Effects of sepsis on mast cells in rat dura mater: influence of L-NAME and VIP.

1. The influence of lipopolysaccharide (LPS)-induced sepsis on the various mast cell phenotypes of rat dura mater were examined both by immunohistochemical and biochemical methods. 2. Three different populations of mast cells were identified in control rats: connective tissue type mast cells (CTMC) which contain rat mast cell protease1 (RMCP1), histamine, serotonin and heparin, mucosal type mast cells (MMC) which contain RMCP2, histamine and serotonin, and intermediate type which contains both RMCP1 and RMCP2 and probably various proportions of amines and heparin. 3. LPS (25 mg kg(-1) i.p.) caused changes in the proportions of the various types of mast cells. The number of MMC and intermediate type mast cells significantly increased and the number of mast cells immunopositive for both heparin and serotonin significantly decreased. Biochemical analysis showed that the histamine concentration of dura increased while its serotonin concentration decreased. 4. While vasoactive intestinal peptide (VIP) (25 ng kg(-1) i.p.) appears to potentiate LPS effects on dura mater mast cells, non-selective inhibition of nitric oxide (NO) synthase by N(g)-nitro-L-arginine methyl ester (L-NAME) (30 mg kg(-1) i.p.) did not influence sepsis-induced mast cell changes. 5. These findings suggest that mast cells of dura mater may play a role in brain protection during sepsis.

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.

Long-term superior cervical sympathectomy induces mast cell hyperplasia and increases histamine and serotonin content in the rat dura mater.

Nerve fibres and mast cells are often described in close morphological and functional interactions in various organs such as the dura mater. The respective roles of mast cell activation and sympathetic impairment in cluster headache and migraine attacks have been repeatedly suggested. We have thus investigated the long-term effects of sympathectomy on mast cell morphology and content in the rat dura mater. Fifteen to 60 days after either sham, unilateral or bilateral superior cervical ganglionectomy, dura were removed for either histochemical or biochemical analysis. In the first case, they were fixed and mast cell heparin was stained by fluorescein isothiocyanate-conjugated avidin. Microscopic examination was followed by digital acquisitions using a tomographic process to assess mast cell density in the whole depth of the dura mater. Unilateral ganglionectomy induced a progressive and significant increase in mast cell density 15-60 days post-surgery in contralateral hemi-dura and 30 days post-surgery in ipsilateral hemi-dura. This increase was significant in both dura 60 days after bilateral ganglionectomy. Following perfusion with saline, we also examined the content of histamine and serotonin, pre-formed amines stored in mast cell granules. Biochemical analysis of dura serotonin and histamine content using high-pressure liquid chromatography and radioenzymatic assays, respectively, revealed under all conditions a serotonin tissue concentration lower than that of histamine. After sham ganglionectomy, the dura serotonin content increased from 15 to 60 days post-surgery, whereas the histamine content remained stable over the same period. After unilateral ganglionectomy, the histamine content increased progressively and significantly 30-60 days post-surgery in both hemi-dura, whereas the serotonin content became significantly different from that of sham only 60 days post-surgery in the ipsilateral dura. After bilateral ganglionectomy, the histamine level significantly increased in both hemi-dura 15-60 days post-surgery, whereas the serotonin level had significantly increased at 60 days post-surgery. These results clearly demonstrate, for the first time, a long-term trophic effect of sympathetic nerve degeneration on mast cells in the dura mater. Since mast cell activation has been described previously on the painful side of cluster headache patients during attack periods, we propose that the sympathetic impairment reported in these patients could be prominent, directly or indirectly inducing mast cell hyperplasia and changes in amine contents in the tissue concerned.

NE inhibits cerebrovascular mast cell exocytosis induced by cholinergic and peptidergic agonists.

