine basilar artery (BA), with a goal to evaluate their vascular safety on angiography and histopathology. Methods: Four mongrel canines (female, mean weight:12.53kg,n=2 each arm) were randomly assigned to receive the Solitaire FR Revascularization device (4mm X 22mm;eV3, Plymouth, MN) or the Enterprise VRD (4.5mm X 22mm ;Codman Neurovascular, Raynham, MA). The canine basilar artery (BA) was accessed through the anterior spinal artery (ASA, control vessel). The device was deployed in the basilar artery and withdrawn in the expanded state to the vertebrobasilar junction. The ASA, in which only the microcatheter was deployed, served as the control. Angiographic scoring of the vasospasm and histopathological assessment using a quantitative grading of the scanning electron microscopy (SEM) and cross-sectional hematoxylin and eosin histology was used to assess for the vascular integrity. Results: The Enterprise VRD and the Solitaire performed equally with grade 3 vasospasm on the first pass, which quickly resolved with intra-arterial Nicardipine administration and minimal vasospasm on subsequent passes. Based on the qualitative histopathological and SEM assessment the Solitaire and the Enterprise VRD device performed comparably when tracked through the canine basilar artery. Based on the luminal surface assessment from the SEM, the most likely damage expected from an intervention with either of these devices is expected to be partial damage to the internal elastic lamina and an exposure of the smooth muscle cells. Microcatheter navigation in the control vessels induced focal endothelial injury along the path in which the catheter was tracked. Conclusion: In an attempt to emergently re-vascularize the ischemic vascular territory, the vulnerability of the cerebrovasculature to manipulation by thrombectomy devices is often overlooked. Deployment of stent-trievers induces intimal damage that does not impact vessel integrity.

Pharmacological evidence that Ca 2+ channels and, to a lesser extent, K + channels mediate the relaxation of testosterone in the canine basilar artery

Testosterone induces vasorelaxation through non-genomic mechanisms in several isolated blood vessels, but no study has reported its effects on the canine basilar artery, an important artery implicated in cerebral vasospasm. Hence, this study has investigated the mechanisms involved in testosterone-induced relaxation of the canine basilar artery. For this purpose, the vasorelaxant effects of testosterone were evaluated in KCl- and/or PGF 2α-precontracted arterial rings in vitro in the absence or presence of several antagonists/inhibitors/blockers; the effect of testosterone on the contractile responses to CaCl 2 was also determined. Testosterone (10–180 μM) produced concentration-dependent relaxations of KCl- or PGF 2α-precontracted arterial rings which were: (i) unaffected by flutamide (10 μM), dl-aminoglutethimide (10 μM), actinomycin D (10 μM), cycloheximide (10 μM), SQ 22,536 (100 μM) or ODQ (30 μM); and (ii) significantly attenuated by the blockers 4-aminopyridine (K V; 1 mM), BaCl 2 (K IR; 30 μM), iberiotoxin ( B K C a 2 + ; 20 nM), but not by glybenclamide (K ATP; 10 μM). In addition, testosterone (31, 56 and 180 μM) and nifedipine (0.01–1 μM) produced a concentration-dependent blockade of the contraction to CaCl 2 (10 μM to 10 mM) in arterial rings depolarized by 60 mM KCl. These results, taken together, show that testosterone relaxes the canine basilar artery mainly by blockade of voltage-dependent Ca 2+ channels and, to a lesser extent, by activation of K + channels (K IR, K V and B K C a 2 + ). This effect does not involve genomic mechanisms, production of cAMP/cGMP or the conversion of testosterone to 17β-estradiol.

Interactive Role of Protein Phosphatase 2A and Protein Kinase Cα in the Stretch-Induced Triphosphorylation of Myosin Light Chain in Canine Cerebral Artery

The interactive role of protein kinase C (PKC) isoforms and protein phosphatase 2A (PP2A) in the mechanisms underlying the gradual reduction in stretch-induced contraction through triphosphorylation of 20-kDa myosin light chain (MLC₂₀) was investigated in the canine basilar artery. In the presence of 5 mM tetraethylammonium, stretching at a rate of 1 mm/s from the initial length (L_i) to 1.5 L_i produced a contraction. Maintaining the stretched state for 15 min (15-min stretch) produced triphosphorylation of MLC₂₀at Ser-19, Thr-18 and Thr-9, and a gradual reduction in the contraction, both of which were reversed by Gö6976 (1 µM), an inhibitor of conventional PKC. The 15-min stretch increased PKCα activity whereas it decreased PP2A activity, both of which were blocked by Y-27632, an inhibitor of rho kinase. Okadaic acid (OA; 1 µM), a PP2A inhibitor, also produced triphosphorylation of MLC₂₀ at the same amino acid residues and activated PKCα, which was inhibited by Gö6976. Stretching and OA increased phosphorylation of 17-kDa PKC-potentiated inhibitory phosphoprotein (CPI-17), and this phosphorylation was inhibited by Gö6976. The present results suggest that activation of PKCα mediated by an inhibitor of PP2A is involved in the stretch-induced triphosphorylation of MLC₂₀, and that this triphosphorylation counteracts the stretch-induced contraction.

