Similar pathological effects of sympathectomy and hypercholesterolemia on arterial smooth muscle cells and fibroblasts.

In a previous study, we showed that after sympathectomy, the femoral (FA) but not the basilar (BA) artery from non-pathological rabbits manifests migration of adventitial fibroblasts (FBs) into the media and loss of medial smooth muscle cells (SMCs). The aim of the present study was to verify whether similar behaviour of arteries occurred in the pathological context of atherosclerosis. Thus, similar experiments were conducted on hypercholesterolemic rabbits, which were chemically sympathectomized with 6-hydroxydopamine (n=4) or treated with vehicle for control (n=5). Cross-sections of BA and FA were immunolabelled for five markers of phenotypic modulation of vascular SMCs and FBs: vimentin, desmin, alpha-smooth muscle actin, beta-isoform of actin, and h-caldesmon and examined using a confocal microscope. Also, 3D images were constructed and morphometric analysis performed using image analysis software. Both intact and sympathectomized BA and FA developed atherosclerotic plaques, but the thickening of the intima was more advanced in sympathectomized animals, as judged by increased plaque frequency and by the phenotypic modulation of SMCs in the intima. Our results show that in the media of FAs hypercholesterolemia induces changes similar to those observed in sympathectomized rabbits in non-pathological conditions, i.e., migration of adventitial FBs to the media and loss of medial SMCs. These latter changes, which can be ascribed to pathological events, were accentuated after sympathectomy in the hypercholesterolemic rabbits. The present study reveals that pathological events, including migration and phenotypic modulation of vascular FBs and loss of SMCs, may be under the influence of sympathetic nerves.

Differing influence of sympathectomy on smooth muscle cells and fibroblasts in cerebral and peripheral muscular arteries.

In the present study, we examined the effect of sympathectomy on the distribution and the relative expression of cytoskeletal proteins used as markers of phenotypic modulation of vascular smooth muscle cells (SMCs) and myofibroblasts (MFBs) in rabbit femoral (FA) and basilar (BA) arteries. Adult rabbits were treated either with repeated 6-hydroxydopamine (6-OHDA) for sympathectomy or with vehicle for control. Cross sections taken from sympathectomized and control arteries 79 days later were immunolabelled for vimentin, desmin, alpha-smooth muscle actin (alpha-SM actin), beta-isoform of actin and h-caldesmon. The distribution of these proteins and the intensity of fluorescent labelled SMCs were examined under a confocal microscope. In the sympathectomized BA, there was no change for desmin, vimentin and h-caldesmon expression, but the expression of both alpha-SM actin and the beta-isoform was significantly higher (+19% and +30%, respectively). In the sympathectomized FA, the expression of the alpha- and beta-isoforms of actin remained unchanged, whereas those of desmin and vimentin were significantly higher (+35% and 17%, respectively) and h-caldesmon expression was lowered by 13%. In contrast to intact FAs, the external layers of sympathectomized FAs revealed migration of fibroblasts from the adventitia and death of SMCs. These results strongly suggest that sympathetic nerves intervene in the cytoskeletal protein remodelling through phenotypic modulation of both SMCs and MFBs during post-natal development, and in pathologies involving similar phenomena, such as atherosclerosis.

Polymorphonuclear leukocyte activation induces cerebral hypoperfusion in rats in the absence of previous ischemia-reperfusion damage.

We determined if activation of circulating neutrophils could influence local cerebral blood flow (lCBF) and cerebrovascular reactivity without previous ischemic endothelial activation. After intracarotid infusion of phorbol myristate acetate (PMA, twice in 30 min), Laser-Doppler measurements of lCBF in the parietal cortex of anesthetised rats showed a fall of 34% (P<0.05) at 30 min, but not in the vehicle group nor the group predepleted in polymorphonuclear leukocytes (PMNLs). Blood gases or arterial pressure did not change significantly in any group. The PMNL count fell by 78% at 30 min and reactivity to systemic hypercapnia by 58% at 30-60 min post infusion in the PMA group. These results show that activated PMNLs reduce lCBF and vasoreactivity in the absence of previous cerebral ischemia.