Autonomic and sensory nerves frequently contact mast cells contained in rabbit leptomeningeal arteries. We have previously shown that parasympathetic and peptidergic neurotransmitters can stimulate mast cell granule exocytosis and serotonin (5-HT) release. In the present study, we examined ex vivo the possible action of the main sympathetic neurotransmitter, norepinephrine (NE), on this exocytotic process. NE, which had no effect on mast cell 5-HT content per se, totally inhibited carbachol-induced 5-HT release and partially reduced neuropeptide-induced 5-HT release. Pretreatment with the alpha 1-adrenergic blocker did not affect the inhibitory effect of NE. Pretreatment with specific beta 1- or beta 2-adrenergic blockers antagonized this action, but the beta 2-blocker exerts a more specific dose-dependent antagonism. Together with our previous data, these results indicate that the equilibrium between autonomic and sensory nerves may determine the release of 5-HT from mast cells (parasympathetic and sensory nerves can trigger exocytosis while the sympathetics can inhibit it). Such a mechanism could be implicated in pathophysiological events in which autonomic dysfunction is likely to be involved, such as vascular headache or other phenomena involving inflammation.

Substance P, calcitonin gene-related peptide, and capsaicin release serotonin from cerebrovascular mast cells.

Rabbit leptomeningeal arteries contain granular cells resembling mast cells that frequently contact autonomic and sensory nerve profiles. In the present in vitro study, we determined whether these cells could be stimulated by substance P (SP) and calcitonin gene-related peptide (CGRP), which are stored and released by sensory C fibers. Immunohistochemistry of the middle cerebral artery showed that 5-HT was stored only in mast cell-like granules. This pool of 5-HT decreased in a dose-dependent manner when exogenous SP and CGRP were added to the incubation solution or when endogenous neuropeptides were released from nerve terminals by capsaicin. The simultaneous administration of CGRP and SP induced a dramatic exocytosis and a 5-HT release significantly greater than the sum of the individual effects of the two neuropeptides. We conclude that, as in classical connective tissue mast cells, the amine content of these granular cells can be released by a degranulation process induced by neuropeptides. The effects of capsaicin suggest that this phenomenon can be triggered by axon reflex of C fibers. The data also provide the first evidence of a synergistic action of SP and CGRP on mast cell degranulation.

Absence of serotonergic innervation from raphe nuclei in rat cerebral blood vessels--II. Lack of tryptophan hydroxylase activity in vitro.

Neurochemical studies performed in vivo have suggested that serotonin-containing and -synthesizing nerves, originating in the raphe nuclei, directly innervate pial blood vessels. Nerve fibres of these vessels have been shown by immunocytochemistry to contain tryptophan hydroxylase (the rate-limiting enzyme of serotonin synthesis) but no serotonin. The present study examines this contradiction by measuring in vitro the tryptophan hydroxylase activity of rat cerebral vessels and femoral arteries (which also contain tryptophan hydroxylase-immunopositive nerves), and comparing them to the tryptophan hydroxylase activity of the rat pineal body, raphe nuclei and brain cortex under identical conditions. Oxygenated incubation solutions contained either [14C]- or "cold" L-tryptophan (2 x 10(-5) to 5 x 10(-4) M) and NSD-1015 (3-hydroxybenzylhydrazine) which inhibits the decarboxylation of 5-hydroxytryptophan, the second step of serotonin synthesis. Tissue fragments were incubated for 35-60 min. High-performance liquid chromatography (on tissue extracts and incubation solutions) as well as determination of 14C activity in the 5-hydroxytryptophan fraction of elution from tissue extracts showed that the pineal body, the raphe nuclei and cortical slices synthesize various amounts of 5-hydroxytryptophan under our experimental conditions. All these tissues contained serotonin. Femoral arteries, but not cerebral vessels, also contained small amounts of serotonin stored before incubation, probably in mast cells. In contrast to brain tissues, no measurable amounts of "cold" or [14C]5-hydroxytryptophan were found in cerebral blood vessel and femoral artery extracts or incubation solutions. Under identical experimental conditions, sympathetic nerves of both types of vessels were able to synthesize large amounts of L-DOPA when incubation solutions contained L-tyrosine instead of L-tryptophan.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbachol induces granular cell exocytosis and serotonin release in rabbit cerebral arteries.

