Adrenergic innervation in the tibial and vagus nerves of rats with streptozotocin-induced diabetes

Adrenergic innervation of tibial and vagus nerves was studied after 1-16 weeks duration of streptozotocin (STZ)-induced diabetes in rats. Sucrose-phosphate glyoxylic acid (SPG) histochemistry and the formaldehyde-induced fluorescence (FIF) method were used to demonstrate adrenergic nerve fibers in the epi-perineurial and endoneurial compartments. Densities of innervation were quantitated with fluorescence microscopy. The density of periarteriolar adrenergic innervation in the epi-perineurium of the tibial and vagus nerves was increased 5 and 12 weeks after STZ injections as compared with control. At 16 weeks, mean densities of periarteriolar innervation in epi-perineurium had returned to or below control levels in both nerve types. In the endoneurium, however, the mean density of adrenergic nerve fibers decreased gradually at 5 weeks after induction of diabetes in both nerves, and was totally absent at 12 weeks. At 16 weeks no sign of recovering innervation in the endoneurium was seen. In conclusion, adrenergic innervation goes through similar pathological alterations both in tibial and vagus nerves shortly after the induction of streptozotocin diabetes. These changes may contribute to diabetic peripheral neuropathy by impairing the regulation of nerve blood flow.     

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.     

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.     

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.     

Distribution of adrenergic innervation of blood vessels in peripheral nerve

The distribution of adrenergic innervation of microvessels in the extrafascicular and endoneurial compartments of rat tibial nerve was examined with glyoxylic acid-induced and formaldehyde-induced histofluorescence methods. Periarterial and arteriolar adrenergic nerves were present in the epineurium-perineurium suggesting that blood flow in the extrafascicular connective tissue is under neurogenic influence. In contrast, blood vessels in the nerve endoneurium were not associated with histofluorescent nerve fibers. The absence of perivascular adrenergic innervation in the endoneurium suggests that regulation of vascular function within the endoneurium is not under neurogenic control.     

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.     

Patterns of adrenergic and peptidergic innervation in human olfactory mucosa: Age-related trends

The distribution and targets of nerves containing the adrenergic markers tyrosine hydroxylase, dopamine β-hydroxylase, and neuropeptide Y in the human olfactory mucosa were investigated by immunohistochemistry. Tissue was obtained at autopsy from the nasal cleft of 16 adults ranging in age from 24 to 90 years, and from one spontaneously aborted 16-week-old fetus. The presence of olfactory receptor neurons in nasal mucosa was confirmed by staining with the antibody to olfactory marker protein. Targets of adrenergic innervation were blood vessels, including the vasa nervorum within the sheaths of olfactory nerve bundles, and Bowman's glands in the lamina propria. Adrenergic fibers penetrated the adventitia of blood vessels and terminated near the media, and were in close proximity to Bowman's glands but did not enter the acini. In the fetal tissue, the vasa nervorum were the major targets of adrenergic fibers. Age-related differences in the pattern and statistically significant differences in the density of innervation of blood vessels were noted between adults under and over 60 years of age. In the younger group, plexuses of nerve fibers containing colocalized dopamine β-hydroxylase and neuropeptide Y occurred adjacent to arterioles and large bundles of fibers adjacent to venules; in older individuals, few fiber plexuses occurred adjacent to arterioles and thin bundles of fibers adjacent to venules. The distribution of adrenergic innervation suggests that vasomotor tone and secretion are regulated by adrenergic nerves. The decrease in adrenergic innervation in older individuals, with resultant effects on perireceptor processes, may be associated with age-related declines in olfactory function. © 1993 Wiley-Liss, Inc.     

Transperineurial arterioles in human sural nerve.

The perineurial sheath of nerve fascicles is a protective cellular layer that separates the endoneurium from the epineurium. Transperineurial arterioles (TPA) connect the endoneurial capillary plexuses to the epineurial arterial nutrient supply. Transperineurial arterioles are defined as any arteriole that is confined to a perineurial cell compartment, which would include all arterioles within the perineurium proper or within perineurial sleeves in the epi- or endoneurium. In this study of biopsied human sural nerves, three-dimensional reconstruction of one micron sections and ultrastructural analysis of step serials, we find that TPA are confined in open-ended perineurial sleeves by which they pass from the epineurium through the perineurial sheath proper into the endoneurium. Most TPA are terminal arterioles as evidenced by size (10-25 microns), morphological characteristics, and the fact that they connect with capillaries. Transperineurial arterioles gradually lose their continuous muscle coat and become post-arteriolar capillaries (PAC). Vascular segments that emerge into the endoneurium from the perineurial sleeves are generally of the PAC variety. Transperineurial arterioles and post-arteriolar capillaries are often associated with a plexus of unmyelinated nerve fibers. Axon varicosities exhibit a variety of morphologically distinct vesicles including dense-cored and a diversity of agranular vesicles. These findings suggest that TPA play a role in the neurogenic control of endoneurial blood flow.     