Effects of chronic L-NAME treatment on rat focal cerebral ischemia and cerebral vasoreactivity.

Nitric oxide has been shown to be involved in the regulation of cerebral blood flow and the consequences of cerebral ischemia. Short-term inhibition of its synthesis induces hypertension and increases the cortical infarct volume in focal ischemia. Our purpose was to investigate the influence of the long-term inhibition of nitric oxide synthase on infarct volume due to middle cerebral artery (MCA) occlusion and on the reactivity of cerebral arteries. Sprague Dawley rats were given N(omega)-nitro-L-arginine methyl ester (L-NAME) for 2 or 6 weeks and compared to untreated normotensive rats and untreated spontaneously hypertensive rats (SHRs). Brain nitric oxide synthase activity was measured by the 14C-L-arginine assay. Arterial blood pressure was measured in each group. Independently, the reactivity of MCA trees was studied in vitro by a perfusion technique. Cortical infarct volume was not significantly modified by either 2-week or 6-week L-NAME treatment, despite induced hypertension, whereas it was significantly higher in SHRs than in normotensive rats. The reactivity of the MCA tree was significantly affected by the treatment with a clearcut time-dependency. Compared to normotensive controls, contractility to noradrenaline and serotonin was reduced, more severely at 6 weeks, and while dilatation to acetylcholine and nitroprusside was moderately reduced at 6 weeks, dilatation to papaverine was then increased. A major difference of treated animals compared to SHRs was the decreased response to 5-hydroxytryptamine. We conclude that infarct expansion may be limited in treated animals by a progressive reduction in cerebral artery response to vasoconstrictory neurotransmitters, concomitant with augmented non-guanylate cyclase dilator responses (cf. papaverine) and some recovery of dilatation to acetylcholine.

Overexpression of cyclooxygenase-2 in rabbit basilar artery endothelial cells after subarachnoid hemorrhage.

OBJECTIVE: We investigated the expression in rabbit basilar arteries of cyclooxygenase (COX)-2, which is the inducible isoform of the enzyme of prostaglandin (PG) production, and the concentrations of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) and representative PGs in the cerebrospinal fluid (CSF) after experimental subarachnoid hemorrhage (SAH). METHODS: Seven sets of basilar arteries were removed from control rabbits and from rabbits killed 1 and 3 days after induced SAH. The arteries were subjected to identical simultaneous immunolabeling for examination with a confocal microscope. One-half of each artery was stained for the constitutive form COX-1 and the other half for COX-2. CSF was sampled in control animals and at 6 hours, 1 day, and 3 days for assays of TNFalpha, PGE2, and 6-keto-PGF1 (metabolite of PGI2). RESULTS: COX-1 immunoreactivity in the endothelial layer was similar in the three groups. Weak endothelial COX-2 immunoreactivity was found in arteries of control animals. COX-2 staining was higher in the group killed at 3 days compared with the control group (P < 0.05). The levels of PGE2 and 6-keto-PGF1alpha in the CSF peaked significantly at 6 hours, then decreased at 3 days to the basal level (PGE2) or significantly lower (6-keto-PGF1). TNFalpha was undetectable in control CSF, significantly higher (P < 0.001) at 6 hours, and undetectable at 3 days. CONCLUSION: After SAH, endothelial COX-1 immunoreactivity does not change, whereas overexpression of COX-2 occurs at 3 days. This induction does not seem linked to TNFalpha production, nor is it responsible for early raised levels of PGE2 and PGI2 in the CSF. We suggest that the role of induced COX-2 may be to modify gene expression and hence alter the properties of the vessel wall after SAH.

Confocal microscopic evidence of decreased alpha-actin expression within rabbit cerebral artery smooth muscle cells after subarachnoid haemorrhage.

