Hypercholesterolemic diet induces vascular smooth muscle cell apoptosis in sympathectomized rats via intrinsic pathway.

In this study, we intend to investigate the role of hypercholesterolemic diet, a high risk factor for atherosclerosis, on vascular cell apoptosis in rats that have been previously sympathectomized. Thus, newborn male Wistar rats received injections of guanethidine for sympathectomy. Sham received injections of vehicle. The two groups were fed 1% cholesterol diet for 3months. Sympathectomy alone group was also exploited. Apoptosis in abdominal aortic tissue was identified by TUNEL method and conventional agarose gel electrophoresis to detect specific DNA fragmentation. Caspases 3 and 9, Bcl-2, Bax and cytochrome c were examined by immunoblotting. Oil Red O staining was used to reveal lipid in the arterial wall. Vascular smooth muscle cells (VSMCs) and macrophages were identified by immunostaining for α-smooth muscle actin and rat macrophage marker (ED1), respectively. The efficacy of sympathectomy was evaluated by analysis of perivascular sympathetic fibers. Our study showed that hypercholesterolemic diet, when performed in rats with neonatal sympathectomy, 1) increased aortic TUNEL-positive cells compared to sham and sympathectomy alone groups, 2) illustrated a typical apoptotic DNA ladder on agarose gel electrophoresis, 3) induced Bax translocation from cytosol to mitochondria, 4) enhanced cytochrome c release from mitochondria to cytosol, 5) increased expression of active caspases 3 and 9, and 6) decreased Bcl-2 expression. VSMCs are identified as the major cell type exhibiting apoptosis in this model. Taken together, it can be concluded that hypercholesterolemic diet, when performed in rats with neonatal sympathectomy, induces vascular cell apoptosis in an intrinsic pathway.

Chemical sympathectomy induces arterial accumulation of native and oxidized LDL in hypercholesterolemic rats.

The aim of the present study was to examine the effect of sympathectomy on plasmatic and arterial native and oxLDL levels, as well as arterial LDL receptors (LDLR) and scavenger receptors in hypercholesterolemic rats, which are normally protected against atherosclerosis. Neonatal Wistar rats received subcutaneous injections of either guanethidine for sympathectomy (Gua+HC) or vehicle (HC), then were fed 1% cholesterol for three months. Intact normocholesterolemic rats were used as control of the HC group. Total cholesterol (TC) and LDL-cholesterol were evaluated in the plasma and the abdominal aorta by an auto-analyzer. Plasmatic and aortic oxLDL and native LDL-apo B100 were assessed by a sandwich ELISA. Aortic and hepatic native LDLR and aortic scavenger receptors (CD36 and SR-A) were quantified at mRNA and protein levels by real time PCR and western immunoblot. The effect of hypercholesterolemia was limited to an increase in plasmatic TC and LDL-cholesterol and a decrease in aortic apoB100 and aortic and hepatic LDLR. Hypercholesterolemia and sympathectomy in combination increased markedly plasmatic and aortic TC, LDL-cholesterol, apo B100 and oxLDL together with aortic scavenger receptors, but reduced markedly aortic and hepatic LDLR. Sympathectomy broke down the rat's protection against hypercholesterolemia by promoting accumulation of native and oxLDL in the aorta via scavenger receptors.

The profile of the extracellular matrix changes in the aorta after sympathectomy in the hypercholesterolemic rats.

