The development of adrenergic innervation in some human foetal blood vessels

The development of adrenergic innervation was examined in saphenous and brachial veins, femoral, brachial and superficial temporal arteries from human foetuses of 7–19 weeks gestation. Fluorescence histochemical and electron microscopic techniques were used on transverse sections of the vessels. No specific adrenergic fluorescence was detected within any of the vessels before 11 weeks gestation, by which time the arteries showed specific adrenergic fluorescence. However, there was no evidence of adrenergic innervation of veins even at 19 weeks gestation. Incubation of the vessels with α-methylnoradrenaline did not lead to earlier detection of fluorescence. Electron microscopy showed that the adrenergic nerves found in the arteries were arranged as bundles of axons partially enclosed within Schwann cell processes, often lying close to capillaries in the outer regions of the tunica adventitia.     

Increased density of perivascular adrenergic innervation in tibial and vagus nerves of spontaneously hypertensive rats

Adrenergic innervation of epiperineurial arterioles and of the endoneurium of the tibial and vagus nerves of spontaneously hypertensive rats (SHR) and normotensive controls (WKY) was studied by glyoxylic acid-induced fluorescence and paraformaldehyde-induced fluorescence methods. Adrenergic perivascular innervation of epiperineurial arterioles in both nerves was denser in SHR than in controls. Mean density in the endoneurium also was higher in SHR in both nerve types. These results suggest that adrenergic perivascular innervation, which may influence nerve blood flow, becomes greater in density in peripheral nerves during chronic hypertension.     

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.     

P2X (purinergic) receptor distributions in rat blood vessels

The distribution of purinergic (P2X1 and P2X2) receptors on smooth muscle cells in relation to autonomic nerve varicosities in rat blood vessels has been determined using immunofluorescence and confocal microscopy. P2X1 and P2X2 receptors were visualised using rabbit polyclonal antibodies against the extracellular domain of the receptors and varicosities visualised using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2X1 receptor clusters were observed on the smooth muscle cells of mesenteric, renal, and pulmonary arteries as well as in the aorta and in veins: a large approximately elliptical cluster 1.32+/-0.21 microm long and 0.96+/-0.10 microm in diameter; and a smaller spherical cluster with a diameter of 0.32+/-0.05 microm. The latter occurred throughout the media of arteries of all sizes, whereas the former were restricted to the adventitial surface of the media and to endothelial cells, except for the pulmonary artery, in which large receptor clusters were found throughout the media of the vessel. At the adventitial surface, the large clusters are in general located beneath SV2 labelled varicosities. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2X and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2X receptor clusters. P2X2 receptors were located on nerves and on endothelial cells. They were also found in low density on the smooth muscle cells in the media. These observations are discussed in relation to the mechanism of purinergic transmission to the smooth muscle cells of blood vessels.     

Age-related changes in the noradrenergic innervation of the coronary arteries in old rats: a fluorescent histochemical study

Age-related changes in the density of the noradrenergic perivascular plexus supplying the coronary vessels in the rat were studied using the glyoxylic acid fluorescence technique. A marked decrease in the density of the noradrenergic innervation of large, medium and small size coronary arteries was observed in 25-month-old rats. In contrast, the fluorescent nerves supplying the coronary veins were not decreased in old animals. The present data are indicative of an age-related reduction of fluorescent noradrenergic nerves in the rat coronary arteries.     

Chronic nerve growth factor treatment of normotensive rats.

