Innervation of the vasa nervorum: changes in human diabetics.

Transperineurial and epineurial vessels are innervated by plexuses of unmyelinated axons. Human sural nerve biopsies were examined ultrastructurally and immunocytochemically with an antibody which recognizes a neuronal and neuroendocrine protein, PGP 9.5, to characterize perivascular axons of these plexuses. Diabetics exhibited a greater degree of abnormal innervation of the vasa nervorum than nondiabetics with and without neuropathy. Abnormal innervation included: a reduction in the percentage of vessels exhibiting perivascular axons and a concomitant increase in the percentage of vessels having denervated Schwann cell units, particularly around vessels confined to perineurial compartments, and remaining axons in nerves from diabetics exhibited fewer varicosities. Denervated arterioles of diabetics also displayed structural changes indicating injury. The arteriolar structural defects and loss of neurogenic control of neural blood flow may lead to or aggravate endoneurial ischemia or hypoxia. The patchy, focal endoneurial fiber loss that is prominent in proximal nerves and associated with the distal myelinated fiber loss of some diabetic patients may be due in part to perivascular denervation of the vasa nervorum.     

The noradrenergic innervation of the vasa nervorum in old rats: a fluorescence histochemical study

The effect of ageing on the density and pattern of noradrenergic nerves in the perivascular nerve plexus supplying the vasa nervorum of the rat sciatic nerve was studied using combined catecholamine histofluorescence and quantitative image analysis techniques. The density of noradrenergic fibres around arteries and arterioles of the rat sciatic nerve vasa nervorum increased in the old animals. In contrast, no changes in perivascular nerve fibres supplying the veins and venules were found in the vasa nervorum of old rats. The increase in old age of noradrenergic innervation of arteries and arterioles of the vasa nervorum may be related to the pathogenesis of some peripheral nerve diseases.     

Adrenergic innervation of the vasa nervorum in the cranial nerves and spinal roots in the subarachnoid space

Fluorescence microscopy revealed that the innervation of the vasa nervorum of both cranial and spinal nerve roots in the subarachnoid space in the monkey is adrenergic in nature. The endoneurium of the cranial nerve roots contained free adrenergic nerve fibers which were not related to the vessel wall.     

Adrenergic innervation of vasa and nervi nervorum of optic, sciatic, vagus and sympathetic nerve trunks in normal and streptozotocin-diabetic rats

Adrenergic nerves were studied in nervi nervorum and perivascular nerve plexus of vasa nervorum in whole-mount nerve sheath preparations of optic, sciatic and vagus nerves and in the paravertebral sympathetic chain in normal and streptozotocin-treated diabetic rats. A substantial or complete loss of fluorescent adrenergic fibres around blood vessels in the optic nerves was observed 8 weeks after induction of diabetes. This was in marked contrast to the increase in perivascular adrenergic fibres in the sciatic, vagus and sympathetic chain nerve trunks of the same animals at the same time. Assays of noradrenaline levels in whole nerve segments also showed that they were not biochemically detectable in the optic nerves but were significantly higher in the vagus of diabetic animals (P less than 0.05). There was also an increase in numbers of mast cells in the vicinity of vasa nervorum of diabetic nerves.     

Light and electron microscopic study of the distribution of substance P-immunoreactive fibers and neurokinin-1 receptors in the skin of the rat lower lip

