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.     

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.     

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.     

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.     

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 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.     

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.     

Presence of a blood-nerve barrier within blood vessels of frog sciatic nerve

The morphological correlates of protein permeability in endoneurial blood vessels of the frog sciatic nerve were investigated. The endothelial cells of these vessels possess numerous free and anastomosing vesicles at both their lumenal and ablumenal surfaces. The cells are joined by junctions characterized by one or two areas of membrane apposition. However, vesicular transport, transcellular channels or open junctions are not found. The protein tracers horseradish peroxidase and microperoxidase remained confined in the vessels after intravascular injection. Likewise, if injected into the endoneurium, horseradish peroxidase did not enter the vascular lumen. The endothelium of frog endoneurial blood vessels forms an effective barrier to protein tracers.     

Chronic constriction model of rat sciatic nerve: nerve blood flow,morphologic and biochemical alterations

We evaluated the nerve blood flow (NBF), light and electron microscopy, and adrenergic innervation of rat sciatic nerve at 2–45 days after the application of four loose ligatures. Ischemia developed at the lesion edge, creating an endoneurial dam. Calcitonin gene-related peptide, norepinephrine and NBF were increased within the lesion. Morphologic alterations consisted of early endoneurial edema, followed by myelinated fiber degeneration, with relative sparing of small myelinated and unmyelinated nerve fibers, and leukocyte adhesion to microvessels. Axonal degeneration predominated over demyelination. At 45 days, profuse regeneration of small myelinated fibers was seen. The mechanism of lesional sensitization is discussed. Received: 30 May 1996 / Revised, accepted: 13 August 1996     

Increased expression of chemokines (MCP-1, MIP-1alpha, RANTES) after peripheral nerve transection

After nerve injury, recruitment of circulating macrophages into the endoneurium is essential for degeneration and subsequently for successful regeneration. However, the factors leading to macrophage recruitment are not known in detail. Chemokines are one of many possible factors influencing recruitment. In this study we wanted to examine, immunohistochemically, the expression of MCP-1, MIP-1alpha and RANTES from 6 hours up to 4 weeks after transection of rat sciatic nerve. An increased expression of MCP-1 was noted already 6 hours after transection, mainly in Schwann cells. Later, the MCP-1 positive staining was seen also in macrophages, fibroblast-like cells and endothelial cells. An increased number of MIP-1alpha positive cells could be noticed after 24 hours, the maximum expression in Schwann cells was noted at the 5-day timepoint. Later, part of the positive cells appeared to be macrophages. RANTES was mainly expressed in inflammatory cells. Endothelial cells in the epi- and endoneurium showed positive staining for every chemokine studied after transection. The contralateral non-operated nerves showed an increased number of positive cells for MCP-1 and MIP-1alpha. In the control nerves MCP-1 and MIP-1alpha positive cells were scattered throughout the endoneurium. This study shows that increased expression of chemokines takes place within endoneurium after peripheral nerve transection. Thus, it is probable that chemokines can take part in the recruitment of macrophages. It further shows that there is an increased expression of the studied chemokines in the non-operated contralateral nerves. Even in normal conditions chemokines are needed, probably to keep resident macrophages within endoneurium.     

An ultrastructural and biochemical analysis of collagen in rat peripheral nerves: the relationship between fibril diameter and mechanical properties

Mechanical features are distributed heterogeneously within nerve tissue, with compliance increased at articulations. This study explored whether differences in stiffness between joint regions (JRs) and non-joint regions (NJRs) of rat median and sciatic nerves were related to localised variation in collagen content or fibril diameter. There was no significant difference in the amount of collagen detected by biochemical assay in JRs and NJRs of either nerve. Ultrastructural analysis showed collagen fibril diameter ranges of 20-80 nm in the endoneurium and perineurium and 30-130 nm in the epineurium. In the median nerve, but not the sciatic nerve, there were significantly smaller fibrils in JRs compared to NJRs. This corresponded to a greater number density of fibrils in JRs compared to NJRs in the epineurium and endoneurium of the median nerve. We report the presence therefore of a population of thinner collagen fibrils in the JR of the median nerve that corresponds to the location of increased compliance in this tissue, suggesting that localised variation in collagen fibril diameter contributes to the longitudinal heterogeneity of tensile properties in this nerve.     