The objectives of this study were to examine the effects of chronic nerve growth factor (NGF) administration on vascular innervation and blood pressure in neonatal rats. Newborn Wistar-Kyoto (WKY) rats bred from normotensive parents were chronically treated with NGF for 8 weeks. Littermate controls received saline. Sympathetic ganglia of treated animals were greatly enlarged and in the superior cervical ganglion neuronal numbers were increased 200% and nuclear areas by 46%. The catecholamine contents of several sympathetically innervated tissues were determined biochemically and found to be significantly elevated in mesenteric arteries, aorta, ileum, adrenal and salivary glands from treated compared to control animals. The catecholamine concentrations were similar to, or exceeded those of the spontaneously hypertensive rat. Histochemically, an aberrant nerve supply was evident occupying a greater volume of the adventitia of the caudal artery and mesenteric arteries. In addition, nerve fibres could be seen penetrating the vessel wall to emerge within the lumen of mesenteric blood vessels. Analysis of the smooth muscle wall of the caudal artery revealed that a small but significantly hyperplastic response had been induced. Systolic blood pressures of NGF-treated and control animals were taken at one week intervals from 5 to 8 weeks of age utilizing the tail cuff method. The blood pressure of treated animals were within the normotensive range. It is concluded that chronic NGF treatment leads to changes in vascular innervation and muscle thickness that are similar to those seen in hypertensive animals. Furthermore, the results suggest the elevated levels of NGF seen in peripheral tissues of the spontaneously hypertensive rat are likely to be responsible for the hyperinnervation and resulting hyperplastic responses within vascular tissues, but not exclusively responsible for the elevated blood pressure.     

Increased density of perivascular nerves to the major cerebral vessels of the spontaneously hypertensive rat: differential changes in noradrenaline and neuropeptide Y during development

Fluorescence and immunohistochemical techniques were used to study the pattern and density of perivascular nerves containing noradrenaline (NA) and neuropeptide Y (NPY) supplying the major cerebral arteries of 4-, 6-, 8- and 12-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar (WIS) controls. Levels of NA and NPY in the superior cervical ganglia were measured. The density of nerves containing NA and NPY was greater in the hypertensive animals at all ages studied. However, the developmental changes in the density of innervation showed similar trends in both SHR and WIS groups. With few exceptions, there was a significant increase in the density of nerves containing NA from 4 to 6 weeks and from 8 to 12 weeks of age. This was in contrast to a low expression, and in some vessels a significant decrease in the number of NPY-containing nerves from 4 to 6 weeks. The density of nerve fibres containing NPY increased significantly in almost all vessels between 6 and 8 weeks of age and then stabilized. Thus there is a differential time course for the appearance of NA and NPY during development. Furthermore, the hyperinnervation of cerebral vessels in SHR by nerves containing NA and NPY precedes the onset of hypertension and associated medial hypertrophy. High-performance liquid chromatography and enzyme-linked immunosorbant assays show that the NA and NPY contents of the superior cervical ganglion do not reflect the changes in innervation pattern seen in the terminal fibres in the cerebral arteries. This tends to support the view that a local neurovascular mechanism is involved in the maintenance of hypertension. The possibility that increase in NPY as well as NA in cerebral perivascular nerves of hypertensive animals is involved in the protection of the blood-brain barrier against oedema and cerebral haemorrhage is raised.     

Elevated concentrations of nerve growth factor in heart and mesenteric arteries of spontaneously hypertensive rats

Considerable evidence indicates an enhanced sympathetic innervation of muscular resistance arteries in the spontaneously hypertensive rat (SHR) compared with its normotensive Wistar-Kyoto (WKY) control. Since nerve growth factor (NGF) is known to affect the growth of sympathetic nerves, we have utilized a sensitive two-site enzyme linked immunoassay for NGF to compare to NGF content of hearts and mesenteric arteries of developing SHR and WKY rats. NGF levels in hearts revealed similar, although not identical, patterns of expression. In both strains, NGF levels declined from postnatal day 15, the earliest age examined, to stabilize at adult levels by postnatal day 32. Adult SHR concentrations were similar to those in age matched WKY controls. In contrast, in the mesenteric vascular bed, NGF levels of SHR were greater than those of WKY controls at all ages above 15 days. Moreover, these changes in NGF occurred concomitantly with increases in vascular mass are medial smooth muscle hyperplasia in the SHR. Whether abnormal NGF levels are a cause or consequence of vascular smooth muscle growth has yet to be determined. These results are consistant with the hypothesis that the hypernoradrenergic innervation of SHR vascular tissues results from an early elevation of NGF gene expression.     

