Jan Erik Hardebo

Moderate homocysteinemia--a possible risk factor for arteriosclerotic cerebrovascular disease.

Highly elevated concentrations of homocysteine measured as homocysteine or cysteine- homocysteine mixed disulfide (MDS) are found in plasma and urine in subjects with inherited abnormalities of the methionine metabolism. These subjects have a high incidence of arteriosclerotic vascular complica- tions during childhood. Homocysteine causes endothelial cell injury and cell detachment that initiates the development of arteriosclerosis. The present study demonstrates a significantly elevated mean plasma MDS concentration in 19 patients with arteriosclerotic cerebrovascular disease compared to 17 controls. Our findings suggest that moderate homocysteinemia might be a risk factor for arteriosclerotic cerebrovascular disease. Stroke Vol 15, No 6, 1984

Origins and Pathways of Cerebrovascular Vasoactive Intestinal Polypeptide-Positive Nerves in Rat

In order to clarify the origins and pathways of vasoactive intestinal polypeptide (VlP)-containing nerve fibers in cerebral blood vessels of rat, denervation experiments and retrograde axonal tracing methods (true blue) were used. Numerous VIP-positive nerve cells were recognized in the sphenopalatine ganglion and in a mini-ganglion (internal carotid mini-ganglion) located on the internal carotid artery in the carotid canal, where the parasympathetic greater superficial petrosal nerve is joined by the sympathetic fibers from the internal carotid nerve, to form the Vidian nerve. VIP fiber bridges in the greater deep petrosal nerve and the internal carotid nerve reached the wall of the internal carotid artery. Two weeks after bilateral removal of the sphenopalatine ganglion or sectioning of the structures in the ethmoidal foramen, VIP fibers in the anterior part of the circle of Willis completely disappeared. Very few remained in the middle cerebral artery, the posterior cerebral artery, and rostral two-thirds of the basilar artery, whereas they remained in the caudal one-third of the basilar artery, the vertebral artery, and intracranial and carotid canal segments of the internal carotid artery. One week after application of true blue to the middle cerebral artery, dye accumulated in the ganglion cells in the sphenopalatine, otic and internal carotid mini-ganglion; some of the cells were positive for VIP. The results show that the VIP nerves in rat cerebral blood vessels originate: (a) in the sphenopalatine, and otic ganglion to innervate the circle of Willis and its branches from anterior and caudally and (b) from the internal carotid mini-ganglion to innervate the internal carotid artery at the level of the carotid canal and to some extent its intracranial extensions.

Calcitonin gene-related peptide is present in mammalian cerebrovascular nerve fibres and dilates pial and peripheral arteries

Calcitonin gene-related peptide (CGRP) is a novel 37-amino acid peptide occurring in neurones within sensory ganglia, in brain stem, as well as in the walls of blood vessels of peripheral organs. Pial arteries of cat showed a well-developed supply of CGRP-positive nerve fibres. The peptide was found to be a potent dilator of both pial and peripheral vessels of rabbit and cat, and of pial vessels from man. The dilatory effect was independent of the vascular endothelium and was not mediated through adrenergic, cholinergic or histaminergic smooth muscle receptors. The neurogenic vasoconstriction induced by electrical field stimulation was temporarily inhibited by CGRP, as studied in central ear arteries from rabbits. The results suggest that CGRP is a transmitter or modulator playing a role in the regulation of vascular tone.

Selective electrical stimulation of postganglionic cerebrovascular parasympathetic nerve fibers originating from the sphenopalatine ganglion enhances cortical blood flow in the rat

Recently, the origins and pathways of cerebrovascular acetylcholine- and vasoactive intestinal polypeptide-containing nerves have been elucidated in detail in the rat: The sphenopalatine ganglion is the major source for postganglionic parasympathetic fibers to the vascular beds of the cerebral hemispheres. To clarify the functional role of the nerves on cerebral blood vessels in vivo, brain cortical microvascular blood flow was measured in rats during electrical stimulation of these particular postganglionic fibers. Animals were subjected to transection of the right nasociliary nerve 2 weeks before the flow measurements to eliminate activation of peptidergic sensory fibers. Relative change in microvascular blood flow was continuously recorded by a laser-Doppler flowmeter system under α-chloralose anesthesia. The postganglionic fibers were electrically stimulated just proximal to the ethmoidal foramen by a bipolar platinum electrode (5 V; 0.5 ms; 3, 10, 30, 60 Hz; as a continuous stimulation for 90 s). Stimulation at 10 Hz induced a marked increase of the cortical blood flow (CoBF) on the ipsilateral side, whereas no change was observed on the contralateral side. It reached a maximum mean value of 42.5% at 46 s, and then slightly declined during the remaining stimulation period. No significant changes were observed in the mean arterial blood pressure or blood gases during or after stimulation. Both atropine and scopolamine failed to alter this flow increase. Electrical stimulation of the postganglionic fibers at different frequencies revealed a maximal increase in the CoBF at 30 Hz in the control situation (47.2%), but at 10 Hz after scopolamine administration (51.6%). This provides the first report showing that selective postganglionic stimulation of the parasympathetic nerve fibers markedly enhances blood flow in the brain, and it supports the view that the neurogenic vasodilatation is primarily noncholinergic.