Cutaneous antidromic vasodilatation and plasma extravasation, two phenomena that occur in neurogenic inflammation, are partially blocked by substance P (SP) receptor antagonists and are known to be mediated in part by mast cell-released substances, such as histamine, serotonin, and nitric oxide. In an attempt to provide a morphological substrate for the above phenomena, we applied light and electron microscopic immunocytochemistry to investigate the pattern of SP innervation of blood vessels and its relationship to mast cells in the skin of the rat lower lip. Furthermore, we examined the distribution of SP (neurokinin-1) receptors and their relationship to SP-immunoreactive (IR) fibers. Our results confirmed that SP-IR fibers are found in cutaneous nerves and that terminal branches are observed around blood vessels and penetrating the epidermis. SP-IR fibers also innervated hair follicles and sebaceous glands. At the ultrastructural level, SP-IR varicosities were observed adjacent to arterioles, capillaries, venules, and mast cells. The varicosities possessed both dense core vesicles and agranular synaptic vesicles. We quantified the distance between SP-IR varicosities and blood vessel endothelial cells. SP-IR terminals were located within 0.23-5.99 microm from the endothelial cell layer in 82.7% of arterioles, in 90.2% of capillaries, and in 86.9% of venules. Although there was a trend for SP-IR fibers to be located closer to the endothelium of venules, this difference was not significant. Neurokinin-1 receptor (NK-1r) immunoreactivity was most abundant in the upper dermis and was associated with the wall of blood vessels. NK-1r were located in equal amounts on the walls of arterioles, capillaries, and venules that were innervated by SP-IR fibers. The present results favor the concept of a participation of SP in cutaneous neurogenic vasodilatation and plasma extravasation both by an action on blood vessels after binding to the NK-1r and by causing the release of substances from mast cells after diffusion through the connective tissue.     

Adrenergic innervation of blood vessels in rat tibial nerve during Wallerian degeneration

Summary Adrenergic innervation of blood vessels in rat tibial nerve during Wallerian degeneration was examined, using the formaldehyde-induced histo-fluorescence method. The left sciatic nerve was transected at the level of the sciatic notch, whereas the right sciatic nerve was left intact and used as control. At 1, 3, 7, 14, 42, 56 or 84 days after transection, the tibial nerves of the transected and contralateral sides were exposed. Pieces of each nerve were used for light microscopy or for examination of adrenergic innervation with the fluorescence microscope. One day after transection, no adrenergic nerve fiber was observed in the endoneurium of the transected nerve. After 3 days, adrenergic innervation of small-and medium-sized arterioles in the epi-perineurium was absent, and after 7 days no fibers were visible around large arterioles. Fluorescent fibers were not detected even at 84 days post-surgery. It is concluded that adrenergic innervation of blood vessels in the rat tibial nerve is irreversibly lost after permanent axotomy, and that adrenergic regulation of nerve blood flow may also be lost.     

Hyperosmotic arabinose solutions open the tight junctions between brain capillary endothelial cells in tissue culture

Noradrenergic, serotoninergic and peptidergic nerves have been demonstrated in perivascular plexuses of vasa nervorum of sympathetic, parasympathetic and somatic nerve trunks. 5-Hydroxytryptamine-, vasoactive intestinal polypeptide- and substance P-containing fibers were found by immunohistochemistry to variable extents in whole mounts of the epineurium of sciatic, vagus and paravertebral sympathetic chains of rabbits. Innervation increased with age. This suggests an hitherto unsuspected role for these vasoactive substances in normal blood flow to nerves and in the genesis of experimental and human neuropathies.     

The nerves to blood vessels supplying blood to nerves: the innervation of vasa nervorum

Noradrenergic, serotoninergic and peptidergic nerves have been demonstrated in perivascular plexuses of vasa nervorum of sympathetic, parasympathetic and somatic nerve trunks. 5-Hydroxytryptamine-, vasoactive intestinal polypeptude- and substance P-containing fibers were found by immunohistochemistry to variable extents in whole mounts of the epineurium of sciatic, vagus and paravertebral sympathetic chains of rabbits. Innervation increased with age. This suggests an hitherto unsuspected role for these vasoactive substances in normal blood flow to nerves and in the genesis of experimental and human neuropathies.     

