Introduction

The importance of perivascular nerves and endothe-lial cells in the local control of cerebral circulation isnow well established.1,2 Immunocytochemical studieshave demonstrated localization of vasoactive agentsin both perivascular nerves and endothelial cells ofcerebral vessels.3–8

Cerebral arteries, particularly those of the circle ofWillis, are innervated by a dense perivascular plexusof sympathetic, parasympathetic and sensory nervefibres.9,10 Sympathetic nerve fibres containing nora-drenaline (NA) and neuropeptide Y (NPY) originatepredominantly from the superior cervical ganglia.4

Parasympathetic nerve fibres containing cholineacetyltransferase (ChAT, the enzyme responsible forthe synthesis of acetylcholine), vasoactive intestinalpolypeptide (VIP) and nitric oxide synthase (NOS,the enzyme responsible for the synthesis of nitricoxide) have their cell bodies in cranial ganglia, mainlythe sphenopalatine ganglia with some contributionsfrom the otic and carotid mini-ganglia.7,11 Thepresence of NPY has been described in some ChAT/VIP-containing neurones of cranial parasympatheticganglia, which do not display any sympatheticmarkers.4,12 Cerebrovascular sensory nerve fibresarise from the ophthalmic division of the trigeminalganglia; these have been shown to contain substance

P (SP) and calcitonin gene-related peptide (CGRP).3

Some NOS-positive cerebrovascular nerve fibres havealso been shown to be sensory.7

There have been no investigations of theimmunolocalization of endothelin-1 (ET-1) in cere-brovascular nerve fibres, although ET has beendetected by immunoassay in the ganglia sendingprojections to cerebral arteries.13 In the present ultra-structural immunocytochemical study we demon-strate that ET-1 is localized in perivascular nerves ofthe rat basilar artery.

Materials and Methods

Electron-immunocytochemical procedure :Adult (3 months old) male Wistar rats (n = 5) were anaes-thetized with sodium pentobarbitone (6 0mg kg–1,i.p.; Sagatal, RMB Animal Health, Harlow, UK) and perfused through the heart (left ventricle) with 4% paraformaldehyde and 0.1% glutaraldehyde (at room temperature) in 0. 1 M phosphate buffer at pH 7.4. Brains were removed and the basilar arteriesdissected. Arteries were immersion-fixed for 5 h at 4°C with the same fixative and then transferred to phosphate buffer and stored overnight at 4°C. The following day the arteries were cut longitudi-nally into strips 3– 4 mm long, and processed for the

Autonomic Nervous System

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THE endothelium is recognized as a major source ofendothelin in the vasculature. In this report we showfor the first time that endothelin can be demonstratedwith immunoelectronmicroscopy in perivascular nerves.About 36% of the axon profiles (varicosity/inter-varicosity) examined in the rat basilar artery showedimmunolabelling with a polyclonal antibody toendothelin-1. Endothelin-labelled axon varicosities werecharacterized by the presence of small spherical agran-ular vesicles (4 2 ± 1 nm); some varicosities also containedlarge granular vesicles (92 ± 5 nm) with labelled cores.Immunolabelling was mostly distributed in the axoplasmand in association with the membrane of synapticvesicles (the lumen of the small agranular vesicles wasimmuno-negative). The presence of endothelin inperivascular nerves of the basilar artery suggests aneuronal as well as endothelial role in the physiologicalcontrol of the vessel wall. NeuroReport 9: 3903–3906 ©1998 Lippincott Williams & Wilkins.

Key words: Basilar artery; Endothelin; Perivascular nerves;Rat; Ultrastructure

Endothelin in perivascularnerves. An electron-immunocytochemicalstudy of rat basilar artery

Andrzej Loesch,1,CA Pamela Milner1,2

and Geoffrey Burnstock1,2

1Department of Anatomy and DevelopmentalBiology and Centre for Neuroscience,University College London, Gower Street,London WC1E 6BT; 2Autonomic NeuroscienceInstitute, Royal Free Hospital School ofMedicine, Rowland Hill Street, London NW3 2PF, UK