We previously showed that sympathectomy induces thickened intima and decreases the expression of cytoskeletal proteins associated with a differentiated smooth muscle cell (SMC) phenotype in hypercholesterolemic rats. In the present study, we sought to determine the effect of sympathectomy on various components of the extracellular matrix (ECM) in the aorta from these animals, since the state of SMC differentiation depends on the nature of ECM components. Collagen types I and III, previously reported to be associated with SMC dedifferentiation, and collagen VI, elastin, laminin and elastin-laminin receptor (E/L-R), previously reported to be associated with SMC differentiation, were analyzed by western immunoblot and confocal microscopy in abdominal aortae from sham rats and hypercholesterolemic rats sympathectomized with guanethidine. Both western immunoblot and immunohistological analysis showed an increase in collagens I and III (more for collagen I), with abundant labeling in the media, adventitia and thickened intima in sympathectomized aortae. Collagen IV labeling was decreased in the media and adventitia and was weak in the thickened intima in sympathectomised aortae. The E/L-R increased and was abundantly labeled in the media and weakly in the thickened intima in sympathectomized aortae. Elastin and laminin decreased and appeared less labeled in the media in the sympathectomised aortae. In the thickened intima, laminin was slightly labeled while elastin was not obviously labeled. These data show that sympathectomy favors the ECM features reported in association with a dedifferentiated/immature SMC phenotype and intimal thickening, probably by actions on both SMCs and fibroblasts.

Influence of antagonist sensory and sympathetic nerves on smooth muscle cell differentiation in hypercholesterolemic rat.

The effect of sympathectomy and sensory denervation on vascular smooth muscle cell (SMC) differentiation was investigated in hypercholesterolemic rats. Newborn rats received injections of guanethidine, capsaicin or both for denervations. Shams received injections of vehicles. The four groups were fed 1% cholesterol diet for 3 months. Intact normocholesterolemic rats were also exploited. Serum total cholesterol and systolic blood pressure (SBP) were measured. Lipid presence in the arterial wall was shown by Red-Oil-O staining. Catecholamine- and CGRP-containing fibres, vimentin and the adult SMC markers alpha-SMC-actin, desmin and h-caldesmon were analysed in abdominal aorta by western blot and confocal microscope. The sympathetic (catecholamine) fibres and SBP increased after sensory denervation while the sensory (CGRP) fibres increased and SBP decreased after sympathectomy. SBP was not changed after double denervation. Total cholesterol increased in sham and rose further after sympathectomy. Vimentin and the three adult SMC markers were not influenced by hypercholesterolemia. However, in the sympathectomized aorta, vimentin increased, desmin did not change, whereas alpha-SMC-actin and h-caldesmon decreased. In the sensory-denervated aorta, vimentin decreased, desmin increased, alpha-SMC-actin did not change and h-caldesmon decreased but less than in sympathectomized aorta. In the doubly denervated aorta, vimentin did not change and the three adult SMC markers decreased, although less than in sympathectomized aorta for alpha-SMC-actin and h-caldesmon. Thickened intima was identified by Red-Oil-O staining in the sympathectomized and (less remarkably) doubly denervated aortas containing SMCs not fully dedifferentiated. Our findings suggest that sympathectomy induces intimal thickening and favours SMC dedifferentiation, whereas sensory denervation favours SMC differentiation.

Randomized, double-blind, placebo-controlled, pilot trial of high-dose methylprednisolone in aneurysmal subarachnoid hemorrhage.

OBJECT: The object of this study was to determine the efficacy of methylprednisolone in reducing symptomatic vasospasm and poor outcomes after subarachnoid hemorrhage (SAH). METHODS: Ninety-five patients with proven SAH were recruited into a double-blind, placebo-controlled, randomized trial. Starting within 6 hours after angiographic diagnosis of aneurysm rupture, placebo or methylprednisolone, 16 mg/kg, was administered intravenously every day for 3 days to 46 and 49 patients, respectively. Deterioration, defined as development of a focal sign or decrease of more than 1 point on the Glasgow Coma Scale for more than 6 hours, was investigated by using clinical criteria and transcranial Doppler ultrasonography, cerebral angiography, or CT when appropriate. The end points were incidence of symptomatic vasospasm (delayed ischemic neurological deficits associated with angiographic arterial narrowing or accelerated flow on Doppler ultrasonography, or both) and outcome 1 year after entry into the study according to a simplified Rankin scale (Functional Outcome Scale [FOS]) in living patients and the Glasgow Outcome Scale in all patients included. RESULTS: All episodes of deterioration and all living patients with a 1-year outcome were assessed by a review committee. In patients treated with methylprednisolone, the incidence of symptomatic vasospasm was 26.5% compared with 26.0% in those given placebo. Poor outcomes according to FOS were significantly reduced in the Methylprednisolone Group at 1 year of follow-up; the risk difference was 19.3% (95% CI 0.5-37.9%). The outcome was poor in 15% (6/40) of patients in the Methylprednisolone Group versus 34% (13/38) in the Placebo Group. CONCLUSIONS: A safe and simple treatment with methylprednisolone did not reduce the incidence of symptomatic vasospasm but improved ability and functional outcome at 1 year after SAH.