Our objective was to determine whether subarachnoid haemorrhage modifies cerebral artery smooth muscle cell phenotype and the contractile protein alpha-actin measured 7 days after haemorrhage. We used a rabbit subarachnoid haemorrhage model and immunofluorescence labelling of alpha-smooth muscle actin, vimentin and desmin. The paired comparison between the haemorrhage and sham rabbits was performed using confocal laser-scanning microscopy. We found in the haemorrhage group significantly less intense alpha-actin immunostaining (p = 0.036) and more intense vimentin immunostaining (p = 0.043) but no significant change in the intensity of desmin staining. Our results indicate an absolute decrease after subarachnoid haemorrhage in the amount of functional alpha-actin and in the light of the literature may suggest a certain degree of dedifferentiation of smooth muscle cells in the cerebral artery wall.

[Vascular wall modifications after disruption of the circulation leading to cerebral ischemia]. / Modifications de la paroi vasculaire après atteinte circulatoire avec ischémie cérébrale.

This article describes the modifications occurring in cerebral vessels during the first 24 h after an ischaemic event followed by reperfusion. The phases of occlusion, no-reflow, post-ischaemic hyperaemia and delayed hypoperfusion are dealt with in terms of haemodynamic, morphological, functional and biochemical data, available especially from animal studies. The interactions of platelets and leucocytes with the endothelium and the major role of reactive oxygen species in lesioning the microvasculature, including the blood-brain barrier, are emphasized.

Influence of the antioxidant quercetin in vivo on the level of nitric oxide determined by electron paramagnetic resonance in rat brain during global ischemia and reperfusion.

We characterized the changes in nitric oxide (NO) levels in the brain during global forebrain ischemia and reperfusion and tested the ability of the natural flavonoid, quercetin, and a synthetic flavonoid, FB277, to increase the amount of available NO by elimination of the superoxide radicals produced during reperfusion. In Sprague-Dawley rats, we used a four-vessel occlusion model of forebrain ischemia (15 min) and reperfusion (30 min). Brain NO was measured on samples of cerebral cortex and cerebellum ex vivo by electron paramagnetic resonance (EPR) spectroscopy. The spin trap used was diethyldithiocarbamate sodium salt (DETC) associated with ferrous citrate. The complex Fe(DETC)2NO was detected at 77 K as a triplet signal at g = 2.035. Groups of animals were treated with quercetin or FB277 (3-morpholinomethyl-3',4',5,7tetramethoxyflavone) or polyethylene glycol-conjugated superoxide dismutase (PEG-SOD). In control (intact anesthetized animals), the signal was about 3 times greater in the cortex than in the cerebellum. During ischemia, the signal rose to 110% in cortex (NS) and 283% in cerebellum (P < 0.05). In reperfusion, it fell again to 91% of control in cerebellum (NS) and 35% in cortex (P < 0.05). Treatment by quercetin (5 mg/kg i.v.) of intact and ischemia-reperfusion groups did not significantly change the signal amplitude in the cerebellum, but did double it in the cortex (to 76% of control) for the ischemia-reperfusion group (P < 0.05). In contrast, FB277 (3.75 mg/kg i.v.) did not increase the signal in the cortex during ischemia-reperfusion, but did do so in the cerebellum (to 152% of control, P < 0.05). The results obtained for PEG-SOD (10,000 U/kg i.v.) were similar to those for FB277. In separate in vitro measurements, we found that quercetin but not FB277 efficiently scavenged superoxide. We hypothesize that quercetin but not FB277 scavenged superoxide anions released in the cortex during reperfusion, thus diminishing the amount of NO removed by the formation of peroxynitrite. The lack of effect of PEG-SOD may be related to the need for chronic treatment to obtain protection.

Effects of oxyhemoglobin in vitro in cerebral arteries from normal animals and animals subject to subarachnoid hemorrhage or indomethacin treatment.