CACorresponding Author

Website publication 1 December 1998 NeuroRepor t 9, 3903–3906 (1998)

pre-embedding peroxidase-antiperoxidase (PAP)electron-immunocytochemistry of ET-1. Initially, thearteries were exposed to 0.3% hydrogen peroxide in50% methanol for 30 min (for blocking of endoge-nous peroxidases and to improve infiltration ofimmunoreagents), washed in 0.05 M Tris-bufferedsaline (TBS; DAKO, Carpinteria, CA, USA), andthen exposed for 1.5 h to normal goat serum (NordicImmunology, Tilberg, the Netherlands) diluted 1:9 in TBS containing 0.1% sodium azide (this buffer was used for the dilution of antibodies). Afterrinsing in TBS, the specimens were incubated for 48 h at 4°C with rabbit polyclonal antibody to ET-1 diluted 1:1000. After washing in TBS, the spec-imens were then (i) exposed for 1.5 h at room temper-ature to goat anti-rabbit IgG serum (Biogenesis,Bournemouth, UK) diluted 1:40, (ii) washed in TBS,(iii) incubated for 3 h at room temperature with arabbit PAP complex (DAKO, Glostrup, Denmark)diluted 1:75, and (iv) treated with 3,3′-diaminoben-zidine (Sigma, Poole, UK) and 0.01% hydrogenperoxide. Specimens were washed 1in TBS and 0.1 M cacodylate buffer (pH 7.4), postfixed in 1%osmium tetroxide for 1 h at 4°C, washed in cacody-late buffer, dehydrated in a graded series of ethanoland propylene oxide embedded in Araldite. Theultrathin sections were stained with uranyl acetateand lead citrate and subsequently examined with aJEM-1010 electron microscope.

Immunocytochemical controls: Rabbit polyclonalET antibody to human/porcine ET-1 was obtainedfrom Cambridge Research Biochemicals (CRB,Cambridge, UK). Preincubation of ET-1 antibodywith 10 nmol human ET-1, ET-2 or ET-3 substancesper ml of optimally diluted antibody was sufficientto abolish immunostaining (CRB). We have previ-ously used this antibody in PAP electron immuno-cytochemistry of endothelial cells14,15 as well as inimmunoassay to detect ET levels in the perfusate of freshly harvested endothelial cells.16 Our ownimmunoabsorption studies showed that preabsorp-tion of ET-1 antibody with its respective antigen(synthetic human ET-1, CRB) eliminated positivelabelling in endothelial cells.14 Immunoassay of ET-1 antibody showed that this antibody cross-reactedwith big endothelin 39 (7%), ET-2 (15%) and ET-3(100%).16 In the present study, the specificity of theimmunolabelling was investigated routinely by omis-sion of the ET-1 antibody and IgG steps, indepen-dently, as well as by replacement of primary antibodyby non-immune normal rabbit serum (NordicImmunology).

Measurements: In order to calculate the diameter of synaptic vesicles that appeared within the ET-1

positive axon profiles (varicosities) in the basilararteries, two measurements, at right angles to eachother, were taken for each vesicle from electronmicrographs. In order to establish the percentage ofperivascular nerve fibres (axon profiles) positive andnegative for ET-1, these were counted in ultrathinsections and the electron micrographs taken fromdifferent levels of specimens. The percentage of ET-positive axon profiles per nerve bundle wascalculated and results expressed as mean ± s.e.m. for n nerve bundles examined.

Results

Perivascular nerve fibres (axon varicosity/intervari-cosity) displaying immunoreactivity for ET-1 wereobserved both within the outer and inner plexusessupplying the basilar artery (Fig. 1a,b). More than30% of the axons were positive for ET-1 (36 ± 5%per nerve bundle, n = 26; 92 of a total 274 axonprofiles examined in three rats). ET-1-positive axonswere seen either in association with, or free from,Schwann cell processes. The narrowest separationobserved between vascular smooth muscle and ET-1-labelled perivascular nerve profiles was about60–70 nm; the widest separation was 4 mm. Smallspherical agranular vesicles predominated in ET-1-positive varicosities. The diameter of these vesicleswas 42 ± 1 nm (n = 50; five varicosities examined; 10vesicles per varicosity were measured). Some ET-1-positive varicosities also contained a few large gran-ular vesicles showing immunoprecipitate associatedwith the granular core (Fig. 1c). The diameter of thesevesicles was 92 ± 5 nm (n = 5; two varicosities exam-ined). Immunoreactivity for ET-1 was associatedwith the cytoplasmic side of the membrane of bothsmall spherical agranular and large granular vesiclesand mitochondria in varicosities (Fig. 1c). In general,ET-1-positive varicosities were intensely labelled, but varicosities displaying a relatively low level ofimmunoreactivity were also observed. By contrast,no immunolabelling was observed in control prepa-rations. An example of control perivascular axonprofiles processed for immunocytochemistry, whereET-1 antibody was omitted from the incubationmedium, showing absence of immunoprecipitate, isillustrated in Fig. 1d.

