A novel method to quantify cutaneous vascular innervation.

INTRODUCTION: To develop a new method to quantify the density of nerves, vessels, and the neurovascular contacts, we studied skin biopsies in diabetes and control subjects. METHODS: Skin biopsies with dual immunofluorescent staining were used to visualize nerves and blood vessels. The density of nerves, vessels, and their neurovascular contacts were quantified with unbiased stereology. Results were compared with examination findings, validated questionnaires, and autonomic function. RESULTS: In tissue from 19 controls and 20 patients with diabetes, inter-rater and intra-rater intraclass correlation coefficients were high (>0.85; P < .001) for all quantitative methods. In diabetes, the nerve densities (P < .05), vessel densities (P < .01), and the neurovascular densities (P < .01) were lower compared with 20 controls. Results correlated with autonomic function, examination and symptom scores. DISCUSSION: We report an unbiased, stereological method to quantify the cutaneous nerve, vessel and neurovascular density and offer new avenues of investigation into cutaneous neurovascular innervation in health and disease.

The Pericyte of the Pancreatic Islet Regulates Capillary Diameter and Local Blood Flow.

Efficient insulin secretion requires a well-functioning pancreatic islet microvasculature. The dense network of islet capillaries includes the islet pericyte, a cell that has barely been studied. Here we show that islet pericytes help control local blood flow by adjusting islet capillary diameter. Islet pericytes cover 40% of the microvasculature, are contractile, and are innervated by sympathetic axons. Sympathetic adrenergic input increases pericyte activity and reduces capillary diameter and local blood flow. By contrast, activating beta cells by increasing glucose concentration inhibits pericytes, dilates islet capillaries, and increases local blood flow. These effects on pericytes are mediated by endogenous adenosine, which is likely derived from ATP co-released with insulin. Pericyte coverage of islet capillaries drops drastically in type 2 diabetes, suggesting that, under diabetic conditions, islets lose this mechanism to control their own blood supply. This may lead to inadequate insulin release into the circulation, further deteriorating glycemic control.

Analysis of nerve supply pattern in human lymphatic vessels of young and old men.

BACKGROUND: The present work deals with innervation patterns along collector lymphatic vessels from cervical, mesenteric, and femoral regions, and lymph capillaries in young and elderly subjects. METHODS AND RESULTS: Morphological and morphometric analysis of nerve fibers along lymph vessels was performed by immunohistochemistry for PGP 9.5, NPY, TH, ChAT, VIP, SP, and dopamine. Nerves containing NPY and TH were frequent, whereas immunoreactivity for ChAT and VIP were few. SP-positive fibers were widely distributed in the medial and endothelial layers. Dopamine neurotransmitters were observed in a few short nerve fibers. A more diffuse presence of nerve fibers in mesenteric and femoral lymph vessels, compared to cervical ones, was detected. In lymph capillary vessels, a few nerve fibers positive for neuropeptides and neurotransmitters were detected, whereas no dopamine and VIP immunoreactive fibers were detected. A wide reduction of all specific nerve fibers analyzed was detected in lymph vessels from elderly subjects. CONCLUSIONS: The presence on lymph vessels of sympathetic and parasympathetic nerve systems can be declared. The differences observed in lymphatic vessel innervation patterns may note the involvement in lymph flow regulation, calling attention in aging, when nerve fibers reduction may cause functional default of lymph vessels.

Optimal dose of plasmid vascular endothelial growth factor for enhancement of angiogenesis in the rat brain ischemia model.

Vascular endothelial growth factor (VEGF) administration has recently been assessed as a therapeutic strategy for ischemic diseases including brain ischemia because of its angiogenic effect. However, VEGF also causes detrimental adverse effects by increasing vascular permeability. This study examined whether plasmid human VEGF (phVEGF) administration induced angiogenic effects in the rat brain ischemia model caused by permanent ligation of both common carotid arteries, and investigated the occurrence of adverse effects. Administration of various doses (0-200 microg) of phVEGF in the temporal muscle was followed by encephalo-myo-synangiosis. Thirty days after treatment, the numbers and areas of capillaries per field in the extracted brains were analyzed with the National Institutes of Health Image software program. The maximal angiogenic effect occurred with a 100 microg dose of phVEGF in the numbers and areas of capillaries in the VEGF-treated brains. Histological examination showed no apparent adverse effects in the brain parenchyma even at the highest administration dose (200 microg) of phVEGF. The maximal angiogenic effect at the optimal dose of phVEGF can be considered under the threshold to cause serious adverse effects in the rat brain.

Excitatory effects of the puberty-initiating peptide kisspeptin and group I metabotropic glutamate receptor agonists differentiate two distinct subpopulations of gonadotropin-releasing hormone neurons.