Origins and pathways of cerebrovascular nerves storing substance P and calcitonin gene-related peptide in rat

Origins and pathways of cerebrovascular substance P- and calcitonin gene-related peptide-positive nerves in rat were studied by immunohistochemistry combined with denervation experiments and retrograde axonal tracer technique. The two peptides have been found to coexist in one and the same neuron. After sectioning of the nasociliary nerve bilaterally the substance P/calcitonin gene-related peptide fibers in the rostral half of the circle of Willis and its branches were eliminated, whereas the number decreased in the caudal half of the circle of Willis and rostral two thirds of the basilar artery. Substance P/calcitonin gene-related peptide fibers in the internal carotid arteries, the caudal third of the basilar artery and the vertebral arteries were not affected by the nerve section. After application of the retrograde axonal tracer True Blue onto the proximal segment of the middle cerebral artery the dye accumulated in several Substance P/calcitonin gene-related peptide-containing cells in the ophthalmic division of the ipsilateral trigeminal ganglion and in a few cells in the maxillary trigeminal division and in the internal carotid miniganglion. No other cranial ganglia accumulating the dye contained any substance P/calcitonin gene-related peptide-positive cells. It is concluded that the rostral portion and part of the caudal portion of the cerebral vessels are innervated by substance P/calcitonin gene-related peptide-containing fibers from the trigeminal ganglion and the internal carotid miniganglion. The great majority of trigeminal fibers reach the vessels via the nasociliary nerve of the ophthalmic division, which enters the cranial cavity through the ethmoidal foramen, whereas fibers from the miniganglion project directly to the bypassing internal carotid artery. A probable pathway for the fibers from the maxillary division is suggested. The caudal portion receives, in addition, a supply from other sensory ganglia (lower cranial and/or upper cervical dorsal root ganglia).

Origins and pathways of choline acetyltransferase-positive parasympathetic nerve fibers to cerebral vessels in rat

The presence of cholinergic nerve fibers in the brain vasculature has been a matter of controversy, partly due to the lack of a reliable histochemical marker. Accordingly, no distinct information about the origin and pathways for such fibers has been available. In the present study on the rat pial vasculature, utilizing a choline acetyltransferase (ChAT) antibody, which is able to demonstrate this enzyme in peripheral nervous tissue, evidence was obtained for an innervation by cholinergic fibers of large pial arteries. Vasoactive intestinal polypeptide (VIP) was present in or in close association with these fibers. By the aid of the retrograde axonal tracer True Blue (TB) applied to the middle cerebral arterial wall, such fibers were shown to originate in a subgroup of ChAT-positive cells in the sphenopalatine, otic, and internal carotid ganglia, which, in addition, contained VIP. The ChAT-positive pial nerve fibers were few in relation to the VIP-immunoreactive fibers, as was also illustrated by the few TB-positive cells in the ganglia that were ChAT positive as compared with the number of cells that were VIP positive. Only a small population of ChAT-containing neurons in these ganglia appeared to project to the pial vessels. The pathway from the sphenopalatine ganglion is via a membranous structure on the medial orbital wall, through the ethmoidal foramen, and along the internal ethmoidal artery to reach the circle of Willis. The fibers from the internal carotid and otic ganglia probably bridge to the internal carotid artery in the carotid canal, those from the otic ganglion after an initial course in the lesser superficial petrosal nerve. In addition, a weak accumulation of ChAT immunoreactivity was observed along thick nerve bundles in the wall of large and smaller pial arteries. VIP fibers were running at some locations along these nerve bundles.

Folic acid responsive postmenopausal homocysteinemia

Homocysteinemia is associated with juvenile arteriosclerosis, recurrent thromboembolic complications and osteoporosis. Plasma homocysteine, measured as homocysteine-cysteine mixed disulfide (MDS), has in other than homocysteinemics been reported to be higher in patients with coronary heart or cerebrovascular disease than in controls, and higher in men than in premenopausal women. Here, in groups of normal men and normal premenopausal and postmenopausal women, we measured plasma MDS in the fasting state and four hours after a methionine load (100 mg/kg body weight), before and after four weeks of folic acid therapy at 5 mg daily. In their fasting plasma, postmenopausal women (n = 5) had significantly (P less than 0.05) higher MDS concentrations than premenopausal women (n = 5) and younger men (n = 5). After the methionine load MDS concentrations in postmenopausal women rose markedly, reaching levels significantly higher than those in younger men (P less than 0.05), and with no overlap with values in premenopausal women (P less than 0.01), or in older men (n = 5, P less than 0.01). Folic acid therapy resulted in substantial reductions (n = 15, P less than 0.01) of MDS concentrations both before the methionine load (-31%) and after (-28%), though subjects had initially had normal concentrations of serum and erythrocyte folates. We speculate that moderate homocysteinemia might contribute to postmenopausal arteriosclerosis and osteoporosis. Should this prove to be the case, folic acid might be a useful prophylactic.