The adrenergic innervation of pulmonary vasculature in the normal and pulmonary hypertensive rat

It would appear that susceptibility to chronic proliferative pulmonary hypertension in response to chronic alveolar hypoxia is most severe in species in which adrenergic innervation of pulmonary arteries is reduced or lacking. Intrapulmonary arteries of the rat have been reported to lack adrenergic innervation by some workers but not others. Since the rat develops severe proliferative pulmonary hypertension in response to prolonged alveolar hypoxia, the different divisions of the lung vasculature of Sprague-Dawley rats were thoroughly examined to determine the presence or absence of an adrenergic innervation. The degree of innervation in normal rats was compared with that of rats developing pulmonary hypertension. Both in normal and experimental pulmonary hypertensive rats the pulmonary arteries, all their branches and the small pulmonary veins with a smooth muscle media were found to be devoid of adrenergic innervation. In contrast, the cardiac-like muscle in the media of large pulmonary veins, the bronchial arteries and the vasa vasorum of larger vessels were richly innervated by adrenergic nerves. Thus the increase in medial smooth muscle which occurs in pulmonary arteries during chronic alveolar hypoxia is independent of a pre-existing adrenergic innervation or of such an innervation newly derived from that of adjacent vessels or structures. This is in contrast to systemic vessels where it has been suggested that increased adrenergic activity and density of innervation may augment hypertrophy of the media in hypertensive animals. Adrenergic nerves are suggested to have a protective action on pulmonary vessels.     

Postnatal development of adrenergic innervation in the tibial and vagus nerves of Fischer-344 rats

Adrenergic innervation of rat tibial and vagus nerves was studied in male Fischer-344 rats between 1 and 84 days of age, using sucrose-phosphate-glyoxylic acid (SPG) histochemistry and the formaldehyde-induced fluorescence (FIF) method. Adrenergic nerve fibers were found in epi-perineurial blood vessels of the vagus nerve at one day of age, whereas blood vessels in the tibial nerve received the first adrenergic nerve fibers at 3 days. A few adrenergic nerve fibers were seen in the endoneurium of both tibial and vagus nerves at 7 days. The densities of adrenergic innervation increased gradually during the first 4 postnatal weeks, and at 21 days the distributions of adrenergic innervation in both nerves resembled those in adult animals. The results suggest that development of adult adrenergic innervation in rat peripheral nerves occurs during the first postnatal month and that sympathetic innervation becomes available to regulate nerve blood flow within this period.     

Innervation of footpads of normal and mutant mice lacking sweat glands

Footpads of normal adult mice are innervated by sympathetic and sensory fibers. The sympathetic fibers associated with sweat glands contain acetylcholinesterase and immunoreactivity for vasoactive intestinal peptide. Although catecholamine histofluorescence is absent, the gland innervation exhibits immunoreactivity for tyrosine hydroxylase. A distinct population of sympathetic fibers, which possess catecholamines and neuropeptide Y as well as tyrosinehydroxylase immunoreactivity, innervates blood vessels. Sensory fibers containing immunoreactivity for substance P and calcitonin gene-related peptide course beneath the epidermis and some form endings in it. Treatment of neonatal mice with the adrenergic neurotoxin, 6-hydroxydopamine, results in loss of sympathetic innervation of sweat glands and blood vessels, permits growth of sensory axons into sweat glands, but does not alter the peptidergic sensory innervation of the dermis and epidermis. Three mouse mutations, Tabby (Ta), crinkled (cr), and downless (dl), disrupt the interactions between the mesenchyme and epidermis that are required for normal development of specific epidermal derivatives, including sweat glands. The sympathetic innervation of blood vessels and sensory innervation of footpad skin of the three mutant mice that lack sweat glands is indistinguishable from normal. The sympathetic fibers that normally innervate sweat glands, however, are not present. These results indicate that in the absence of their normal target, the sympathetic fibers that innervate sweat glands are lacking. Furthermore, they suggest that, although sensory fibers may sprout into sympathetic targets in the footpad, the domains occupied by sensory fibers are not normally accessible to sympathetic axons. © 1994 Wiley-Liss, Inc.     