Experiments were performed to test the hypothesis that subarachnoid hemorrhage (SAH) causes functionally relevant perturbations of cyclooxygenase activity in cerebral arteries. Four groups of rabbits were formed: (I) controls; (II) sham injected animals (2 ml physiological solution in the cisterna magna); (III) SAH group (2 ml blood in cisterna magna); (IV) indomethacin group (4 mg/kg i.v. 30 min before sacrifice). Animals of groups II and III were used 3 days after injection. The basilar arteries (BAs) were removed and perfused at a constant flow rate (after electrocoagulation of all branches) in vitro in a 2-ml bath at 37 degrees C. After 45 min equilibration, the arteries were subjected to a fixed protocol: first, in Krebs solution, contraction to increasing extraluminal concentrations of histamine (HA), followed by a single maximal extraluminal concentration of acetylcholine (ACh); then, after 30 min rest, the same tests were repeated in oxyhemoglobin (oxyHb) solution (extraluminal, 10-4 M). Perfusion pressure changes reflected changes in artery resistance. Although oxyHb alone increased pressure, indicating contraction of the arteries, its most important effect was to increase contraction to HA (in groups II, III, and IV but not controls) and to strongly inhibit ACh-induced relaxation in the SAH (-66.3%) and indomethacin (-46.9%) groups (III and IV) but not the control (-27.6%) group. The latter result suggests that a relaxing factor released by ACh in oxyHb solution in the control group was not present in groups III and IV. In conjunction with the results on HA, which is known to normally release prostacyclin (PGI2) from the endothelium, it is concluded that PGI2 was not or little released from arteries of the SAH group when they bathed in oxyHb solution. Alternatively, in the SAH group constrictor prostaglandins were released in response to HA and ACh in place of PGI2.

Reduced cortical vasodilatory response to stimulation of the nucleus basalis of Meynert in the aged rat and evidence for a control of the cerebral circulation.

In earlier studies we showed that electrical stimulation of the rat nucleus basalis of Meynert (NBM) induces large increases in cerebral blood flow, mainly through cholinergic mechanisms. We then investigated the effect of aging on this influence by measuring cortical blood flow (CoBF) and tissue gas partial pressures (PtO2, PtCO2) in the conscious young adult and aged rat. NBM stimulation increased frontal (+101%) and parietal (+29%) CoBF in young rats. The effects were halved in aged rats. Moreover, PtO2 was significantly increased in young but not in aged rats. By contrast, the corticovascular reactivity to hypercapnia did not differ between young and aged rats, nor did the potentiating vasodilator effect of physostigmine. In combined autoradiographic measurements of cerebral blood flow and cerebral glucose utilization, we recently found that the cortical circulatory response to NBM stimulation was not accompanied by significant metabolic change. Thus, the blood flow changes observed in the cortex cannot be ascribed to increased metabolic activity. The distribution of this uncoupling coincides with that of cholinergic NBM projections directly impinging on cortical microvessels. These data support the cortical microcirculation and suggest the possible involvement of NBM dysfunction in the pathology of cortical microcirculation.

Cerebrovascular reactivity: role of endothelium/platelet/leukocyte interactions.