The relaxant effect of okadaic acid on canine basilar artery involves activation of PKCα and phosphorylation of the myosin light chain at Thr-9

Vasodilator responses induced by okadaic acid were investigated in canine basilar artery precontracted with 80 mM KCl. Okadaic acid (1 µM) relaxed the artery and this relaxant effect was partially inhibited by Gö6976, a conventional protein kinase C inhibitor, and calphostin C, an inhibitor of conventional and novel PKCs. Rottlerin, a specific inhibitor of PKCδ, did not influence okadaic acid's effect. KCl increased phosphorylation of 20,000-Dalton myosin light chain (MLC 20) at Ser-19. Okadaic acid additionally increased MLC 20 phosphorylation at Thr-18 and Thr-9, resulting in triphosphorylation of MLC 20. This phosphorylation was inhibited by Gö6976. Okadaic acid stimulated phosphorylation of PKCα and 17,000-Dalton PKC-potentiated inhibitory phosphoprotein (CPI-17), and Gö6976 inhibited these phosphorylations. These results suggest that okadaic acid's relaxant effect involves MLC 20 triphosphorylation through a direct phosphorylation by PKCα and an indirect phosphorylation by inhibition of myosin light chain phosphatase through PKCα-mediated CPI-17 phosphorylation.

Inhibitory Effects of Eicosapentaenoic Acid on Chronic Cerebral Vasospasm after Subarachnoid Hemorrhage: Possible Involvement of a Sphingosylphosphorylcholine-Rho-Kinase Pathway

Background and Purpose: Rho-kinase (ROK)-mediated Ca²⁺ sensitization of vascular smooth muscle (VSM) contraction plays a pivotal role in cerebral vasospasm (CV). We previously demonstrated that sphingosylphosphorylcholine (SPC) induces Ca²⁺ sensitization through sequential activation of the Src family protein tyrosine kinases (Src-PTKs) and ROK in vitro, and that Ca²⁺ sensitization is inhibited by eicosapentaenoic acid (EPA) through the selective inactivation of Src-PTK. In this study, we examined whether SPC induced CV in vivo, and, if it did, whether EPA would inhibit CV, as induced by SPC or in an in vivo model of subarachnoid hemorrhage (SAH). Methods: Changes in the diameter of the canine basilar artery were investigated by angiography after administering SPC into the cisterna magna. Then, Y27632, a specific Rho-kinase inhibitor, or EPA was injected intracisternally and the effects of both agents were investigated. In another experiment using a single-hemorrhage model, Y27632 or EPA was injected on day 7 after SAH and the changes in the diameter of the canine basilar artery were investigated. Results: At cerebrospinal fluid concentrations of 100 and 300 µmol/l, SPC induced severe vasoconstriction (maximum vasoconstriction by SPC (100 µmol/l): 61.8 ± 8.2%), which was markedly reversed by Y27632 (96.3 ± 4.4%) or EPA (92.6 ± 12.8%). SAH caused severe vasospasm on day 7 (67.6 ± 7.8%), which was significantly blocked by Y27632 (95.5 ± 10.6%) or EPA (90.0 ± 4.4%). Conclusions: SPC is a novel mediator of ROK-induced CV in vivo. The inhibition of CV induced by SPC or after SAH by EPA suggests beneficial roles of EPA in the treatment of CV. Our findings are compatible with the notion that the SPC-ROK pathway may be involved in CV.