Activation of the G-protein-coupled receptor GPR54 by kisspeptins during normal puberty promotes the central release of gonadotropin-releasing hormone (GnRH) that, in turn, leads to reproductive maturation. In humans and mice, a loss of function mutations of GPR54 prevents the onset of puberty and leads to hypogonadotropic hypogonadism and infertility. Using electrophysiological, morphological, molecular, and retrograde-labeling techniques in brain slices prepared from vGluT2-GFP and GnRH-GFP mice, we demonstrate the existence of two physiologically distinct subpopulations of GnRH neurons. The first subpopulation is comprised of septal GnRH neurons that colocalize vesicular glutamate transporter 2 and green fluorescent protein and is insensitive to metabotropic glutamate receptor agonists, but is exquisitely sensitive to kisspeptin which closes potassium channels to dramatically initiate a long-lasting activation in neurons from prepubertal and postpubertal mice of both sexes. A second subpopulation is insensitive to kisspeptin but is uniquely activated by group I metabotropic glutamate receptor agonists. These two physiologically distinct classes of GnRH cells may subserve different functions in the central control of reproduction and fertility.

In vitro culture of rat neuromicrovascular endothelial cells on polymeric scaffolds.

Polyphosphazenes are polymers possessing a skeleton composed of alternating phosphorous and nitrogen atoms, and two side-moieties linked to each phosphorous atom. Polyphosphazenes with amino acid esters as side-moieties are biocompatible and biodegradable polymers. Two polyphosphazenes, poly[bis(ethyl alanate) phosphazene] and poly[(ethyl phenylalanate)0.8(ethyl alanate)0.8(ethyl glycinate)0.4 phosphazene] (PPAGP) were synthesized, and processed to form small fibers. Their ability to support rat neuromicrovascular endothelial cell (EC) adhesion and growth has been studied, using poly(D,L-lactic acid) as reference compound. Scanning electron microscopy revealed that both poly[bis(ethyl alanate) phosphazene] and PPAGP fibers were thinner than poly(D,L-lactic acid) fibers, and possessed a more irregular and porous surface. All polymers increased EC adhesion, compared with polystyrene, but only polyphosphazenes were able to improve EC growth. The highest increase in EC proliferation was induced by PPAGP, which, as revealed by environmental scanning electron microscopy, was also able to induce ECs to arrange into tubular structures. The conclusion is drawn that PPAGP may provide the best scaffold for engineered blood vessels, because it promotes adhesion, growth, and organization of ECs into capillary-like structures.

Immunohistochemical study of immunophilin 1-15 fragment in intact frog brain, and in the brain and spinal cord of intact and spinal cord hemisectioned rats.

Previously by immunohistochemical technique the distribution of immunophilin 1-15 fragment (IphF) isolated from bovine hypothalamus was examined in various tissues (heart, lung), including immune system organs (spleen and thymus) of intact rats. IphF-like immunoreactivity (IphF-LI) was revealed in several cell types: lymphocytes, monocytes, macrophages and mast cells. In the present study the immunohistochemical localization of IphF was examined in intact rat and frog brains. In rat brain several cell groups concentrated particularly in the supraoptic nucleus (SON) of hypothalamus, medulla oblongata (reticular formation, olives, hypoglossal and facial motor nuclei) and cerebellum (lateral cerebellar nucleus) demonstrated IphF-LI. In frog hypothalamus (SON) the same working dilution (1:5000) of IphF-antiserum revealed very strong immunoreactivity. In the paraventricular nucleus (PVN) IphF-LI varicosities were scattered around the immunonegative cells. The second cell groups showing IphF-LI in the frog brain were gliocytes (mainly the astrocytes). Besides, IphF distribution was investigated in rats subjected to hemisection of spinal cord (SC) with and without administration of proline-rich polypeptide (PRP). PRP was isolated from bovine neurohypophysis neurosecretory granules, produced by magnocellular nuclei of hypothalamus. Hemisection of SC led to changes of IphF distribution in the hypothalamus. In PRP treated animals IphF showed no immunoreactivity. PRP is suggested to act as a neurotransmitter and neuroregulator.

[Basic classical, peptidergic and nitrergic innervation pattern of human nasal glands--a histochemical and immunohistochemical study]. / Die klassisch-vegetative, peptiderge und nitrerge Innervation der Drüsen der menschlichen Nasenschleimhaut1.