Both ischemic preconditioning and ghrelin administration protect hippocampus from ischemia/reperfusion and upregulate uncoupling protein-2.

BACKGROUND: A major endogenous protective mechanism in many organs against ischemia/reperfusion (I/R) injury is ischemic preconditioning (IPC). By moderately uncoupling the mitochondrial respiratory chain and decreasing production of reactive oxygen species (ROS), IPC reduces apoptosis induced by I/R by reducing cytochrome c release from the mitochondria. One element believed to contribute to reduce ROS production is the uncoupling protein UCP2 (and UCP3 in the heart). Although its implication in IPC in the brain has been shown in vitro, no in vivo study of protein has shown its upregulation. Our first goal was to determine in rat hippocampus whether UCP2 protein upregulation was associated with IPC-induced protection and increased ROS production. The second goal was to determine whether the peptide ghrelin, which possesses anti-oxidant and protective properties, alters UCP2 mRNA levels in the same way as IPC during protection. RESULTS: After global forebrain ischemia (15 min) with 72 h reperfusion (I/R group), we found important neuronal lesion in the rat hippocampal CA1 region, which was reduced by a preceding 3-min preconditioning ischemia (IPC+I/R group), whereas the preconditioning stimulus alone (IPC group) had no effect. Compared to control, UCP2 protein labelling increased moderately in the I/R (+39%, NS) and IPC+I/R (+28%, NS) groups, and substantially in the IPC group (+339%, P < 0.05). Treatment with superoxide dismutase (10000 U/kg ip) at the time of a preconditioning ischemia greatly attenuated (-73%, P < 0.001) the increase in UCP2 staining at 72 h, implying a role of oxygen radicals in UCP2 induction.Hippocampal UCP2 mRNA showed a moderate increase in I/R (+33%, P < 0.05) and IPC+I/R (+40%, P < 0.05) groups versus control, and a large increase in the IPC group (+333%, P < 0.001). In ghrelin experiments, the I/R+ghrelin group (3 daily administrations) showed considerable protection of CA1 neurons versus I/R animals, and increased hippocampal UCP2 mRNA (+151%, P < 0.001). CONCLUSION: We confirm that IPC causes increased expression of UCP2 protein in vivo, at a moment appropriate for protection against I/R in the hippocampus. The two dissimilar protective strategies, IPC and ghrelin administration, were both associated with upregulated UCP2, suggesting that UCP2 may often represent a final common pathway in protection from I/R.

Inhibition of caspase-9 activation and apoptosis is involved in ischemic preconditioning-induced neuroprotection in rat brain.

OBJECTIVES: Cerebral ischemic pre-conditioning (IPC) is capable of protecting hippocampal neurons from ischemia/reperfusion (I/R) injury. In the current study, we investigated the role of activated caspase-9 in the protective process induced by IPC and related it to cytochrome c release and apoptosis. METHODS: I/R injury was induced by a four-vessel occlusion model in Wistar rats which were randomly divided into ischemia/reperfusion group (I/R), ischemic pre-conditioning + I/R group (IPC + I/R) and control group. Histologic changes in the pyramidal layer of the hippocampal CA1 region were determined by hematoxylin and eosin (H&E) staining. The relative proportion of apoptotic neurons in this area was assessed with TUNEL staining. The redistribution of cytochrome c and activation of caspase-9 were detected in the same area with immunohistochemistry and Western blotting respectively. RESULTS: Compared to the I/R group, IPC increased the number of surviving neurons in the hippocampal CA1 region (p<0.001), markedly reduced the number of apoptotic pyramidal neurons (p<0.001), inhibited the release of cytochrome c from mitochondria to cytoplasm (p<0.001 for positively stained neurons) and decreased the amount of activated caspase-9 (p<0.001). DISCUSSION: These findings confirm that IPC is capable of protecting neurons from injury by apoptosis. The release of cytochrome c to the cytosol demonstrates that the mitochondrial pathway was involved, and the reduction in this release caused by IPC was clearly associated with reduced caspase-9 activation. Together, these results suggest that IPC protects neurons via action on the mitochondrial/caspase-9 pathway of apoptosis.