Chondroitinase applied to peripheral nerve repair averts retrograde axonal regeneration

Antegrade, target-directed axonal regeneration is the explicit goal of nerve repair. However, aberrant and dysfunctional regrowth is commonly observed as well. At the site of surgical nerve coaptation, axonal sprouts encounter fibrotic connective tissue rich in growth-inhibiting chondroitin sulfate proteoglycan that may contribute to misdirection of axonal regrowth. In the present study, we tested the hypothesis that degradation of chondroitin sulfate proteoglycan by application of chondroitinase at the site of nerve repair can decrease aberrant axonal growth. Adult rats received bilateral sciatic nerve transection and end-to-end repair. One nerve was injected with chondroitinase ABC and the contralateral nerve treated with vehicle alone. After 28 weeks, retrograde axonal regeneration was assessed proximal to the repair by scoring neurofilament-immunopositive axons within the nerve (intrafascicular) and outside the nerve proper (extrafascicular). Intrafascicular retrograde axonal growth was equivalent in both control and chondroitinase treatment conditions. In contrast, chondroitinase treatment caused a pronounced (93%) reduction in extrafascicular retrograde axonal growth. The decrease in axon egress from the nerve was coincident with an increase in antegrade regeneration and improved recovery of motor function. Based on these findings, we conclude that chondroitinase applied at the site of nerve transection repair averts dysfunctional extrafascicular retrograde axonal growth.     

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.     

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.     

BDNF overexpression induces differential increases among subsets of sympathetic innervation in murine back skin

Besides their recognized dependence on nerve growth factor (NGF) during development, the dependence of mature sympathetic ganglion neurons on other neurotrophins is still unclear. Here, we have investigated the sympathetic innervation of back skin in mice overexpressing brain‐derived neurotrophic factor (BDNF) under the alpha‐myosin heavy‐chain promoter, as well as in BDNF knockout (–/–) mice. Compared with wild‐type controls, the dorsal skin of BDNF overexpressing mice displayed a significantly enhanced number of adrenergic, tyrosine hydroxylase‐immunoreactive (IR) nerve fibres, while cholinergic or peptidergic sensory nerve fibres appeared unaltered. The adrenergic hyperinnervation in dorsal skin of BDNF overexpressing mice was most pronounced in the arrector pili muscle of hair follicles, while no increase of tyrosine hydroxylase‐or neuropeptide Y‐IR fibres associated with subcutaneous blood vessels was found. Instead, back skin of BDNF knockout (–/–) mice contained significantly fewer tyrosine hydroxylase‐IR dermal nerve fibres than wild‐type animals. This suggests that BDNF plays an important role in the control of different subsets of adrenergic innervation in murine back skin, and indicates that paravertebral sympathetic ganglia display a previously unrecognized differential BDNF‐dependence in vivo.     

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.     

Re-innervation of submucosal arterioles by myenteric neurones following extrinsic denervation.

We used a combination of selective lesions, immunohistochemistry and video monitoring of arteriolar diameter to determine the source of the changes in vasodilator innervation to guinea pig ileal submucosal arterioles which occur following removal of their extrinsic sympathetic and sensory nerve fibre input. A non-cholinergic neurogenic vasodilation appeared in arterioles in which extrinsic denervation was performed 50-90 days previously. The non-cholinergic innervation did not result from regrowth of extrinsic fibres because the neurogenic response was not altered by combining long-term denervation with capsaicin treatment or re-denervation 7 days prior to examination. However, non-cholinergic neurogenic vasodilations were not observed in arterioles which had been subjected to long-term denervation combined with a myectomy 7 days prior to examination. Immunohistochemical co-localization of SP and CGRP in these vessels confirmed previous findings that a prominent SP perivascular nerve plexus appeared after long-term denervation. Perivascular SP-containing fibres that appeared after long-term denervation were unaffected by capsaicin or re-denervation but were absent from preparations in which long-term denervation and myectomy were performed. These results demonstrate that myenteric neurones are the source of the non-cholinergic innervation which appears after extrinsic denervation and support our previous conclusion that SP is the neurotransmitter responsible for this non-cholinergic vasodilation in submucosal arterioles of the small intestine.     