Quantitative assessment of the innervation of epineurial arteries in the peripheral nerve by immunofluorescence: differences between controls and patients with peripheral arterial disease

The peripheral nerve is supplied by the vasa nervorum. The epi- and perineurial vessels are innervated by an autonomic plexus, which plays a role in regulation of the endoneurial blood flow. This innervation is decreased in diabetes and alcohol polyneuropathy and seems to precede the development of diabetic polyneuropathy. A decreased innervation may therefore play a role in the development of polyneuropathy. In peripheral arterial disease (PAD) clinical and morphological features are present, related to severity of ischemia. To investigate the innervation of the vasa nervorum in severe ischemia, we performed immunofluorescence staining with the general neural marker protein gene product (PGP) 9.5 in whole mount preparations of epineurial vessels of nine sural nerves taken from patients with legs amputated because of severe PAD (59+/-15 years, mean +/- SD) and ten age-matched controls (61+/-24 years). In patients with PAD the nerve density of the perivascular plexus was decreased in comparison with controls (mean intercept density/mm +/- SD) 26.0+/-6.9 in PAD and 39.9+/-10.7 in controls, area% 6.0+/-1.6 in PAD and 9.9+/-2.6 in controls, both P<0.01, t-test). A decreased perivascular plexus may result in a diminished regulation of the endoneurial blood flow in patients with severe PAD.     

Localization of galanin immunoreactivity in the opossum esophagus

Galanin-like immunoreactivity was studied at 7 levels of the opossum esophagus, lower esophageal sphincter (LES) and adjacent portion of the stomach by indirect immunofluorescence; it was restricted to nervous structures. The majority of myenteric and submucous neurons were galanin-positive and received positive axo-somatic terminations. They also sent out axons staining positively. Galanin-positive fibers and a few atypically located neurons formed a mucous plexus at the bases of mucous glands. Varicose galanin fibers innervated the muscularis mucosae, circular and longitudinal muscle layers, while thick fascicles traversed the muscularis mucosae and circular muscle, possibly interconnecting the myenteric, submucous and mucous plexuses. Galanin-positive fibers did not supply blood vessels. There was no obvious gradient of innervation density along the esophagus, but the sphincter appeared to be more densely innervated than the esophageal body. There was no galanin-positive input to striated muscle. In view of its widespread distribution, this neuropeptide may serve multiple functions in the esophagus.     

Demonstration of terminalis,olfactory, trigeminal and perivascular nerves in the rat nasal septum

The innervation of the nasal septum and around the olfactory bulb has been investigated in rats by means of whole-mount preparations and histological sections. Silver staining, OsO₄ staining, PAS staining, cholinesterase reaction and fluorescence for catecholamine-containing nerves were used. The nervus terminalis forms on the medial side of the olfactory bulb a ganglionated plexus, from which branches are given off which course peripherally with the vomeronasal nerves. From a dorsal part of the terminalis nerve plexus an anterior branch is given off which runs along the anterior ethmoidal nerve to the nasal vestibule where it connects with a group of ganglia. The peripheral branches of the nerve run from here along two epithelial cristae formed histologically like dermal papillae. Ventrally in the respiratory region at the junction of the nasal cavity and the nasopharynx is a 1 × 2 mm area with olfactory epithelium, glands of Bowman and an independent innervation from the olfactory bulb. This is the so-called septal olfactory organ. Trigeminal nerves form a plexus in the respiratory region and in the vestibule, but do not supply the olfactory region. Catecholamine-containing and cholinesterase-positive nerves run along the meningeal arteries on the cribriform plate and accompany their branches to the vascular plexus in the olfactory and respiratory regions. Double innervation is found not only of this vascular plexus but of the venous sinuses in the swell bodies of the vestibule. The glands of the nose are not surrounded by catecholamine-containing nerves.     

Regeneration of perivascular adrenergic innervation in rat tibial nerve after nerve crush

Summary Adrenergic innervation of blood vessels in the rat tibial nerve during degeneration and regeneration was studied using the formaldehyde-induced fluorescence method. The left sciatic nerve was crushed with suture threads to produce a 4-mm length of crushed nerve. At 1, 3, 7, 14, 28, 56 and 84 days after nerve crush, degenerative and regenerative changes in the nerve were verified using light microscopy. At each time point, adrenergic innervation was examined in epi-perincurial whole mount and nerve cross-section preparations. One day after nerve crush, fluorescence of adrenergic nerve fibers in the endoneurium was absent. Fluorescent adrenergic nerve fibers reappeared in the endoneurium at day 56 and reached the control density by 84 days. In the epi-perineurium, adrenergic innervation of small and medium-size arterioles was absent at 3 days, in large arterioles at 7 days. At 56 days, all epi-perineurial arterioles were reinnervated by a faint, sparse adrenergic network, which reached the control density at 84 days. The results suggest that adrenergic innvervation in the rat peripheral nerve is lost during nerve degeneration, but recovers when the nerve has regenerated.     