Sympathectomy causes aggravated lesions and dedifferentiation in large rabbit atherosclerotic arteries without involving nitric oxide.

Previously [Histochem J 1997;29:279-286], we found that sympathectomy induced neointima formation in ear but not cerebral arteries of genetically hyperlipidemic rabbits. To clarify the influence of sympathetic nerves in atherosclerosis, and whether their influence involves vascular NO activity, we studied groups of normocholesterolemic intact (NI) and sympathectomized (NS), and hypercholesterolemic intact (HI) and sympathectomized (HS) rabbits (diet/6-hydroxydopamine for 79 days). Segments of basilar (BA) and femoral (FA) arteries were studied histochemically, to evaluate differentiation (anti-desmin, anti-vimentin, anti-h-caldesmon, and nuclear dye), by confocal microscopy, and by in vitro myography. In BAs, staining of NI and NS groups was similar. In hypercholesterolemic groups, a small neointima developed, more frequently in HS segments where smooth muscle cells (SMCs) positive for all antibodies appeared to be migrating into the neointima. In FAs, SMCs stained for the three antibodies in the NI group, but we observed desmin- and h-caldesmon-negative, vimentin-positive cells in some external medial layers of the NS, HI and HS groups, identical to adventitial fibroblasts. Large neointimas of the HS group contained vimentin-positive and largely desmin- and h-caldesmon-negative cells. Relaxation of BA or FA segments to acetylcholine was not decreased by sympathectomy. Sympathectomy increased the contraction of resting FAs to nitro-L-arginine (p = 0.0379). Thus, sympathectomy aggravates the tendency for FA SMCs to migrate and dedifferentiate, increasing atherosclerotic lesions, without decreasing NO activity, but has only minor effects on BAs.

Dexamethasone preventing contractile and cytoskeletal protein changes in the rabbit basilar artery after subarachnoid hemorrhage.

OBJECT: The aim of this project was to study the perturbations of four smooth-muscle proteins and an extracellular protein, type I collagen, after subarachnoid hemorrhage (SAH) and to examine the possible preventive effects of dexamethasone. METHODS: Using a one-hemorrhage rabbit model, the authors first examined the effects of SAH on the expression of alpha-actin, h-caldesmon, vimentin, smoothelin-B, and type I collagen; second, they studied whether post-SAH systemic administration of dexamethasone (three daily injections) corrected the induced alterations. Measurements were obtained at Day 7 post-SAH. The proteins were studied by performing immunohistochemical staining and using a laser-scanning confocal microscope. Compared with control (sham-injured) arteries, the density of the media of arteries subjected to SAH was reduced for alpha-actin (-11%, p = 0.01) and h-caldesmon (-15%, p = 0.06) but increased for vimentin (+15%, p = 0.04) and smoothelin-B (+53%, p = 0.04). Among animals in which SAH was induced, arteries in those treated with dexamethasone demonstrated higher values of density for alpha-actin (+13%, p = 0.05) and h-caldesmon (+20%, p = 0.01), lower values for vimentin (-55%, p = 0.05), and nonsignificantly different values for smoothelin-B. The density of type I collagen in the adventitia decreased significantly after SAH (-45%, p = 0.01), but dexamethasone treatment had no effect on this decrease. CONCLUSIONS: The SAH-induced alterations in the density of three of four smooth-muscle proteins were prevented by dexamethasone treatment; two of these proteins--alpha-actin and h-caldesmon--are directly related to contraction. This drug may potentially be useful to prevent certain morphological and functional changes in cerebral arteries after SAH.