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.     

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.     

Vascular innervation in human skeletal muscle with and without neuromuscular disease

Summary A quantitative ultrastructural study has been made of the innervation of 461 arterioles in 114 skeletal muscle biopsies of patients with or without neuromuscular disease excluding diabetes and autonomic neuropathy. In 18 controls the number of nerves and Schwann cells around each vessel was related to the size of the vessel, whether the vessel was within a muscle fascicle or between muscle fascicles. The innervation of arterioles increased with increased diastolic blood pressure. There was no statistically significant change in innervation with increased systolic blood pressure or with age, from 4 to 85 years. In 96 cases of neuromuscular disease and especially in motor neurone disease, axonal varicosities in cross section tended to be larger, more often contained no vesicles or only a few and had altered satellite cell cover depending on the location of the arteriole. Whilst the numerical density of Schwann cells did not change with disease, fewer varicosities were identified within Schwann cells in motor neurone disease, metabolic myopathy and neuropathy and myopathy due to toxins or vascular disease. Preterminal axons in nerve fascicles adjacent to arterioles were lost in polymyositis and muscle disease due to toxins or vascular disease. In polymyositis, metabolic myopathy and motor neurone disease there was some evidence of compensatory nerve sprouting, either in the nerve fascicles or in the adventitia of the arterioles. These structural changes may be related to the changes in blood flow or vascular reactivity described by others in motor neurone disease, polymyositis and metabolic myopathy. It is concluded that the ultrastructure of the vascular innervation of human skeletal muscle is similar to that in other mammals [12, 19] and is changed more with increased diastolic blood pressure and neuromuscular disease than with age.     

Changes in the density and distribution of sympathetic nerves in spleens from Lewis rats with adjuvant-induced arthritis suggest that an injury and sprouting response occurs

Previously we demonstrated reduced norepinephrine concentrations in spleens from Lewis rats with adjuvant-induced arthritis (AA), an animal model of rheumatoid arthritis. This study extends these findings, examining the anatomical localization and density of sympathetic nerves in the spleen with disease development. Noradrenergic (NA) innervation in spleens of Lewis rats was examined 28 days following adjuvant treatment to induce arthritis or vehicle for the adjuvant by using fluorescence histochemistry for catecholamines, with morphometric analysis and immunocytochemistry for tyrosine hydroxylase. In AA rats, sympathetic nerve density in the hilar regions, where NA nerves enter the spleen, was increased twofold over that observed in vehicle-treated rats. In contrast, there was a striking twofold decline in the density of NA nerves in splenic regions distal to the hilus in arthritic rats compared with nonarthritic rats. In both treatment groups, NA nerves distributed to central arterioles, white pulp regions, trabeculae, and capsule. However, NA nerve density was reduced in the white pulp but was increased in the red pulp in AA rats compared with non-AA rats. These findings indicate an injury/sprouting response with disease development whereby NA nerves die back in distal regions and undergo a compensatory sprouting response in the hilus. The redistribution of NA nerves from white pulp to red pulp suggests that these nerves signal activated immune cells localized in the red pulp in AA. Although the mechanisms of this redistribution of NA nerves into the red pulp are not known, it may be due to migration from white pulp to red pulp of target immune cells that provide trophic support for these nerves. The redistribution of NA nerves into the red pulp may be critical in modulating immune functions that contribute to the chronic inflammatory stages of arthritis.     

Enhanced inflammatory response via activation of NF-kappaB in acute experimental diabetic neuropathy subjected to ischemia-reperfusion injury

Reperfusion following ischemia increases ischemic fiber degeneration (IFD) in diabetic nerves compared to control normoglycemic nerves. The mechanism of this excessive susceptibility is unclear. Since reperfusion injury results in an inflammatory response, we tested the hypothesis that the diabetic state increases the inflammatory cascade. We used an animal model of unilateral ischemia-reperfusion (IR) injury to streptozotocin (STZ)-induced diabetic nerve to evaluate the density and localization of mediators of the inflammatory response using selective immunolabeling methods (for nuclear factor kappa B (NF-kappaB), intercellular adhesion molecule-1 (ICAM-1), cytokines and inflammatory cells). We studied a 1-month diabetic group and an age-matched control group (n=6 each). The right limb underwent 3 h ischemia at 35 degrees C and 7 days reperfusion. This was achieved by ligating the supplying arteries and collaterals to the right sciatic-tibial nerve for 3 h, followed by releasing the ties. Immunohistochemistry was performed on proximal sciatic and mid tibial nerves. NF-kappaB expression in diabetic sciatic endothelial cell and Schwann cell (SC) was significantly increased over that of controls subjected to identical IR injury. We observed a nearly 2-fold increase in density of NF-kappaB and ICAM-1 expression in microvessels of diabetic nerve compared with control nerve. Extensive infiltration of monocyte macrophages (1C7) was observed in the endoneurium of diabetic nerves, while only mild infiltration of granulocytes (HIS 48) occurred in the endoneurium of diabetic tibial nerves. This study provides evidence for an enhanced inflammatory response in diabetic nerves subjected to IR injury apparently via NF-kappaB activation.     