In the last two decades, a tremendous amount of knowledge has been accumulated in various fields of biomedical research that discloses mechanisms of platelet/leukocyte/endothelium interactions. Occupying a strategically important location between circulating blood and underlying tissues, the endothelium effectively modulates both the functional state of the blood cells and the tone of vascular smooth muscle by generating or metabolizing a host of humoral substances. Under normal conditions, the endothelium releases agents with predominantly vasodilator and antiaggregant/anticoagulant activity that prevent thrombotic and angiospastic disorders. However, a variety of pathophysiological stimuli may trigger endothelial reorganization with the expression of different prothrombotic factors and activation of platelets and leukocytes that, combined, leads to blood cell adhesion to the endothelial monolayer, aggregation as thrombi, and the formation of numerous spasmogenic substances. Activation of the blood cells in the vicinity of the endothelium may induce endothelial dysfunction/injury, resulting in impairment of normal endothelial antispasmodic control. Within the microcirculatory bed, intravascular activation of the blood cells leads to scattered microvessel plugging, increased vascular permeability, edema formation, and cytotoxic actions of blood cell-released agents on the underlying tissue. A growing body of evidence suggests that these processes may be involved in pathophysiological cerebrovascular reactions including symptomatic angiospasm following subarachnoid hemorrhage, segmental occlusive constriction in atherosclerotic cerebral arteries, and constrictive vasomotion in microvessels. A perturbation in the delicate equilibrium between blood cells and endothelium in the microcirculation seems to be a factor aggravating ischemic brain damage or even primarily causing focal cerebral ischemia and scattered microinfarctions. Increased predisposition to these pathophysiologic events might influence unfavorably the effects of risk factors such as hypercholesterolemia, hypertension, and diabetes on cerebrovascular morbidity and mortality. Although the importance of blood cell/endothelium imbalance appears to be clear, its pharmacologic regulation is not sufficiently established. Some drugs have been demonstrated to limit platelet and/or leukocyte activity and protect the endothelial defense mechanisms, but the optimal therapeutic strategy has yet to be elaborated.

Leukocyte-induced endothelial dysfunction in the rabbit basilar artery: modulation by platelet-activating factor.

We studied the effects of polymorphonuclear leukocytes (PMNLs) activated by N-formyl-methionyl-leucyl-phenylalanine on the endothelium-dependent relaxation of the rabbit basilar artery (BA). In the presence of activated PMNLs the maximal vessel relaxation to acetylcholine (ACh) and bradykinin (endothelium-dependent dilators) was decreased from 62 +/- 7 and 48 +/- 6% to 23 +/- 9 and 19 +/- 7, respectively, (p < 0.05). The endothelium-independent relaxation to nitroprusside was not affected by PMNLs. When PMNLs were activated in the organ chamber in the presence of a low concentration of platelet-activating factor (PAF, 10(-10) mol/l), the depression of ACh- and bradykinin-induced relaxation increased by 27 +/- 9 and 23 +/- 7%, respectively (p < 0.05), though at this concentration PAF alone did not cause PMNLs to induce endothelial dysfunction. In addition, in the presence of PAF, activated PMNLs inhibited endothelium-dependent relaxation at lower cell concentrations and shorter periods of contact with the endothelium. PMNL effects on the endothelium were correlated with the level of cell exocytosis as tested by accumulation of beta-glucuronidase activity. In the presence of PAF, accumulation of this activity increased from 46 +/- 6 to 79 +/- 8 U/ml (p < 0.05). Examination of BA segments by scanning electron microscopy revealed that, after the treatment with activated PMNLs, the endothelium was morphologically preserved, but in the presence of PAF PMNLs caused more apparent microlesions in the endothelial layer. We conclude that small quantities of PAF potentiate the activation of marginated PMNLs. These cells then become more aggressive towards the endothelium, producing significant depression of the endothelium-dependent relaxation.

A new synthetic flavonoid protects endothelium-derived relaxing factor-induced relaxation in rabbit arteries in vitro: evidence for superoxide scavenging.

A new synthetic flavone derivative, 6,7-dimethoxy-8-methyl-3',4',5-trihydroxyflavone, was studied for its capacity to protect the acetylcholine-induced relaxation of rabbit ear and cerebral arteries from inhibition by superoxide anion. This property was evaluated via two types of in vitro experiments, using rabbit ear or basilar arteries mounted in organ baths equipped for isometric tension measurement. When a high level of superoxide anion was generated by adding 3 x 10(-4) M pyrogallol to the bath, the relaxation to acetylcholine was substantially inhibited. This inhibition was significantly reversed by both superoxide dismutase (25 and/or 50 U/mL) and the flavonoid (3 x 10(-6) M and/or 10(-5) M) in both types of arteries. In the presence of the basal level of superoxide anion, the responses to acetylcholine were significantly potentiated by the flavonoid (10(-5) M) in the ear but not the basilar artery. Thus this flavonoid protects endothelium-dependent relaxation from high levels of superoxide anion possibly by scavenging superoxide anion and may have a certain therapeutic value as an agent capable of promoting natural vasodilatation.