Previously, we reported that rabbit cerebral arteries contain mast cells that frequently establish close contacts with parasympathetic-like nerve fibers. Here we have examined the possible function of this link by comparing the effects of carbachol and compound 48/80 on mast cell morphology and on the serotonin (5-HT) and histamine content of these arteries. In vivo, 2 micrograms/min of compound 48/80 or 1 micrograms/min of carbachol was infused for 30 min into one internal carotid artery of pentobarbital anesthetized rabbits, the contralateral artery being infused with vehicle. In vitro, the action of 10(-6) M carbachol was tested on isolated middle cerebral artery trees (MCAs) in the presence or absence of 10(-7) M atropine. The effects of carbachol were also tested in vitro on sympathectomized arteries. The 5-HT and histamine contents of all MCAs were measured by radioenzymatic assay, and fragments were prepared for electron microscopy. No histamine was detectable in any artery studied. The 5-HT content of arteries from control animals and those perfused with vehicle (in vivo) or incubated in the physiological solution (in vitro) was 250-300 pmol/mg protein. Both compound 48/80 and carbachol reduced this amount by approximately 50% and induced a marked degranulation of mast cells. Both secretion and degranulation were dramatically blocked in vitro by atropine. No difference in the 5-HT content was observed between intact and sympathectomized arteries under any condition. We conclude that a large proportion of rabbit cerebrovascular 5-HT is stored in mast cells and that cholinergic nerve activation could theoretically release this pool by acting on mast cell muscarinic receptors.

Effects of methylene blue on hypoxic cerebral vasodilatation in the rabbit.

The present studies were conducted to examine the role of cerebrovascular guanylate cyclase in hypoxic cerebral vasodilatation. In arteries mounted in vitro for measurements of isometric tension, 20 min of hypoxia (bath oxygen partial pressure, approximately 15 Torr) significantly increased cyclic GMP levels from 16 to 32, from 15 to 25 and from 20 to 38 pmol/g in rabbit common carotid, internal carotid and basilar arteries. These increases were blocked either by pretreatment with 3 microM methylene blue, or by removal of the vascular endothelium. Methylene blue also significantly delayed hypoxic relaxation in the basilar and internal carotid arteries, and blocked transient hypoxic vasoconstriction in the common carotid. Together, these in vitro results demonstrate that vascular cytosolic guanylate cyclase participates in an endothelium-dependent manner in the direct effects of hypoxia on cerebral arteries, and that the nature of this participation varies significantly between arteries. When methylene blue (20 mg/kg) was administered in vivo, however, it had no effect on the magnitude of hypoxic cerebral vasodilatation as determined by both local (mass spectrometry) and global (venous outflow) methods of blood flow measurement. This latter finding suggests that: 1) large and small cerebral arteries may differ significantly in terms of either endothelial function or sensitivity to methylene blue; or 2) feedback regulation of other mechanisms of hypoxic cerebral vasodilatation compensate for the effects of guanylate cyclase inhibition. Additional experiments using other inhibitors of cytosolic guanylate cyclase and/or vessels isolated from the cerebral microcirculation will be necessary to distinguish between these possibilities.

Nonuniformity of CBF response to NE- or ANG II-induced hypertension in rabbits.

The regional response of brain vasculature to moderate hypertension was investigated using two hypertensive drugs, norepinephrine (NE) and angiotensin II (ANG II), infused intravenously at low concentrations (increase in blood pressure 15-40 mm Hg). Regional cerebral blood flow (rCBF) was measured in unanesthetized and anesthetized rabbits using the [14C]ethanol saturation technique. In both groups of animals, NE and ANG II induced regional differences in the flow changes as compared with controls, confirming a regional (or segmental) heterogeneity in the regulatory mechanisms to hypertension. The responses to identical rises in blood pressure (BP) in most of the structures analyzed depended on the drug used. In the unanesthetized rabbits, the increase in vascular resistance induced by NE was greater than that induced by ANG II. With the two drugs, there was no correlation between the flow changes in any of the structures considered and either the BP increase or the BP level in unanesthetized animals. However, these flow changes were correlated with the BP increase in anesthetized animals, although differences between the effects of NE and ANG II were again observed. This study suggests that cerebrovascular regulatory mechanisms in hypertension are probably more complex than a simple myogenic reaction. Their heterogeneity and their dependence both on the cause of hypertension and on the presence of anesthetics suggest the intervention of an integrating pathway.

Intrinsic and extrinsic mechanisms involved in the cerebrovascular reaction elicited by immobilization stress in rabbits.