Acetylcholine-induced relaxation of rabbit basilar artery in vitro is rapidly reduced by reactive oxygen species in acute hyperglycemia: role of NADPH oxidase.

We examined the effects of acute hyperglycemia on the function of rabbit cerebral arteries in vitro. It was hypothesized that increased formation of reactive oxygen species (ROS) could occur, which could explain how hyperglycemia aggravates certain pathologic situations such as cerebral ischemia. Three-millimeter basilar artery segments were incubated in either normoglycemic (NG, 5.5 mM D-glucose) or hyperglycemic (HG, 25 mM D-glucose) solution containing 3.10(-6) M indomethacin. After 90 minutes equilibration, a test (=T1) of relaxation to acetylcholine (Ach) at three concentrations was performed on histamine-precontracted segments. Three further identical tests were performed (T2-T4), after 30-minute rest periods. Ach responses in NG solution were stable, whereas those in HG solution, although greater at T1, fell progressively from one test to the next (P < 0.0001 versus NG), whereas nitroprusside responses did not change. In separate experiments, this time-dependent fall in Ach responses was significantly prevented by superoxide dismutase (SOD) plus catalase (P = 0.0003), but not by SOD alone. It was also significantly prevented by the NAD(P)H oxidase inhibitors diphenyleneiodonium (P = 0.020) and apocynin (P = 0.0179), but not by allopurinol (xanthine oxidase inhibitor). Control experiments with l-glucose ruled out a hyperosmotic or non-specific glucose effect. We conclude that, in HG solution in vitro, rapidly increasing ROS production largely derived from NAD(P)H oxidase reduced relaxation to acetylcholine. The rapidity of this effect suggests that the function of these arteries may be affected during brief periods of hyperglycemia in vivo.

Microvascular remodeling after occlusion-recanalization of a branch retinal vein in rats.

PURPOSE: To describe the time course of microvascular changes after transient branch retinal vein occlusion (BRVO) in rats. METHODS: BRVO was induced in pigmented rats by focal laser photocoagulation. The subsequent changes in the retinal angiogram were followed up, both in vivo by confocal scanning laser ophthalmoscopy and ex vivo by confocal microscopy. RESULTS: At day 1, capillary closure affected the three microvessel layer differentially, the intermediary layer being the most affected. Collateral veins, which were initiated by the dilation of deep-layer venules, pursued their course below adjacent arteries. These microvascular changes peaked between days 1 and 3. After recanalization at day 3, microvascular changes regressed gradually but incompletely, and at day 30 capillary closure and venule dilation persisted. CONCLUSIONS: Transient occlusion of a retinal vein in rats leads to short- and long-term microvascular remodeling upstream of the occlusion site. This study describes a model for the tridimensional arrangement of retinal microvessel that accounts for the topography of the early capillary closure and collateral vessel formation that occur after BRVO. In the long term, these changes regressed incompletely, with recanalization of the occluded vein, suggesting that after a short period of occlusion, microvascular changes may become at least partially independent of flow. Despite the intrinsically limited applicability of this model to human vein occlusion, the results suggest that even if therapeutic decompression of an occluded vein is performed early, it may not reverse capillary dropout completely.

Structural and hemodynamic analysis of the mouse retinal microcirculation.

PURPOSE: In the holangiotic retina, little is known about the connections between and the circulation within microvessel layers. The goal of the present study was to explore the three-dimensional arrangement and hemodynamics of mouse retinal microvessels. METHODS: Confocal microscopy was performed on fluorescein dextran-filled retinal flatmounts. Capillary velocity in the deep layer was measured by epifluorescence intravital microscopy. The changes in the studied parameters after branch retinal vein occlusion were evaluated. RESULTS: The superficial and intermediate layers are both asymmetric crossroads for capillary blood flow, with approximately 70% of the capillary connections directing the flow from the arterioles into the deep layer. The venous flow from the deep layer joins the major veins in the superficial layer through transverse venules, indicating that major veins are directly connected to the deep layer. Red and white blood cell velocities +/- SD in the deep layer were 1.26 +/- 0.34 and 0.8 +/- 0.32 mm/sec respectively. After branch vein occlusion, venule dilation and decreased velocity were observed in the deep layer. CONCLUSIONS: In the mouse retina, a tridimensional model of retinal microcirculation was established, showing that most microvessel connections on the arteriolar side direct the flow from the superficial to the deep layer, and vice versa on the venular side. However, the presence of direct arteriovenous connections in the superficial layer and the longer vessel length in the deep layer offer the possibility of actively modulating intraretinal flow. Compared with other capillary beds, both the capillary velocity and microhematocrit are high, a situation that favors nutrient delivery to the inner retina.