BACKGROUND: Seromucous glands are important components of the human nasal mucosa. The innervation patterns are relevant for understanding the control of the different physiological and pathophysiological glandular functions. Beside classic neurotransmitters some neuropeptides seem to influence the glandular secretion. METHODS: Tissue samples of 35 human inferior turbinates were taken during nasal surgery and preserved. Serial cryosections or paraffin sections were cut and incubated with antibodies either to Tyrosinhydroxilase or to Vasoactive Intestinal Peptide (VIP), Calcitonin Gene-Related Peptide (CGRP) and endothelial or brain Nitric Oxide Synthase (eNOS or bNOS). AChE- and NADPH-diaphorase-histochemistry were performed. RESULTS: Immunoreactive nerve fibers were found in the periglandular tissue around the acini, ducts and in the periglandular connective tissue. The density of positive immunoreactive structures depended on the different antibodies. VIP was found in contact to acinus cells, CGRP in the connective tissue around glandular cells. Particular immunoreactions to VIP and CGRP-antibodies could be detected near the glandular duct system. The eNOS-reactions were found in small capillaries near the acinus cells. CONCLUSIONS: Immunohistochemical and histochemical methods allow a detailed marking of nerval structures in nasal mucosa. The localization of neurons with different neurotransmitters and neuropeptides in the periglandular tissue confirms the direct nerval control of the diverse glandular functions. The detection of bNOS- and NADPH-d-positive structures around glandular cells and eNOS in the endothelium of periglandular capillaries suggests that NO takes an additional part in the regulation of nasal glands.

Absence of cholinergic sympathetic innervation from limb muscle vasculature in rats and mice.

Although the existence of cholinergic sympathetic vasodilatory innervation in limb muscle vasculature is well established for some species, previous pharmacological studies have failed to reveal the presence of such innervation in rats. Recently, Schafer and colleagues [Schafer, M.K., Eiden, L.E., Weihe, E., 1998. Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system. Neuroscience 84(2), 361-376] reported that vesicular acetylcholine transporter immunoreactivity (VAChT-IR), a marker for cholinergic terminals, is present in the innervation of the microvasculature of rat hindlimb skeletal muscle and concluded that rats possess cholinergic sympathetic innervation of limb muscle vasculature. Because of our interest in identifying targets of cholinergic sympathetic neurons, we have analyzed the transmitter properties of the innervation of muscle vessels in rat and mouse limbs. We found that the innervation of vasculature in muscle is noradrenergic, exhibiting robust catecholamine histofluorescence and immunoreactivity for tyrosine hydroxylase (TH) and the peptide transmitters, neuropeptide Y (NPY) and occasionally vasoactive intestinal peptide (VIP). In contrast, cholinergic phenotypic markers,VAChT-IR and acetylcholinesterase (AChE) activity, are absent. Neuron cell bodies in sympathetic ganglia, retrogradely labeled with injections of tracer into limb muscles, also lacked VAChT but contained TH-IR. The innervation of large extramuscular feed arteries in hindlimbs was also devoid of cholinergic markers, as were the cell bodies of sympathetic neurons innervating extramuscular femoral arteries. These results, like those of previous physiological studies, provide no evidence for the presence of cholinergic sympathetic innervation of muscle vasculature in rats or mice.

NK-1 receptor desensitization and neutral endopeptidase terminate SP-induced pancreatic plasma extravasation.

Substance P (SP) induces plasma extravasation and neutrophil infiltration by activating the neurokinin-1 receptor (NK1-R). We characterized the mechanisms regulating this response in the rat pancreas. Anesthetized rats were continuously infused with SP, and plasma extravasation was quantified using Evans blue (EB) dye. Continuous infusion of SP (8 nmol. kg(-1). h(-1)) resulted in a threshold increase in EB at 15 min, a peak effect at 30 min (150% increase), and a return to baseline by 60 min. The NK1-R antagonist CP-96,345 blocked SP-induced plasma extravasation. After 60 min, the NK1-R was desensitized to agonist challenge. Resensitization was first detected at 20 min and increased until full recovery was seen at 30 min. Inhibition of the cell-surface protease neutral endopeptidase (NEP) by phosphoramidon potentiated the effect of exogenous SP; therefore endogenous NEP attenuates SP-induced plasma extravasation. Thus the continuous infusion of SP stimulates plasma extravasation in the rat pancreas via activation of the NK1-R, and these effects are terminated by both desensitization of the NK1-R and the cell-surface protease NEP.

An immunohistochemical study on the innervation of two peptidergic (NPY and VIP) nerves in the cerebral arterial tree and choroid plexus of the newt (Amphibia: Urodela).