Variations in cerebral blood flow and partial pressures of oxygen and carbon dioxide (pO2, pCO2) were studied in rabbits during short-duration (1 min) immobilization stress. The techniques used enabled us to determine these variables locally in the caudate nucleus in a continuous, simultaneous and quantitative fashion. It could be shown that cerebral blood flow and arterial blood pressure increased in parallel immediately after inducing the stress reaction, and that pO2 increased further, indicating that cerebral oxygen supply is maintained by the hyperaemia. Previous administration of a beta-receptor blocker or of a cholinergic receptor blocker significantly diminished the cerebrovascular reaction to stress, inducing a decrease in pO2 during the reaction. Administration of both blockers nearly abolished the cerebral vasodilation studied. Previous administration of an alpha-receptor blocker enhanced the reactive hyperaemia. No disturbance of the blood-brain barrier could be observed in rabbits subjected to stress. Intravenous injection of adrenaline, as well as angiotensin II inducing similar increases in blood pressure, had no comparable effect on the blood flow. The conclusion is that in this model of anxiety, neurogenic mechanisms are involved in the provision of a sufficient oxygen supply to the brain.

Long-term effects of superior cervical ganglionectomy on cortical blood flow of non-anesthetized rabbits in resting and hypertensive conditions.

The long-term effects of sympathectomy on cerebral cortical blood flow (CBF) were studied in the conscious rabbit. The quantitative, repeated measurements of blood flow were made by determination of helium clearance by mass spectrometry and were obtained simultaneously with measurements of local tissue pO2 and pCO2. Eight to 10 weeks after unilateral sympathectomy, resting blood flow in the homolateral cortex was decreased by a mean of 17% compared to the heterolateral cortex. In two animals in which blood flow was recorded both before and after the sympathectomy, the same phenomenon was observed from 8 to 30 days after the operation. The response to i.v. infusion of noradrenaline was identical in both hemispheres: doses inducing a 40% rise in blood pressure did not significantly modify CBF. The responses to angiotensin II-induced hypertension were also identical. Histochemical verifications demonstrated the effectiveness of the denervation in the pial arteries and the intraparenchymal arteries in the region studied. Thus the decrease in CBF induced by chronic sympathectomy cannot be attributed to the development of hypersensitivity to catecholamines. This decrease remained stable whatever the value of resting flow and was maintained under anesthesia. It is concluded that, as in the peripheral circulation, chronic sympathectomy affects the equilibrium of the vascular smooth muscle fibers, but that circulating amines play no compensatory role in the cerebral circulation because of the blood-brain barrier.

Generalized cerebral vasoconstriction induced by intracarotid infusion of angiotensin II in the rabbit.

This study investigated the influence of angiotensin II, perfused into one common carotid artery at a dose of 0.065 micrograms/kg/min, on the cerebrovascular resistance of the anesthetized rabbit by means of complementary in vivo methods. Heat clearance and mass spectrometry measurements indicated that in the homolateral caudate nucleus angiotensin induced a significant decrease in local blood flow (18.2 +/- 9%), a fall in pO2 (14.2 +/- 5.3%) and no significant change in pCO2. The [14C]ethanol tissue sampling technique revealed a significant decrease in flow in all 10 structures sampled in the brain. This decrease was similar in magnitude in both the ipsilateral and the contralateral hemisphere with regard to the site of injection. When expressed in terms of cerebrovascular resistance (CVR) and allowing for a slight increase in blood pressure (less than 10%), these results show that angiotensin II infusion induced an increase in CVR of 18-32%. We conclude that: A unilateral intracarotid infusion of a low dose of angiotensin II induces an increased vascular tone in all cerebral structures. This action, being bilateral, cannot readily be explained by a direct action of angiotensin II on the cerebral vessels in view of the very low recirculating concentration of angiotensin II (less than 10(-9) M). The hypothesis of a cerebral vasomotor influence of angiotensin II by action on a central structure is discussed.

Quantitative changes in regional cerebral blood flow of rats induced by alpha- and beta-adrenergic stimulants.