Axon-tracing properties of indocyanine green.

OBJECTIVE: It has been shown recently that the application of indocyanine green (ICG) over the retinal surface is followed by prolonged staining of the optic disc. This study was performed to analyze the diffusion of ICG in the optic tract. METHODS: Anterograde diffusion of ICG was evaluated after injection into the vitreous of rabbits. Retrograde diffusion was evaluated after microinjection into the lateral geniculate nucleus of rats. RESULTS: Anterograde and retrograde diffusion occurred along the axons at a rate of about 2 mm per hour when ICG was injected. Anterograde staining of the visual pathway persisted for several weeks. After injection into the lateral geniculate nucleus, fluorescent retinal ganglion cells could be visualized for at least 7 days in conscious rats by conventional infrared photography. Microscopic examination findings of retrograde-labeled retinas showed the presence of ICG vesicles inside the axons, cytoplasm, and dendrites of retinal ganglion cells. No evidence of toxic effects was detected by optical microscopy. CONCLUSIONS: Indocyanine green is a fast bidirectional axonal tracer. Injection into normal vitreous results in long-term staining of the visual pathway. In vivo counting of ICG-labeled retinal ganglion cells in rats can be performed for several days after injection. Indocyanine green is therefore potentially of interest for use in experimental neurophysiological studies. CLINICAL RELEVANCE: The present results suggest that in humans, epiretinal application of ICG results in prolonged staining of the visual pathway. Therefore, additional studies of long-term toxic effects of ICG on neural cells are warranted before recommending its use in humans as an intraoperative tool for vitreoretinal surgery.

Caveolin-1 and -3 dissociations from caveolae to cytosol in the heart during aging and after myocardial infarction in rat.

OBJECTIVE: Caveolins, the structural proteins of caveolae, modulate numerous signaling pathways including Nitric Oxide (NO) production. Among the caveolin family, caveolin-1 and -3 are mainly expressed in endothelial and muscle cells, respectively. In this study, we investigate whether (i) changes in caveolin abundance and/or distribution occur during cardiac aging and failure in rat, and (ii) the process could influence NO synthase (NOS) activity. METHODS: Using immunohistolabelling and Western blot approaches, expression and distribution of caveolins were analysed in adult (Ad), senescent (S-Sh) and myocardial infarction-induced failing (S-MI) hearts. NOS3/caveolin-1 interactions were evaluated by immunoprecipitation assays. RESULTS: At the microscope level, caveolin-1 distribution in the endothelial cells was unchanged between the groups. Conversely the typical distribution of caveolin-3 in myocyte sarcolemma was dramatically altered in S-MI rats, resulting in a heterogeneous pattern throughout the septum. Total abundance of caveolin-1 and -3 remained stable whatever the group. In the fractions free of caveolae (Triton X-100 soluble), the levels of caveolin-1 alpha and -3 increased with aging (+20%, and +104%, P<0.05 versus Ad, respectively) and were further enhanced in S-MI (+25%, +30%, P<0.05, P<0.001 versus S-Sh respectively). In these fractions, NOS3/caveolin-1 alpha complexes increased as well. In addition, NOS activity was negatively correlated to caveolin-1 level in the cytosolic fractions. CONCLUSIONS: We demonstrate that dissociation of caveolin from caveolae is associated with aging and heart failure, the process being related to the decreased NOS activity.