Fluorescence histochemical observations on the adrenergic innervation of the cardiovascular system in the aged rat

The Falck-Hillarp fluorescence technique was applied to tissue samples of the heart and certain vessels of both 4 and 24 month Wistar rats. It appeared that with increasing age, the density and fluorescence intensity of the cardiac sympathetic innervation was maintained at a level comparable to the young adult (4 month) in the atria, left ventricle and in the region of the sino-atrial node. Similarly the adrenergic innervation of the coronary vasculature, particularly in the atrium was maintained into old age. In contrast the adrenergic innervation of the common and internal carotid arteries and abdominal aorta was very sparse in the 24 month rat compared to the young adult in which these vessels were surrounded by dense fluorescent plexuses. Peripheral arteriolar innervation in the pancreas was apparently similar in both age-groups. The present study suggests that the density of adrenergic innervation varies within the constituents of the cardiovascular system in aged rats.     

Congenital erythropoietin over-expression causes "anti-pulmonary hypertensive" structural and functional changes in mice, both in normoxia and hypoxia

Acute alveolar hypoxia causes pulmonary vasoconstriction that matches lung perfusion to ventilation to optimize gas exchange. Chronic alveolar hypoxia induces pulmonary hypertension, characterized by increased muscularization of the pulmonary vasculature and right ventricular hypertrophy. Elevated erythropoietin (EPO) plasma levels increase hematocrit and blood viscosity and may affect structure and function of the pulmonary circulation. To differentiate between the direct effects of hypoxia and those linked to a hypoxia-induced increase in EPO/hematocrit levels, we investigated the lung vasculature in transgenic mice constitutively over-expressing EPO (termed tg6) upon exposure to normoxia and chronic hypoxia. Despite increased hematocrit levels (approximately 0.86),tg6 mice kept in normoxia did not develop selective right ventricular hypertrophy. The portion of vessels with a diameter of 51-95 microm and >155 microm was increased whereas the portion of small vessels (30-50 microm) was decreased. Pulmonary vascular resistance and the strength of hypoxic vasoconstriction measured in isolated perfused lungs were decreased. Vasoconstrictions induced by the thromboxane mimetic U46619 tended to be reduced. After chronic hypoxia (FiO2 = 0.10, 21 days), vascular resistance and vasoconstrictor responses to acute hypoxia and U46619 were reduced in tg6 mice compared to wildtype controls. Chronic hypoxia increased the degree of pulmonary vascular muscularization in wildtype but not in tg6 mice that already exhibited less muscularization in normoxia. In conclusion, congenital over-expression of EPO exerts an "anti-pulmonary hypertensive" effect, both structurally and functionally, particularly obvious upon chronic hypoxia.     

Ultrastructural studies of cerebral arteries and collateral vessels in moyamoya disease

Moyamoya disease was originally defined as a characteristic syndrome of recurrent headaches, occlusion of the distal internal carotid arteries and the foggy (moyamoya) clusters of collateral vessels at the base of the brain as demonstrated by cerebral angiography. The etiology is unknown and pathobiology is poorly understood. We examined the intracranial arteries in 3 patients to demonstrate characteristic changes and to obtain a better understanding of the basis mechanisms of the disease. Controls were obtained from 3 normotensive patients who died as a result of cancer. Occluded internal carotid arteries were characterized by severe thickening of the intima with a dense luminal array of smooth muscle cells, a deeper less cellular zone, pronounced tortuosity of the internal elastica and thinning of the media. Collateral vessels were arterial in structure and were affected by similar proliferative changes in the intima, thinning of the media, and contorted internal elastica. Stainable lipids were not part of the typical components. Severe contortion of the internal elastica, medial damage and intimal proliferation may result from recurrent and sustained spasticity of the cerebral arteries. The distal lenticulostriate arteries showed severe medial damage similar to what is termed as a moth-eaten change in hypertensive patients dying of massive cerebral hemorrhage.     

Development and maturation of noradrenaline-containing nerves of the rat uterine artery. Effects of acute and chronic oestrogen treatment.

The development and maturation of noradrenaline-containing nerves of the rat uterine artery was investigated, histochemically and biochemically, at seven different postnatal age-stages and following acute and chronic treatment with oestradiol. Morphological changes in the vessel were quantitatively evaluated on toluidine blue-stained semithin sections and low magnification electronmicrographs. In summary, the uterine artery is innervated at birth; the adult pattern of innervation is established at two weeks of age; the innervation density increases progressively between the infantile and prepubertal periods, accompanying proliferation and growth of smooth muscle cells in the tunica media; changes in the innervation are followed by an increase in the tissue concentration of noradrenaline and neither the endocrine changes characterizing puberty nor acute or chronic treatment with oestradiol have an effect on the pattern of development of the uterine artery and associated noradrenaline-containing nerves. Results are interpreted considering the differential susceptibility of urinogenital organs to sex hormones.     