Differences between the peripheral and the central nervous system in permeability to sodium fluorescein

Sodium fluorescein (SF) was used as a very small tracer (mol wt 376; 5 A° diameter) to examine diffusion barriers in peripheral nerves and to compare them to those in other regions of the nervous system. The technique involved immobilization of the tracer by rapid freezing, followed by freezedrying and vacuum embedding in paraffin. The localization of the SF was then determined in tissue sections using fluorescence microscopy. Even at the highest doses of intravenously (IV) injected tracer, no extravasation could be detected in the cerebral cortex. On the other hand, SF penetrated very rapidly into peripheral ganglia and into the epineurium and perineurium of large peripheral nerves. The penetration of SF into the endoneurium of large nerves was, however, much more restricted with tracer detectable within the endoneurium only at high doses and long survival times. Even in such cases, the level of SF fluorescence was much lower within nerve fascicles than in the epineurium and the perineurium, and a sharp gradient in fluorescence intensity persisted at the inner border of the perineurium. The extent of extravasation into the endoneurium varied markedly between different fascicles of the same nerve and between different nerves in the same animal. Experiments involving injection of high doses of SF adjacent to the nerve indicated relatively little movement of SF across the perineurium, which indicates that the observed accumulation of tracer within the endoneurium was the result of direct extravasation of SF from the endoneural blood vessels. Small nerve branches (< 100 μ in diameter) showed an earlier and more extensive penetration of SF into the endoneurium than large nerves like the sciatic, hypoglossal, or ventral tail nerve. This may be due to a diffusion of SF along the extracellular space of the endoneurium from nerve terminals where the perineurial barrier is open-ended. In experiments involving IV injection of a solution containing both green fluorescent SF and red fluorescent Evans Blue (Evans Blue-serum albumin complex, EBA = mol wt 69,000), the distribution of SF could be directly compared at various sites and sacrifice times to that of EBA, a much larger tracer. SF appeared more rapidly and extensively than EBA in the various compartments in ganglia and peripheral nerve. The distribution of EBA was the same as is typically seen when this tracer is injected alone, indicating that there was no change in vascular permeability associated with IV injection of SF. Since SF is of very small size, freely diffusible, nontoxic, and detectable at very low concentrations, it should be a useful complement to existing tracers. When tissues are processed according to the indicated procedure, one can obtain a very sensitive and reliable localization of this tracer which should be of value for studies in the nervous system concerning various pathological conditions associated with permeability alterations.     

Perivascular noradrenergic and peptide-containing nerves show different patterns of changes during development and ageing in the guinea-pig

The development of noradrenergic and peptide-containing perivascular nerves in common carotid, mesenteric, renal and femoral arteries of the guinea-pig was studied using the glyoxylic acid fluorescence and indirect immunofluorescence techniques on whole-mount stretch preparations at 6 stages between 6 weeks in utero and two years after birth. The noradrenergic plexus was more dense than the peptide-containing nerve plexuses in all the blood vessels, and, in general, calcitonin gene-related peptide-containing nerves were more numerous than substance P-containing nerves which in turn were more numerous than vasoactive intestinal polypeptide-containing nerves. In mesenteric and carotid arteries, noradrenergic nerve density reached a peak at about 4 weeks after birth that was maintained to old age, whereas the peptide-containing vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP) nerve plexuses reached a peak at birth and declined thereafter to about half maximum density in old age. In contrast, in the renal and femoral arteries, peptide-containing nerves reached a maximum density at 4 weeks after birth, while noradrenergic nerve density reached a peak around birth; both noradrenergic and peptide-containing nerve plexuses declined in density in old age. Of the 4 vessels studied, the mesenteric artery showed the greatest density of innervation for both noradrenergic and peptide-containing nerves at all stages of development, while the femoral artery was the least innervated. The possibility that some perivascular peptide-containing nerves play a trophic role during development is discussed.     