Critical role of endothelial nitric oxide synthase and cyclooxygenase in response of rabbit basilar artery to serotonin.

The modes of action of serotonin (5-HT) on the tone of the rabbit basilar artery were investigated in vitro with the aim of determining the exact role of the endothelium. After sacrificing the animal under pentobarbital anesthesia, 3-mm segments of the artery were removed and mounted in a 5-ml myograph for isometric tension recording. Vessels precontracted by histamine were relaxed by acetylcholine. Mean maximum relaxation at 10(-4) M was reduced from 79% to 22% (P < 0.001) by 10(-5) M N-nitro-L-arginine (L-NA), and from 73% to 63% (NS) by 3.12(-6) M indomethacin. Intact non-precontracted vessels were contracted by 5-HT (10(-9) M to 10(-5) M): 10(-5) M L-NA significantly increased the contractile force (approximately twofold), whereas 3.10(-6) M indomethacin significantly decreased it (to approximately 35%). In histamine-precontracted vessels, 5-HT induced at low concentrations (3.10(-9) M to 3.10(-8) M) a reduction in tone and induced an increase in tone at higher concentrations. At 10(-5) M, L-NA abolished the relaxant phase of the response, whereas 3.10(-6) M indomethacin potentiated it. In uridine triphosphate-precontracted segments, there was not a net reduction in tone under 5-HT at 3.10(-9) to 3.10(-8) M, but further contraction appeared at higher concentrations. The presence of 10(-5) M L-NA significantly increased the contraction to 5-HT, but 3.10(-6) M indomethacin did not significantly reduce it. Endothelial lesion reduced by about 50% the contractile response of L-NA-treated arteries to 5-HT; and conversely, endothelial lesion increased approximately twofold the contraction of indomethacin-treated arteries to 5-HT. We conclude that 5-HT causes the release from the endothelium of two vasoactive factors, one of which is probably the vasodilator nitric oxide, but the size of the relaxation may depend on the prevailing level of nitric oxide synthase activation. The second factor is a cyclooxygenase-dependent contractile agent. However, the contraction to 5-HT was not modified by the presence of the thromboxane synthase inhibitor CGS 13080 (10(-4) M), suggesting that thromboxane A2 is not the main contractile agent released.

Cerebrovascular inflammation following subarachnoid hemorrhage.

Aneurysmal subarachnoid hemorrhage frequently results in complications including intracranial hypertension, rebleeding and vasospasm. The extravasated blood is responsible for a cascade of reactions involving release of various vasoactive and pro-inflammatory factors (several of which are purported to induce vasospasm) from blood and vascular components in the subarachnoid space. The authors review the available evidence linking these factors to the development of inflammatory lesions of the cerebral vasculature, emphasizing: 1) neurogenic inflammation due to massive release of sensory nerve neuropeptides; 2) hemoglobin from lysed erythrocytes, which creates functional lesions of endothelial and smooth muscle cells; 3) activity, expression and metabolites of lipoxygenases cyclooxygenases and nitric oxide synthases; 4) the possible role of endothelin-1 as a pro-inflammatory agent; 5) serotonin, histamine and bradykinin which are especially involved in blood-brain barrier disruption; 6) the prothrombotic and pro-inflammatory action of complement and thrombin towards endothelium; 7) the multiple actions of activated platelets, including platelet-derived growth factor production; 8) the presence of perivascular and intramural macrophages and granulocytes and their interaction with adhesion molecules; 9) the evolution, origins, and effects of pro-inflammatory cytokines, especially IL-1, TNF-alpha and IL-6. Human and animal studies on the use of anti-inflammatory agents in subarachnoid hemorrhage include superoxide and other radical scavengers, lipid peroxidation inhibitors, iron chelators, NSAIDs, glucocorticoids, and serine protease inhibitors. Many animal studies claim reduced vasospasm, but these effects are not always confirmed in human trials, where symptomatic vasospasm and outcome are the major endpoints. Despite recent work on penetrating vessel constriction, there is a paucity of studies on inflammatory markers in the microcirculation.