Nerve growth factor treatment of adult rats selectively enhances innervation of urinogenital tract rather than vascular smooth muscle

Following treatment of adult rats with nerve growth factor (0.5 mg/rat, three times a week for 3 weeks), the innervation of cardiovascular and urinogenital tract smooth muscle was investigated using immunoassay and immunohistochemical techniques. Substance P and calcitonin gene-related peptide levels were increased in the vas deferens, but not in the atria or femoral artery. Neuropeptide Y and vasoactive intestinal polypeptide levels were unchanged. In penile tissues, there was a marked increase in the density of substance P-, calcitonin gene-related peptide-, neuropeptide Y-, tyrosine hydroxylase- and vasoactive intestinal polypeptide-containing nerves innervating the urethra and in SP-containing nerves in the tunica with little change in the innervation of the deep dorsal vein and artery and corpus cavernosum. In the bladder, there was increased innervation of the detrusor by neuropeptide Y- and vasoactive intestinal polypeptide-containing nerves, but a decrease in innervation by substance P-containing nerves in the trigone. There were no changes in the density of innervation of the femoral artery after nerve growth factor treatment. Thus, in the mature rat, sensory and sympathetic nerves innervating urinogenital tract smooth muscle appear to be more responsive to exogenous nerve growth factor than those innervating cardiovascular smooth muscle. This may reflect an ongoing requirement of plasticity of innervation in the urinogenital tract of the sexually mature animal.     

A histochemical study on the innervation of cerebral blood vessels in the bullfrog

Specific histochemical techniques for the demonstration of catecholamine and acetylcholinesterase have been used to study the distribution of adrenergic and cholinergic nerves on the cerebral blood vessels of bullfrog, Rana catesbeiana. The adrenergic nerve meshworks on the cerebral arteries of bullfrog were less dense, had a more elongate appearance along the arterial axis as compared with those of mammals and were rather similar to those of snakes. The nerve plex-uses on the cerebral carotid artery and its main branches were somewhat denser than those on the basilar artery. The most characteristic feature of innervation in the bullfrog cerebral vessels was that no acetylcholinesterase-positive fibres were observed on the extraparenchymal arteries, whereas, in all higher vertebrates investigated so far, the cerebral arteries have been found to be dually innervated although differences in the density of innervation of the two nerves may exist. This suggests that the peripheral adrenergic innervation on the cerebral blood vessels appeared earlier than the cholinergic one in the evolution of vertebrates. On the other hand, both adrenergic and acetylcholinesterase-positive fibres were observed in close contact with parenchymal arterioles and capillaries suggesting the possible existence of a dual central innervation. This feature, however, was by no means common. Thus, the central neurons have a significant influence on the cerebral circulation in the bullfrog is somewhat equivocal. Most of the pial and the parenchymal small vessels and the parenchymal capillaries exhibited a heavy acetylcholinesterase activity on the vascular walls. Although the significance of the enzyme is obscure as yet, this has to be considered in relation to the regulatory mechanism of the cerebral circulation.     

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.     

The morphometry of consecutive segments in cerebral arteries of normotensive and spontaneously hypertensive rats

The media cross-sectional area, the media thickness, the internal radius and the ratio between media thickness and internal radius were determined in consecutive sections of extraparenchymal cerebral arteries of 7- and 12-month-old normotensive and spontaneously hypertensive rats. The study included intracranial pial and basal arteries as well as extracranial cervical arteries. In the chronically hypertensive rats the media to radius ratio was consistently higher than in normotensive rats over the entire calibre spectrum investigated (radius 5-400 micron). The increase of the ratio in the extracranial arteries of the hypertensive rats was exclusively due to a thicker media. In the basal intracranial arteries the increase of ratio was due to a thicker media and/or a smaller internal radius in 7- and 12-month-old rats with moderate hypertension (mean arterial pressure, MAP 171 +/- 8 and 177 +/- 7 mm Hg respectively). In 7-month-old rats with severe hypertension (MAP 204 +/- 11 mm Hg) the increase of ratio was mainly due to a smaller internal radius. The observed structural alterations are likely to be of hemodynamic importance.     