Ultrastructural and biochemical evidence for a sympathetic neural influence on the choroid plexus

The possibility of a sympathetic innervation of the choroid plexus was studied ultrastructurally and functionally. Electron microscopy of cat and rabbit plexuses after adminstration of 5-hydroxydopamine showed nonmyelinated axon terminals in close relation both to epithelial cells (distance 20 nm) and to peripheral smooth muscles (distance less than 100 nm) of small arterioles, indicating a true innervation of both structures. Both adrenergic and nonadrenergic (probably cholinergic) terminals could be distinguished on the basis of the different electron density in the small vesicles of the varicosities. As a functional parameter, carbonic anhydrase activity was determined radiometrically in blood-free rabbit choroid plexuses. The enzyme activity (which was reduced by acetazolamide) was increased by elimination of the norepinephrine transmittor from the choroid plexuses either by reserpine treatment or sympathetic denervation. These observations show that the choroid plexus is innervated by secretion-inhibitory sympathetic nerves from the superior cervical ganglia.     

Tangier disease. A case with sensorimotor distal polyneuropathy and lipid accumulation in striated muscle and vasa nervorum

Summary A 65-year-old man with Tangier disease (analphalipoproteinemia) had had a progressive sensorimotor distal neuropathy with sensory ataxia for 1 year. Muscle biopsy demonstrated excess lipid vacuoles on histochemical and electron-microscopic techniques. Sural nerve biopsy showed a marked loss of large fibers and an increase in small myelinated fibers, with presence of remyelinating fibers and clusters of regeneration; a few aspects of active demyelination and some onion-like formations were also present. Lipid accumulation chiefly affected the Schwann cells of unmyelinated fibers and, to a lesser degree, of myelinated fibers, endoneurial fibroblast, and vasa nervorum. Teased fibers showed prevalent aspects of de-/remyelination and, often in association, marked myelin wrinkling suggesting axonal atrophy.This Tangier patient differs from known cases for the presence of a distal symmetrical sensorimotor polyneuropathy (not previously reported in Tangier disease) and because of the morphological findings of de-/remyelination coexisting with aspects of axonal atrophy and previous degeneration, and of lipid accumulation within striated muscle and vasa nervorum. This latter finding contrasts with the assumption that in Tangier disease vessel walls are not a site of lipid storage: probably the vasa nervorum are different, in this respect, from other vessels, because of the intense lipid metabolism of the nervous tissue. Thus we suggest that involvement of vasa nervorum in Tangier disease may be more important than previously suspected, possibly playing a role in the causation of neuropathy.     

Studies on vascular permeability in peripheral nerves

Summary Permeability changes of vasa nervorum and exudation of serum albumin in a nutritional peripheral neuropathy induced by isonicotinic acid hydrazide (INH) in rats were studied by menas of fluorescence microscopic tracing of intravenously injected albumin tagged with Evans blue or with fluorescein isothiocyanate.All rats given INH for fourteen days which developed histologically demonstrable neuropathy also showed abnormal vascular permeability and exudation of the albumin conjugate into the endoneurium. Once outside the blood vessels the conjugate had a predilection for spreading in the endoneurial interstices along the nerves.Thus, like traumatic peripheral neuropathies, this nutritional neuropathy is associated with permeability changes of vasa nervorum and exudation of serum albumin in the endoneurium. The simultaneous occurrence of nerve fibre lesions and permeability changes of vasa nervorum indicates that these two phenomena are in some way related to each other.Supported by grants from the Swedish Multiple Sclerosis Society and from the Swedish Medical Research Council, Project B 68-12X-82-04.