Immunoelectronmicroscopical demonstration of major histocompatibility class II antigen: expression on endothelial and perivascular cells but not Schwann cells in human neuropathy

A pre-embedding technique for identifying major histocompatibility (MHC) class II antigen with monoclonal antibody LN3 at the electron microscope level by immunogold silver enhancement was applied to sections from 12 human nerve biopsies. Many perivascular mononuclear cells in the epineurium, perineurium and endoneurium expressed MHC class II antigen and had the morphological appearance of macrophages. Cell processes expressing MHC class II antigen extended throughout the endoneurium, often close to Schwann cells. No MHC class II expression was identified on myelinating or non-myelinating Schwann cells. The endothelial cells of epineurial blood vessels expressed MHC class II antigen on their luminal surfaces more often and more strongly than those of the endoneurial vessels. These observations indicate that perivascular cells in both the endoneurium and perineurium commonly express the molecules necessary to present antigen to CD4⁺ T lymphocytes, but Schwann cells do not.     

Spontaneous Incomplete Recovery of Peroneal Palsy After a Sealbite

Incomplete recovery of peroneal palsy, after a sealbite during swimming has not been reported. A 58-year-old woman was bitten into the right knee by a seal during swimming, resulting in incomplete division of the right profound branch of the peroneal nerve and complete division of the superficial branch of the peroneal nerve. Initial drop-foot, absent foot eversion, and hypaesthesia of the lateral lower leg were followed by almost complete clinical recovery of motor functions within four month by spontaneous re-innervation from the proximal nerve stump, despite severely abnormal electrophysiological findings. Re-evaluation 6 years after the accident revealed only discrete weakness for foot and toe extension, slight distal hypaesthesia, but still increased distal latency, reduced compound muscle action potential, slowed nerve conduction velocity of the right peroneal nerve, and neurogenic electromyography of the right anterior tibial and long peroneal muscles. Nerve conduction studies were hampered by co-innervation of the extensor digitorum brevis muscles by the tibial nerves. This case shows that peroneal palsy from a sealbite may recover spontaneously almost completely, but may go along with residual, subclinical, electrophysiological abnormalities. The later may be enhanced by innervation variants.     

Stimulation of an intracranial trigeminally-innervated structure selectively increases cerebral blood flow

The cranial circulation, both extracerebral and cerebral, is innervated by fibers from the trigeminal nerve. This system is known as the trigeminovascular system. The large venous sinuses and dura mater are pain-sensitive and are innervated primarily by branches of the ophthalmic division of the trigeminal nerve. Studies were conducted in the α-chloralose anaesthetised cat to examine bulk carotid and cerebral blood flow responses to electrical stimulation of the trigeminal ganglion and superior sagittal sinus. Bulk carotid blood flow was measured using an ultrasonic flow probe and meter applied to the common carotid artery while cerebral blood flow was measured using laser Doppler flowmetry. Vascular resistance was calculated using simultaneously collected blood pressure data. Stimulation of the trigeminal ganglion resulted in a frequency-dependent reduction in both bulk carotid and cerebral vascular resistance. The mean maximal reduction was 39±5% at 20/s for the carotid bed and 37±6% at 20/s for the cerebral circulation. Stimulation of the superior sagittal sinus resulted in a frequency-dependent reduction in resistance that involved the cerebral circulation with little effect on bulk carotid resistance. The mean maximum reduction was 37±6% at 20/s for the cerebral circulation and 11±3% at 2/s for bulk carotid resistance. The more focused effects of superior sagittal sinus suggest a highly organised somatotopic arrangement of the trigeminal innervation of the cranial circulation. Such a physiological schema fits the known anatomy as reflected by the differential peptidergic innervation from the trigeminovascular system to cranial vessels and may be important in understanding the pathophysiology of migraine, cluster headache and subarachnoid haemorrhage. ©1997 Elsevier Science B.V. All rights reserved.