White matter lesions and alteration of vascular cell composition in the brain of spontaneously hypertensive rats.

There have been few studies on the white matter lesions of spontaneously hypertensive rats (SHR). From the point of view of hypertension and arteriosclerosis, white matter lesions were examined in SHR and stroke-prone SHR (SHRSP), and were then compared with Wistar-Kyoto (WKY) rats. The vasculopathy was analyzed by morphometric immunohistochemistry for collagen and smooth muscle actin. Both SHR and SHRSP had hypertension at > or = 12 weeks of age, and the latter developed severe white matter lesions at 20 weeks. Immuno- histochemistry revealed proliferation of microglia in the white matter and an increase in smooth muscle actin in the vessels of SHRSP compared with the WKY rats and SHR, but there were no changes in the collagen. These results indicate a role of hypertension in the pathogenesis of white matter lesions. However, genetic difference may also be responsible since SHR and SHRSP showed similar hypertension.     

Arterial sympathetic innervation and cerebrovascular diseases in original rat models

The role of the arterial sympathetic innervation in cerebrovascular pathology was investigated in new experimental models using Brown Norway (BN) and Long-Evans (LE) rats. The BN rat is susceptible to intracerebral hemorrhage (ICH) within the cerebral cortex when rendered hypertensive whereas the LE rat is prone to cerebral aneurysms (CAs) in arteries of the circle of Willis with hypertension and carotid ligation. Noradrenaline (NA) content, determined by high performance liquid chromatography (HPLC), was lower both in the caudal and cerebral arteries in the BN than in the LE rat. Denervation of cerebral arteries by superior cervical ganglionectomy did not increase ICH lesion incidence in BN hypertensive rats. A possible link between the level of caudal artery NA content and the occurrence of ICH lesions and CAs was studied in rats from two distinct BNXLE crosses: back-cross (BC) rats (F1XBN) and F2 rats (F1XF1) which respectively display, with hypertension and carotid ligation, a high incidence of either ICH lesions or CAs. In BC rats, the level of caudal artery NA content was not related to ICH lesion occurrence. However, in F2 rats a low caudal artery NA content was associated with a high incidence of ruptured CAs. Thus, a low arterial sympathetic innervation may participate in mechanisms leading to rupture of CAs.     

Analysis of innervation of human mesenteric vessels in non-inflamed and inflamed bowel – a confocal and functional study

Abstract  We investigated the distribution and density of perivascular nerves in human mesenteric arteries and veins and their responses to noradrenaline (NA), ATP and neuropeptide Y (NPY) in control (non-inflamed) and inflamed bowel, using confocal microscopy and in vitro pharmacology. The density of innervation at the adventitial-medial border of arteries and within the medial muscle coat of veins was increased in inflammatory bowel disease (IBD). Expression of markers for both sympathetic nerves and sensory-motor nerves was significantly increased in IBD. Calcitonin gene-related peptide-containing sensory-motor nerves were present in control arteries and IBD, but not in control veins. The density of 5-hydroxytryptamine-containing nerves was variable in controls, but consistently increased (three to four times) in IBD. Vasoactive intestinal peptide (VIP) expression increased (doubled) in arteries and veins. Arteries and veins contracted to NA and ATP, but only veins constricted to NPY. ATP contractions were reduced in arteries and veins in IBD, while contractions to NA were only slightly reduced. Neuropeptide Y induced significantly greater (20%) contractions of IBD veins. In summary, the density of sympathetic and sensory-motor innervation of both mesenteric arteries and veins was increased in IBD. Both 5-hydroxytryptamine and VIP immunoreactivity were also increased. The responses of both arteries and veins to ATP, and to a lesser extent NA, were reduced in IBD while responses to NPY were greater in veins. Decreased responses to ATP indicate changes in purinergic-mediated transmission in the pathological state.