Extrinsic pathways of the adrenergic innervation of the guinea-pig trachealis muscle

Origins and extrinsic pathways of the adrenergic innervation of the guinea-pig trachealis muscle were studied using fluorescence histochemical techniques. Bilateral superior cervical ganglionectomy caused a marked reduction in the adrenergic innervation of the extra-thoracic region, which suggests that these ganglia are a major source of adrenergic innervation to this muscle. Combined anterior and posterior transection of the recurrent laryngeal nerves also caused a marked reduction in the density of adrenergic fibres in the extra-thoracic trachealis muscle. Crushing of these nerves revealed adrenergic fibres running both anteriorly and posteriorly. The majority of these adrenergic nerves were lost after superior cervical ganglionectomy and thus the fibres running in both directions originate in the superior cervical ganglion. Antero-posteriorly directed fibres entered the recurrent laryngeal nerve from the superior cervical ganglion via an anastomosis at the level of the cricoid cartilage, while those running postero-anteriorly entered the recurrent laryngeal nerve posteriorly from the vagus nerve and these adrenergic fibres were lost after cervical vagotomy.     

Effect of oestrogen and progesterone on noradrenergic nerves and on nerves showing serotonin-like immunoreactivity in the basilar artery of the rabbit

The effects of oestrogen and progesterone on noradrenergic nerves and nerves with serotonin (5-HT)-like immunoreactivity in the basilar artery were investigated in the rabbit using whole-mount stretch preparations. The noradrenergic nerves were demonstrated by glyoxylic acid fluorescence histochemistry and nerves with 5-HT-like immunoreactivity by indirect immunofluorescence techniques. Quantitative image analysis of fluorescent nerve fibres revealed that nerve density and varicosity diameter of nerves with 5-HT-like immunoreactivity were significantly (P less than 0.01 and P less than 0.05 respectively) reduced after 4-week administration of oestrogen and the intensity of fluorescence was also reduced. However, there were no significant changes after progesterone. Neither oestrogen nor progesterone had any effect on noradrenergic innervation. The findings are discussed in relation to higher incidence of migraine headaches in females taking oral contraceptives.     

Trans-synaptic regulation of the development of end organ innervation by sympathetic neurons.

To examine the regulation of development of end organ innervation the superior cervical ganglion (SCG), and two of its target organs, the iris and pineal gland, were studied using biochemical and histofluorescent approaches. During postnatal ontogeny the activity of tyrosine hydroxylase (T-OH), which is localized to adrenergic neurons, increased 50-fold in iris, and 34-fold in pineal nerve terminals of the rat. These increases paralleled the in vitro rise in iris [3H]norepinephrine ([3H]NE) uptake, a measure of the presence of functional nerve terminal membrane. These biochemical indices of end organ innervation correlated well with developmental increases in density of innervation, adrenergic ground plexus ramification and nerve fiber fluorescence intensity as determined by fluorescence microscopy. Unilateral transection of the presynaptic cholinergic nerves innervating the SCG in 2-3-day-old rats prevented the normal development of end organ innervation: T-OH activity, [3H]NE uptake, innervation density, plexus ramification and fluorescence intensity failed to develop normally in irides innervated by decentralized ganglia. It is concluded that trans-synaptic factors regulate the maturation of adrenergic nerve terminals, and the development of end organ innervation by SCG.     

自发性高血压大鼠脑底动脉神经肽Y能神经纤维的分布

应用免疫组织化学技术ＡＢＣ法,对１０只雄性自发性高血压大鼠脑底动脉神经肽Ｙ能神经纤维分布进行了观察。在自发性高血压大鼠脑底血管的大脑前动脉、大脑中动脉、大脑后动脉和基底动脉壁上均可见棕褐色的免疫反应阳性纤维,纤维较粗,曲线状,呈网状分布,密度较高。与正常血压大鼠同一部位脑底动脉血管壁上的阳性纤维密度相比明显增加。结果表明：高血压大鼠脑底动脉各主要分支较正常血压大鼠脑底动脉各主要分支有更高密度的神经     

An increase in the expression of neuropeptidergic vasodilator, but not vasoconstrictor, cerebrovascular nerves in aging rats

Perivascular nerve fibres containing noradrenaline (NA), serotonin (5-HT), substance P (SP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) were localized in whole-mount stretch preparations of the arteries of the rat circle of Willis using fluorescence and immunohistochemical techniques. Changes in the pattern and density of these perivascular nerves were studied from birth to 27 months of age. All perivascular nerve types reached a peak density of innervation at 1 month of age. This was followed by a general fall in the density of fluorescent nerve fibres. However, with aging, there was a decrease in the expression of vasoconstrictor neurotransmitters (NA and 5-HT) in cerebrovascular nerves, whereas the expression of vasodilator neurotransmitter (VIP and CGRP) in perivascular nerve fibres supplying the rat cerebral arteries was strikingly increased in old age. The density of NPY- and SP-containing nerve fibres was not significantly altered in old age. These changes are discussed in relation to the increased incidence of cerebrovascular disorders in the elderly.