Discussion

The present electron-immunocytochemical studydemonstrates that in the Wistar rat basilar artery, a subpopulation of perivascular axons exhibitsimmunoreactivity to ET-1. This new finding extendsprevious immunocytochemical observations of theinnervation to cerebral vessels and adds ET-1

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FIG. 1. Perivascular nerve fibres of the rat basilar artery labelled for ET-1 (a–c) and a control specimen (d). (a) Several ET-1-positive (whiteasterisks) and ET-1-negative (Ax) axon profiles are seen in the nerve bundle of an outer plexus distant from the vascular smooth muscle(sm) of the media. col Collagen fibres; fib fibroblast. ×18 700. (b) A nerve bundle of an inner plexus close to smooth muscle displays ET-1-positive and ET-1-negative axon profiles. m, Mitochondrion. ×27 000. (c) A higher magnification example of an ET-1-positive varicosityshowing immunolabelling attached to the membrane of small agranular vesicles (av) and in association with cores of large granular vesi-cles (gv). ×92,000. (d) Perivascular nerve fibres in control specimen (omission of ET antibody) showing no immunolabelling. ×19 000

to the list of vasoactive agents identified in cerebro-vascular nerves.

A question raised by the present results is whetherfunctional implication may be associated with thepresence of ET-1 in a subpopulation of perivascularaxons of cerebral blood vessels. It has been knownfor some time that the cerebrovascular endothelialcells contain ET-1, as demonstrated immunocyto-chemically in rabbit basilar and posterior communi-cating arteries,5 rat basilar artery17 and human middlecerebral artery.15 Release of ET-1 from rabbit cere-bral vessels has been shown following perfusion ofthe cerebral circulation with a perfluorocarbon emul-sion at increased flow.18 As a potent vasoconstrictorto vascular smooth muscle, ET-1 released fromendothelial cells may be implicated in vasomotorcontrol of the vessel wall.19 The present findingssuggest a possibility of perivascular nerves as a sourceof ET-1 release and therefore in endothelium-inde-pendent (neuronal) control of vasoconstriction.

The possibility cannot be excluded that the syner-gistic action of ET-1 derived from the endotheliumand perivascular nerves could cause vasospasm inpathophysiological conditions and eventually lead to cerebral ischaemia. A pathophysiological role forET-1 has been implicated in delayed vasospasmfollowing subarachnoid haemorrhage.20 Pharmaco-logical studies have already shown that hypoxia andanoxia cause Ca2+-dependent constrictions of isolatedcerebral arteries (basilar artery) and that removal ofthe endothelium may attenuate these constrictions.21

A role for neuronal ET-1 has not yet been explored.A hypoxia-inducible factor has been identified on the ET-1 gene.22 In addition to its action as a potentvasoconstrictor, ET-1 has a wide spectrum of phar-macological effects; it is mitogenic and trophic tosmooth muscle cells and has been proposed as ananti-apoptotic agent.23,24

There are several lines of evidence that ET-1 actsas a neuromodulator in the central nervous systemand may play a significant role in the central controlof fluid balance and autonomic activity.25,26 ET-1immunoreactivity and/or ET-1 mRNA expressionhas previously been localized in human spinal cord,dorsal root ganglia and enteric plexuses.27,28 It isknown that ET-1 may influence the autonomicnervous system in that it inhibits NA release fromperivascular sympathetic nerves and potentiates theeffects of sympathetic nerve stimulation.29,30 It has

also been suggested that ET-1 may play a role in themodulation of SP/CGRP release from primaryafferent neurones.31 In addition to the endothelium,perivascular nerves must now be considered as apotential source of ET.

Conclusion

A proportion of axon profiles in the rat basilar arteryimmunostain for ET-1. The presence of ET inperivascular nerves suggests a neuronal role for ETin the physiological control of the vessel wall inadditon to its known endothelial role.

References

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ACKNOWLEDGEMENTS: The authors thank Mr R. Jordan for editorial assis-tance and Mr. M. Miah for skilful technical assistance. The support of the BritishHeart Foundation is gratefully acknowledged.

Received 12 August 1998;

accepted 23 September 1998

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