Actions of platelet-activating factor on isolated rabbit basilar artery: modulation by activated polymorphonuclear leukocytes.

In segments of rabbit basilar artery (BA) platelet-activating factor (PAF) initiated a concentration-dependent transitory contraction which was unaffected by indomethacin or nordihydroguaiaretic acid (NDGA). N omega-nitro-L-arginine (NLA) did not change the magnitude of PAF-induced contraction, but in the presence of NLA the contraction tended to be more prolonged. In precontracted BA segments, PAF caused a concentration-dependent relaxation which was unaffected by NDGA. Indomethacin and NLA decreased PAF-induced relaxation by 10-30 and 70-90%, respectively; in combination these agents totally eliminated PAF-induced vessel reactions. Treatment of BA segments by rabbit peritoneal polymorphonuclear leukocytes (PMNLs) activated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) for 20 min led to PMNL adhesion to the vascular endothelium and a significant decrease (60-100%) in acetylcholine-induced endothelium-dependent vessel relaxation. After the treatment of BA segments, PAF induced strong, slow, tonic contraction of either non-precontracted or precontracted vessels which was unaffected by indomethacin or NLA. NDGA decreased this contraction by 30-60%. These results indicate that PAF is an endothelium-dependent vasodilator that can also produce an insignificant constrictor effect. However, when the vessels are affected by activated PMNLs, PAF is transformed to a strong vasoconstrictor, presumably due to the generation of as yet unknown vasoconstrictor stimuli resulting from PMNL-endothelium interactions. Under these conditions the PAF-induced contraction is partly mediated by 5-lipoxygenase metabolites.

Leukocyte-induced acute endothelial dysfunction in middle cerebral artery in rabbits. Response to aggregating platelets.

BACKGROUND AND PURPOSE: Recent evidence suggests a possible role for leukocytes in angiospastic reactions of large cerebral arteries. This study examined the effect of activation of endogenous circulating leukocytes on endothelium-dependent relaxation in the middle cerebral artery in rabbits. METHODS: Leukocytes were activated by rapid injection of either 40 micrograms/kg phorbol 12-myristate 13-acetate, or 0.2 mg/kg N-formyl-methionyl-leucylphenylalanine into the left carotid artery. Control rabbits received an equal volume of vehicle. Concentration-dependent isometric tension responses of the left and right middle cerebral artery to the dilators acetylcholine, ADP, sodium nitroprusside, or calcium ionophore (A23187), as well as to aggregating platelets, were compared in vitro in control animals and in animals killed 10 minutes after the injection of leukocyte activators in normal and leukocyte-depleted rabbits. RESULTS: In the control animals there was no significant difference in the reactivity of the left and right middle cerebral arteries. The injection of the leukocyte activators led to enhanced contractile responses to aggregating platelets and a significant reduction in the endothelium-dependent relaxation in response to acetylcholine, ADP, and A23187 in the left middle cerebral artery (the injected side), whereas the effect of an endothelium-independent dilator sodium nitroprusside remained unchanged. In leukocyte-depleted rabbits the injection of either of the leukocyte activators used did not induce significant changes in the reactivity of the left middle cerebral artery. CONCLUSIONS: Intravascular leukocyte activation appears to induce an acute disturbance of the endothelium-dependent relaxation. Under these conditions, platelet activation might result in marked angiospastic reactions of large cerebral arteries.

Endothelial dysfunction in cerebral vessels following carotid artery infusion of phorbol ester in rabbits: the role of polymorphonuclear leukocytes.