Cerebral blood flow was measured with the 14C-ethanol technique in 8 regions (frontal, parieto-temporal and occipital cortex, caudate nucleus, thalamus, cerebellum, mesencephalon, and pons) of rats. The highest flow values (83-89.5 ml/100 g/min) were found in cortical areas, whereas pons had the lowest flow (48 ml/100 g/min). Intravenous infusion of noradrenaline or adrenaline markedly reduced rCBF (by 22-48% of control levels) in all regions except thalamus, mesencephalon, and pons. The noradrenaline-induced reduction was blocked, and the effect of adrenaline reversed, after pretreatment with the alpha-receptor antagonist, phentolamine. Isoprenaline infusion markedly augmented rCBF in thalamus, mesencephalon, pons, and also in the caudate nucleus. The response was reduced by the beta-receptor antagonist, propranolol. The experiments show the presence and heterogenous distribution in the cerebrovascular bed of slpha- and beta-adrenoceptors that can be activated by sympathomimetics given systematically. If noradrenaline was allowed to pass the blood-brain barrier after osmotic opening with urea, an increased regional flow was obtained, probably due to a mechanism where the vasodilator effect secondary to activation of cerebral metabolism predominated over the direct vasoconstrictor effect of the amine.

Multiregional cerebral blood flow changes induced by a cholinomimetic drug.

The effect of parasympathomimetic drug, carbachol, on regional cerebral blood flow was tested in the rabbit with the 14C-ethanol tissue sampling technique. Intracarotid injection of carbachol significantly increased flow with respect to untreated control animals in 6 out of 10 structures sampled. However, compared with animals to which atropine was also administered, the flow increases were greater with carbachol alone. These effects are discussed in terms of muscarinic and nicotinic activation.

Is there an active mechanism limiting the influence of the sympathetic system on the cerebral vascular bed? Evidence for vasomotor escape from sympathetic stimulation in the rabbit.

The influence of the cervical sympathetic chain on cerebral circulation in the rabbit was studied by means of 3 complementary techniques. Two dynamic techniques involving chronically implanted probes were used: blood flow in the caudate nucleus (CN) was measured by thermal clearance; tissue PO2 and PCO2 in the same structure were measured by mass spectrometry. Other variables measured continuously and simultaneously included arterial blood pressure (BP), PaO2 and PaCO2. The third technique was a tissue sampling method based on the Fick principle and using 14C1 ethanol as tracer. Blood flow in 7 regions was measured at stable BP, PaO2 and PaCO2. Stimulation of the sympathetic chain at 15 Hz induced mean maximal decreases in CN blood flow of 23.9% (thermal clearance) and 24.4% (ethanol technique). Mean decrease of PO2 in the CN at 15 Hz was 16.6%. Significant falls in blood flow were observed with the ethanol technique in all 7 structures measured. During prolonged stimulation (greater than 1 min) CN blood flow and PO2 were found to escape towards the baseline level, which was sometimes even exceeded during the stimulation (blood flow). Stimulation frequency had only a very moderate influence on the rate of escape, and no evidence of a metabolic mechanism was found, although injection of barbiturate decreased the escape. These results are discussed with respect to the conflicting evidence on the effects of sympathetic stimulation in the brain, and to possible mechanisms for the escape phenomenon.

Quantitative multiregional blood flow measurements during cervical sympathetic stimulation.

The ethanol tissue sampling method for rCBF measurement was used to obtain information on the effects of cervical sympathetic stimulation in 8 cerebral structures in the non-anaesthetized rabbit. Sympathetic stimulation induced flow decreases of 12-29% according to structure, confirming the capability of this nerve to significantly reduce rCBF. Furthermore, a regional differentiation of cerebral structures into an 'anterior' group (mean decrease 22%) and a 'posterior' group (mean decrease 12%) with different reactivity to stimulation was established, thus confirming previous work in this laboratory with a local thermoclearance technique, and histochemical studies on sympathetic innervation to cerebral arteries and arterioles. These results provide strong evidence of the functional nature of the sympathetic nervous system in CBF regulation. The complementary nature of the ethanol technique (quantitative, multiregional measurement) and the local thermoclearance technique (continuous, semiquantitative measurements in 2-3 regions), and the absence of anaesthesia and significant trauma, means that combination of these two techniques offers considerable advantages in research on dynamic phenomena of the kind studied here.