Gene expression in a canine basilar artery vasospasm model: a genome-wide network-based analysis

To investigate the changes of gene expression on the cerebral vasospasm after subarachnoid hemorrhage, we used genome-wide microarray for a canine double-hemorrhage model and analyzed the data by using a network-based analysis. Six dogs were assigned to two groups of three animals: control and hemorrhage. The effects were assessed by the changes in gene expressions in the artery 7 days after the first blood injection. Among 23,914 genes, 447 and 66 genes were up-regulated more than two- and fivefold, respectively, and 332 and 25 genes were down-regulated more than two- and fivefold, respectively. According to gene ontology, genes related to cell communication (P = 5.28E-10), host–pathogen interaction (7.65E-8), and defense–immunity protein activity (0.000183) were significantly overrepresented. The top high-level function for the merged network derived from the network-based analysis was cell signaling, revealing that the subgroup that regulates the quantity of Ca2+ to have the strongest association significance (P = 4.75E-16). Canine microarray analysis followed by gene ontology profiling and connectivity analysis identified several functional groups and individual genes responding to cerebral vasospasm. Ca2+ regulation may play a key role in these gene expression changes and may be involved in the pathogenesis of cerebral vasospasm.

Vasorelaxing effect of the Rho-kinase inhibitor, Y-27632, in isolated canine basilar arteries

Objectives: Increased calcium sensitization mediated by Rho/Rho-kinase may be important in the pathogenesis of cerebral vasospasm. The effects of a highly selective Rho-kinase inhibitor, Y-27632, were investigated on spasmogen-induced contractions of canine basilar artery.Methods: Typical spasmogenic substances present after subarachnoid hemorrhage (SAH), including prostaglandin F2a (PGF2a), 12-deoxyphorbol 13-isobutyrate (DPB), sphingosylpho-sphorylcholine (SPC) and high K+, were used in the study. Isometric tension was recorded in canine basilar artery rings in vitro. Intracellular calcium concentration ([Ca2+]i) and contraction force were measured simultaneously in fura-2-loaded canine basilar artery strips. The myosin light chain (MLC) phosphorylation levels were measured by glycerol gel electrophoresis followed by Western blotting.Results: Isometric tension recording revealed that the Rho-kinase inhibitor, Y-27632, dose-dependently inhibited vasocontraction induced by PGF2a and SPC, but not that induced by DPB. Simultaneous recordings of [Ca2+]i and tension revealed that the vasorelaxing effect of Y-27632 was not associated with changes in [Ca2+]i, suggesting that Y-27632 may inhibit calcium sensitization. Vasocontraction induced by DPB was not inhibited by Y-27632, but was inhibited by staurosporine. Phosphorylation of MLC was increased by PGF2a and SPC, and significantly inhibited by Y-27632, whereas such phosphorylation was increased by DPB, but not significantly inhibited by Y-27632.Discussion: Several spasmogenic mediators released after SAH may cause vasospasm through Rho-kinase-mediated increase in calcium sensitization. Rho-kinase inhibitors, including Y-27632, may be effective for the prevention of cerebral vasospasm after SAH.

Safety and Pharmacokinetics of Sirolimus-eluting Stents in the Canine Cerebral Vasculature: 180 Day Assessment

We evaluated local and systemic pharmacokinetics and pharmacodynamics of sirolimus-eluting stents (SES) in canine cerebral vessels. SES (1.5 x 8 mm, 79 microg/479 microg sirolimus) and control stents (1.5 x 8 mm stainless steel with or without polymer) were implanted in canine basilar and ventral spinal arteries. Animals were sacrificed for local pharmacokinetic (36 animals at 1, 3, 8, 30, 90, 180 days) and pharmacodynamic (60 animals at 3, 30, 90, 180 days) assessment. Postrecovery adverse clinical events were not serious, requiring no unscheduled treatment. Histologically, brain and spinal cord sections revealed scattered microinfarcts and minimal gliosis consistent with postprocedure changes in all four stent-treatment groups. All stented vessels at all time points demonstrated good luminal patency with low injury and inflammation scores and no thrombosis of either stented or branch arteries. Endothelialization was complete in all stent groups by 30 days. Intimal smooth muscle cell scores were reduced in both SES groups at 30, 90, and 180 days. Systemic sirolimus levels peaked between 1 and 7 hours postimplant (maximum concentration, 1.2 +/- 1.47, 79 microg; 4.5 +/- 1.23 ng/ml, 479 microg), then declined rapidly to 1 ng/ml or less by 96 hours. Peak local tissue sirolimus levels were 41.5 ng/mg (79 microg) and 65 ng/mg (479 microg). SES in canine cerebral vessels were associated with good luminal patency to 180 days, with complete endothelialization and no evidence of acute thrombosis. This model has shown that SES deployed within the brain do not cause neurotoxicity during a 180-day time course, even when exaggerated doses are used. The findings support the contention that SES are safe to use and maintain patency in cerebral vessels.