The effect of 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (PMA) on endothelium-dependent and endothelium-independent vasoconstriction and vasodilation was studied in isolated segments of rabbit middle cerebral artery (MCA). Concentration-dependent responses of the left and right MCA to the constrictors KCl, noradrenaline, uridine 5'-triphosphate, serotonin, and histamine, as well as to the dilators acetylcholine, bradykinin, sodium nitroprusside, and calcium ionophore (A23187), were compared in control animals and after PMA injection into the left common carotid artery. In the control animals there was no significant difference in the responses of the left and right MCA to either the constrictors or the dilators studied. After PMA injection the endothelium-dependent relaxation in response to acetylcholine, bradykinin, and A23187 was reduced in the left MCA (PMA-injected side), whereas the effect of the endothelium-independent dilator sodium nitroprusside remained unchanged. Simultaneously greater contractile responses of the left MCA to serotonin and histamine were obtained. Neither infusion of L-arginine in vivo before the PMA injection nor incubation of the isolated MCA segments with L-arginine affected this difference in MCA reactivity. Platelet depletion did not change the PMA-induced reduction in the endothelium-dependent relaxation, whereas after leukocyte depletion this reduction practically disappeared. These results suggest that the PMA-induced brain microembolia causes acute endothelial dysfunction, which is possibly mediated by intravascular activation of leukocytes and is independent of nitric oxide synthesis from L-arginine. This phenomenon might play an important role in cerebral angiospastic disorders after intravascular activation of leukocytes in cerebral ischemia and reperfusion.

Basal forebrain control of cortical blood flow and tissue gases in conscious aged rats.

Cholinergic projections from the basal forebrain are capable of influencing local cortical blood flow (CoBF). The effect of age on this influence was investigated by measuring CoBF and tissue gas partial pressures (PtO2, PtCO2) by mass spectrometry in conscious young adult (2-4 months) and aged (22-28 months) Fischer 344 rats. Electrical stimulation (50 microA) of the substantia innominata (SI) increased frontal (+100.9%) and parietal (+28.4%) CoBF in young rats, but the effects were less in aged rats (frontal, +48.6%, P < 0.05; parietal, +18.9%, difference N.S.). Frontal PtO2 was increased in young but not aged rats (P < 0.01.). During standard hypercapnia, changes in CoBF, PtO2 and PtCO2 did not differ between young and aged rats. Under physostigmine infusion (0.15 mg/kg/h, i.v.), the CoBF increases to SI stimulation were approximately doubled in both cortices, in young and aged rats, and PtO2 increases were also significantly greater. However, frontal PtO2 increases were significantly smaller in aged (+7.6%) than in young (32.7%) rats, as were frontal PtCO2 reductions. We conclude: (i) the influence of the SI on frontal CoBF and PtO2 is substantially reduced with age; (ii) although physostigmine treatment potentiates this influence in both groups, the beneficial effects are relatively limited for aged rats.

Time course of variations in rabbit cerebrospinal fluid levels of calcitonin gene-related peptide- and substance P-like immunoreactivity in experimental subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Cerebral vasospasm after subarachnoid hemorrhage may result partially from the imbalance between vasodilator and vasoconstrictor factors. The vasodilator peptides substance P and calcitonin gene-related peptide contained in the trigeminovascular system are involved in the vasomotor phenomenon occurring after subarachnoid hemorrhage. The delayed arterial narrowing may reflect the time course of the release of these peptides. Therefore, we followed the time course of the changes in cerebrospinal fluid immunoreactivity of substance P and calcitonin gene-related peptide in a model of experimental subarachnoid hemorrhage. METHODS: Cerebrospinal fluid samples were taken in the basal state and at 30 minutes, 24 hours, and 3 days after a single injection of 1 mL autologous arterial blood into the cisterna magna of rabbits using a percutaneous suboccipital route. Substance P-like and calcitonin gene-related peptide-like immunoreactivities were determined in centrifuged cerebrospinal fluid and plasma by use of enzyme immunoassay. RESULTS: Early (30 minutes) after induced subarachnoid hemorrhage, there was a large increase in cerebrospinal fluid substance P-like immunoreactivity (P < .01) and calcitonin gene-related peptide-like immunoreactivity (P < .01). Arterial and hemorrhagic cerebrospinal fluid levels of substance P-like immunoreactivity were different (P < .03), indicating that the increased cerebrospinal fluid level did not result only from the blood contamination. Twenty-four hours after induced subarachnoid hemorrhage, the immunoreactivities of substance P and calcitonin gene-related peptide remained significantly higher than the basal level (P < .01). At day 3, both immunoreactivities had decreased to a level nonsignificantly different from the basal level. CONCLUSIONS: The early high values of the cerebrospinal fluid immunoreactivities for substance P and calcitonin gene-related peptide, apart from the contamination by arterial blood, probably resulted from the depletion of neurotransmitter peptides from the trigeminovascular fibers.