Neuropeptide Y Co-Exists with Vasoactive Intestinal Polypeptide and Acetylcholine in Parasympathetic Cerebrovascular Nerves Originating in the Sphenopalatine, OTIC, and Internal Carotid Ganglia of the Rat

Neuropeptide Y co-exists with noradrenaline in the majority of the sympathetic nerves supplying cerebral blood vessels. However, after sympathectomy in the rat the number of cerebrovascular neuropeptide Y nerve fibers are only reduced in number despite a complete disappearance of the adrenergic markers. The origin of these non-sympathetic neuropeptide Y fibers was studied by nerve transections and retrograde axonal tracing utilizing True Blue. Three days after bilateral superior cervical sympathectomy, the number of neuropeptide Y-containing nerve fibers decreased to about 40% of that in non-treated animals. One week after True Blue application on the proximal portion of the middle cerebral artery, the tracer accumulated in neurons of the sphenopalatine, otic, and internal carotid ganglia. Of these cells 80%, 95% and 5%, respectively, were neuropeptide Y-positive. Some of the True Blue/neuropeptide Y-positive cells displayed immunoreactivity for vasoactive intestinal polypeptide and some were positive for choline acetyltransferase. Two weeks after bilateral removal of the sphenopalatine ganglion or transection of postganglionic fibers from the ganglion reaching the pial vessels through the ethmoidal foramen, together with subsequent sympathectomy, no neuropeptide Y-containing nerve fibers could be observed on the anterior cerebral and internal ethmoidal artery or the distal portion of the middle cerebral artery, whereas a few nerve fibers remained on the proximal portion of the middle cerebral artery, internal carotid artery, and the rostral portion of the basilar artery. In conclusion, neuropeptide Y in cerebrovascular nerves is co-stored not only with noradrenaline in sympathetic nerves from the superior cervical ganglion, but also with acetylcholine (reflected in the presence of choline acetyltransferase) and vasoactive intestinal polypeptide in parasympathetic nerves originating in the sphenopalatine, otic, and internal carotid ganglia.

Trigeminal fibre collaterals storing substance P and calcitonin gene-related peptide associate with ganglion cells containing choline acetyltransferase and vasoactive intestinal polypeptide in the sphenopalatine ganglion of the rat. An axon reflex modulating parasympathetic ganglionic activity?

In immunohistochemical studies on rat two types of nerve fibres, both showing substance P and calcitonin gene-related peptide-like immunoreactivity, have been localized in the sphenopalatine ganglion, the principal cells of which contain both vasoactive intestinal polypeptide and choline acetyltransferase. One fine-calibre fibre type forms basket-like arrangements around approximately 3-5% of the principal neurons, whereas another, more coarse type traverses the ganglion without making contacts with the ganglion cells. By transection of nerves connecting with the ganglion, in combination with retrograde tracing experiments, it was concluded that the fine-calibre fibres exclusively come from the trigeminal ganglion, whereas the second type in addition, and mainly, originate in the internal carotid ganglion which is situated along the greater superficial petrosal nerve and the pterygoid nerve at their junction with the internal carotid nerve. The brain vasculature was shown to be one target structure for the innervated principal cells in the sphenopalatine ganglion. The arrangement provides the functional possibility for a modulatory interaction between the autonomic and sensory systems, thus resembling an axon reflex mechanism in the peripheral nervous system.

Enzymes related to monoamine transmitter metabolism in brain microvessels.

Abstract: The activities of tyrosine hydroxylase, aromatic l‐aminoacid decarboxylase, monoamine oxidase, and catechol‐O‐methyltransferase were measured in microvessel fractions (capillaries and venules), parenchymal arterioles, and pial vessels from rat brains, and the decarboxylase activity was compared in brain microvessels from rabbit, cat, dog, pig, cow, baboon, and man. Cranial sympathectomy was performed to estimate the neuronal contribution to the enzyme activities. All vascular regions had substantial activities of the various enzymes studied. The activity of aromatic l‐aminoacid decarboxylase in cerebral microvessels was high in rat, dog, pig, cow, and man; intermediate in rabbit and cat; and low in baboon. In addition to this enzyme, cerebral microvessels also contained tyrosine hydroxylase and monoamine oxidase. Aromatic aminoacid decarboxylase and monoamine oxidase serve an enzymatic barrier function at the microvascular level, whereas the main function of tyrosine hydroxylase is probably to synthesize monoamines within nerve terminals that remain in close association with microvessels under the conditions used for preparation of the microvascular fraction. In larger intracerebral and pial vessels monoamine oxidase was present both in the wall itself and in perivascular sympathetic nerves; the remaining two enzymes had a primarily neuronal localization. The latter types of vessels also contained catechol‐O‐methyltransferase in their walls.