Stretch-induced triphosphorylation of myosin light chain and myogenic tone in canine basilar artery

The relationship between phosphorylation of 20,000 Da myosin light chain (MLC 20) and contraction in response to mechanical stretch was investigated in the canine basilar artery. A slow stretch (at a rate of 1 mm/s and a stimulus period for 15 min) increased triphosphorylated MLC 20 despite lowered intracellular calcium concentration and mechanical activities, such as myogenic tone, shortening velocity and stiffness of the artery. Nicardipine, a Ca 2+ channel blocker, and ML-9, a myosin light chain kinase (MLCK) inhibitor, partially inhibited the stretch-induced MLC 20 phosphorylation. The remained phosphorylation was further reduced by calphostin C, a protein kinase C (PKC) inhibitor. Y-27632, a Rho-kinase inhibitor, inhibited phosphorylation of myosin light chain phosphatase and attenuated MLC 20 phosphorylation. These results suggest that slow stretch induces triphosphorylation of MLC 20, which is mediated by MLCK, PKC, and Rho-kinase, and that the triphosphorylation of MLC 20 does not result in myogenic contraction, rather seems to counteract it.

Stretch-induced triphosphorylation of myosin light chain despite low myogenic tone in canine basilar artery

Stretch-induced triphosphorylation of myosin light chain despite low myogenic tone in canine basilar artery

Halothane does not protect against vascular injury in isolated cerebral and mesenteric arteries

This study was designed to examine regional differences in the vascular injury induced by hydrogen peroxide (H2O2) and to determine its modulation by halothane in canine basilar and mesenteric arteries.Rings of canine basilar and mesenteric arteries with intact endothelium were mounted in Krebs bicarbonate solution for isometric tension recording. The relaxation responses to substance P (10(-8) M) and sodium nitroprusside (SNP; 10(-8) to 10(-5) M) were examined before and after exposure to H2O2 (1 mM) for eight minutes in the presence or absence of halothane (2%), to evaluate the effects of oxidative injury on the endothelium-dependent and -independent relaxation. The contractile responses to KCl (30 mM) and prostaglandin (PG) F(2alpha) (3 x 10(-6) M) were also compared in rings with and without exposure to H2O2.After exposure to H2O2 the relaxant responses to substance P were significantly inhibited in basilar arteries (P < 0.01), but not in mesenteric arteries. Exposure to H2O2 also attenuated SNP-induced relaxation in basilar (P < 0.05), but not in mesenteric arteries. The attenuation of the contractile responses to KCl and PGF(2alpha) after H2O2 exposure was observed only in basilar arteries (P < 0.01). Simultaneous exposure to halothane did not affect the attenuation of these relaxant and contractile responses. Scanning electron microscopy revealed that H2O2 resulted in marked disruption of the endothelial layer in basilar arteries, compared to almost no morphological changes in mesenteric arteries.These results indicate that the endothelium and vascular smooth muscle of the basilar artery are more susceptible to oxidative stress than those of the mesenteric artery. Halothane, at clinically relevant concentrations, exerts no significant influence on this vascular injury.

Nicotine Exposure, Mimicked Smoking, Directly and Indirectly Enhanced Protein Kinase C Activity in Isolated Canine Basilar Artery, Resulting in Enhancement of Arterial Contraction

Nicotine Exposure, Mimicked Smoking, Directly and Indirectly Enhanced Protein Kinase C Activity in Isolated Canine Basilar Artery, Resulting in Enhancement of Arterial Contraction

Biomechanical and phenotypic changes in the vasospastic canine basilar artery after subarachnoid hemorrhage