Increased influence of calcium and nicardipine on rabbit basilar artery reactivity after brief subarachnoid hemorrhage.

We studied the changes in reactivity of basilar arteries immediately after a subarachnoid hemorrhage (SAH) in response to serotonin (5-HT), uridine 5'-triphosphate (UTP), and extracellular Ca2+. Although much evidence suggests that an early phase of vasoconstriction occurs after SAH, no direct data exist on changes in the role of extracellular Ca2+ shortly after in vivo contact with subarachnoid blood. Ten minutes after injection of blood (SAH) or physiologic solution (sham SAH) into the cisterna magna, rabbits were killed and their basilar arteries were removed for isometric tension measurements on 3-mm segments. Responses to UTP, 5-HT, and Ca2+ (with addition of 100 mM K+) were compared between SAH, sham SAH, and control arteries. SAH arteries showed substantially increased responses to all agents as compared with the other two groups. The calcium entry blocker nicardipine (10(-10)-10(-8) M) inhibited all responses to Ca2+ in a concentration-dependent manner; the most sensitive arteries were the SAH arteries. At 10(-9) M nicardipine and 1.5 mM Ca2+, the inhibition attained 52.4% for control, 39.7% for sham SAH, and 70.5% for SAH (p < 0.05, SAH vs. sham SAH). The results suggest that calcium entry into smooth muscle cells is facilitated by SAH, and this might be explained by an increased number of operational calcium channels. This change, in the presence of spasmogens such as the platelet-derived factors we tested, should result in very early large-scale Ca2+ entry, which might contribute to development of the delayed arterial narrowing known as vasospasm which is a major complication of SAH.

Spreading depression induces prolonged reduction of cortical blood flow reactivity in the rat.

The purpose of the present study was to examine the dynamic aspects of the cerebrovascular events occurring during and up to 2 h following cortical spreading depression (CSD) in the rat, using the mass spectrometry technique which enables continuous measurements of the cortical tissue PO2 and PCO2 and repeated blood flow measurements (CoBF) by helium clearance. We mostly sought to determine whether cortical perforation by a stimulation electrode induced long-lasting perturbation of the cortical vasoreactivity to hypercapnia and basal forebrain electrical stimulation. Cortical perforation in the animal under alpha-chloralose anesthesia, chronically implanted with mass spectrometry probes, was associated with biphasic changes in tissue gases. PO2 first briefly decreased (-7.8%) and then strongly increased (+79%) while PCO2 changed in the opposite direction (+7%, -13%) in the ipsilateral frontal cortex. Qualitatively similar changes occurred in the ipsilateral parietal cortex. The CoBF measurements showed a marked vasodilation (131 and 108% in the frontal and parietal cortex, respectively) in parallel with the PO2 increase, followed by a prolonged (60 min), moderate hypoperfusion (maximum -17% at 20 min after CSD). There was a pronounced reduction of vascular reactivity to both hypercapnia (20.3% of the control response) and substantia innominata stimulation (1/6 of the response obtained 80 min later) at 10 min after CSD. Both reactivities progressively recovered in approximately 2 h. Since the issue of CSD in human has become a popular hypothesis for migraine, the reduced cerebrovascular reactivity could constitute the basis of a test for the involvement of CSD in migraine.