Because it has been argued that active myogenic tone prolongs cerebral vasospasm for >2 wk after subarachnoid hemorrhage (SAH), we attempted to identify the mechanism that plays the main role in sustaining the prolonged cerebral vasospasm. We especially focused on the roles of biomechanical and phenotypic changes in the cerebral arteries in the mechanisms of prolonged vasospasm after SAH. We used the basilar arteries from a "two-hemorrhage" canine model to make serial measurements of maximal contraction capacity and arterial stiffness (papaverine-insensitive tone) until day 28. We also examined hematoxylin-eosin-stained vasospastic canine basilar arteries for histological changes and immunohistochemically examined them for expression of myosin heavy chain isoforms (SMemb, SM1, and SM2), which are markers of smooth muscle phenotypic changes. Changes in collagen concentration in canine basilar arteries were also measured. Angiographic cerebral vasospasm persisted until day 14 and then gradually diminished; artery diameter returned to the control diameters on day 28. Maximal contraction capacity decreased until day 21 and showed some recovery by day 28. Arterial stiffness, on the other hand, progressed until day 28. Histological examination revealed medial thickening and increased connective tissue until day 21 and a return to control findings by day 28. The increased connective tissue was not accompanied by changes in collagen concentration, suggesting a role of some other protein in the increase in connective tissue. Immunohistochemical studies with anti-SMemb, anti-SM1, and anti-SM2 antibodies showed enhanced expression of SMemb from day 7 to day 21 and disappearance of SM1 and SM2 on days 14 and 21. The changes in myosin heavy chain isoform expression returned to normal on day 28. The above results indicate that biomechanical and phenotypic changes may play a pivotal role in sustaining cerebral vasospasm for >2 wk after SAH, with minimal changes in active myogenic arterial tone.

Nicotine Exposure, Mimicked Smoking, Directly and Indirectly Enhanced Protein Kinase C Activity in Isolated Canine Basilar Artery, Resulting in Enhancement of Arterial Contraction

Cigarette smoking is a significant risk factor in the incidence of cerebrovascular disorders. Among the many compounds in cigarette smoke, nicotine is considered to most significantly affect cerebral arterial tone. The purpose of this study is to investigate precise pharmacological effects of nicotine on the regulation of cerebral arterial tone. To mimic smoking, a low concentration of nicotine (10(-6) mol/L), which is equivalent to the serum level of habitual smokers, was treated for 1 hour in an isometric tension study and for 24 hours in a study using cultured vascular endothelial cells (VECs). Using the canine basilar artery, the effect of nicotine on uridine 5'-triphosphate (UTP)-induced vasoconstriction was examined in the isometric tension study. Protein kinase C (PKC) activity in the canine basilar artery was measured by enzyme immunoassay. Endothelial function was assessed by endothelium-dependent vasodilatation and endogenous nitric oxide (NO) synthesis in VECs using a fluorescent indicator, diaminofluorescein-FM diacetate (DAF-FM/DA). Nicotine significantly enhanced UTP-induced contraction and PKC activity in the artery, and attenuated endothelium-dependent vasodilatation and NO synthesis in VECs. Because PKC activity was increased by de-endothelialization itself, endothelial dysfunction by nicotine enhances PKC activity. Because PKC was further activated by nicotine even in the de-endothelialized artery, nicotine directly affects PKC activities in smooth muscle. These results indicate that nicotine potentiates contractile response through direct and indirect PKC activation in the canine basilar artery.

Interactive Role of Protein Kinase C-δ with Rho-Kinase in the Development of Cerebral Vasospasm in a Canine Two-Hemorrhage Model

Background: We previously reported that protein kinase C (PKC)-δ was initially translocated from the cytosol to the membrane fraction (on day 4), followed by PKC-α, with the progression of cerebral vasospasm after subarachnoid hemorrhage (SAH) on day 7. Rho/Rho-kinase pathways have also been proposed to be involved in the vasospasm. Thus we investigated the interactive role of Rho-kinase and PKC in the development of cerebral vasospasm after SAH. Methods: The cerebral vasospasm was produced using a ‘two-hemorrhage’ canine model. The animals were treated with Y-27632, a Rho-kinase inhibitor, and rottlerin, a PKC-δ inhibitor, both injected into the cisterna magna. Results: Y-27632 inhibited the vasospasm, 20-kDa myosin light chain (MLC₂₀) phosphorylation, and PKC-δ translocation after the second injection of autologous blood on day 4. In contrast, Y-27632 did not affect the vasospasm on day 7. Rottlerin also inhibited the vasospasm on day 4, but had no effect on MLC₂₀ phosphorylation and RhoA translocation. The vasospasm was accompanied with the phosphorylation of caldesmon (CaD), an actin-linked regulatory protein, which was strongly attenuated by Y-27632 and rottlerin. The application of PKC-δ to skinned strips of isolated canine basilar arteries caused a contraction and an increase in CaD phosphorylation. Conclusion: The development of cerebral vasospasm after SAH (on day 4) is caused by at least two mechanisms: one involves MLC₂₀ phosphorylation mediated by the inhibition of MLC₂₀ phosphatase by Rho-kinase, and the other CaD phosphorylation mediated by the activation of PKC-δ by Rho-kinase, which results in the alleviation of the inhibition by CaD of myosin Mg²⁺-ATPase activity.