The pattern of neuropeptide Y (NPY)- and vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) innervation was investigated in the cerebral arterial tree and choroid plexus of the newt by the use of an indirect immunofluorescence technique combined with chemical sympathectomy (6-OHDA). The data presented here, in conjunction with histochemical data reported previously, showed the following characteristic features of cerebrovascular innervation in this urodelan species. (1) The cerebral arterial tree and choroid plexus had unbalanced NPY-IR and VIP-IR innervation, characterized by the absence or a markedly lesser density of VIP-IR nerves. (2) All or nearly all of the NPY-IR nerves were sympathetic in nature. (3) A few cerebral perivascular NPY-IR nerves in some individuals originated from the sympathetic NPY-IR nerve cells intrinsic to the major cerebral arteries of the anterior circulatory system. (4) Acetylcholinesterase-positive neurons lacking both NPY and VIP immunoreactivities are a major nerve type in cerebrovascular parasympathetic innervation. The preferential NPY-IR innervation of the plexus microvascular-epithelial regions must be considered in relation to its special functions, such as the regulation of microcirculation, and cerebrospinal fluid (CSF) production and transportation. CSF is vital for the movement of nutrients and metabolites in the newt brain.

Effects of hypnotic suggestions on ultraviolet B radiation-induced erythema and skin blood flow.

Results from both animal and human studies have indicated that inflammatory skin reactions such as the flare response to histamine prick test involve a neurogenic regulatory component. It is still unknown to which degree inflammation induced by ultraviolet (UV) radiation may be mediated by the central nervous system. To investigate this, the effect of hypnotic suggestions to increase and decrease the response to UVB radiation on erythema and cutaneous blood flow was investigated in 10 highly hypnotizable subjects. The results showed a significant effect of hypnotic suggestions on cutaneous blood flow compared with the responses of a control group. For erythema no conclusive evidence was found. The results indicate the possibility of separate regulatory mechanisms behind central nervous system influence on UVB-induced erythema and skin blood flow respectively, and further investigations are needed.

A low frequency, high amplitude rhythmic fluctuation of laser-Doppler skin blood flow after subarachnoid phenol block.

A 51-year-old male with a huge chondrosarcoma received subarachnoid dorsal root blocks with 10% phenol in glycerine to treat severe pain along the left leg. The dermatomes below the Th9 lost all somatic sensation on the left side after the nerve blocks, but the patient was not completely relieved from the pain. Laser-Doppler flowmetry on the toe of the left foot disclosed an increased blood flow and an abnormal fluctuation of the cutaneous capillary blood flow, i.e. a high amplitude rhythmic (HAR) wave with 2.5 to 3 cycles.min-1. The low frequency HAR wave persisted for the subsequent 3 months until a tingling sensation returned to the left leg. It would seem that some travelling roots of the sympathetic nerve were preserved from the chemical neurolysis and the remaining efferent and afferent nerve fibers were responsible for the persisting low frequency HAR wave and pain in the left leg.

Neuronal control of brain microvessel function.

Cerebral capillary endothelium forms a barrier limiting and controlling the movement of ions and solutes between blood and brain. Recent anatomical, physiological and biochemical studies have suggested the possibility that capillary function may be directly controlled by neuronal structures. Alterations in neuronal systems involved in the regulation of microcirculation may account for microvascular dysfunctions which occur in different pathologic conditions.

Neurohemal contact in the internal zone of the rabbit median eminence.

The concept of neurosecretion as the mechanism by which neural control of adenohypophyseal function is accomplished was based on the observation that long capillary loops penetrate deeply into the supraopticohypophyseal tract as it passes through the median eminence internal zone. However, neural contact upon these capillary loops has not been demonstrated in the mammalian median eminence. The present transmission electron microscopic investigation of the rabbit median eminence demonstrates neurohemal contact in the median eminence internal zone. Axons containing small lucent vesicles 53.3 +/- 3.28 nm in diameter (mean +/- SEM) or small lucent and large granular vesicles with a mean diameter of 122.4 (+/- 3.28 nm) in their terminals make neurohemal contact with capillary loops in the internal zone and form a cuff about them. These terminals resemble terminals found in the external zone. Intravenous injection of the false neurotransmitter 5-hydroxydopamine (5-OH-DA) renders small lucent vesicles granular in both the external and internal zone. The effect of 5-OH-DA injection is abolished by concurrent reserpine administration. Whereas large granular vesicles in many terminals become lucent after reserpine administration, in others they remained electron dense. Viewed in the light of previous studies our findings suggest that the internal plexus arises from the external plexus and invaginates the neuropil carrying connective tissue and parvicellular axon terminals of aminergic and peptidergic systems from the external zone into the internal zone, that some elements making neurohemal contact with long capillary loops are terminals of the noradrenergic reticular infundibular tract arising outside the hypothalamus in the brainstem, and that long capillary loops form a system of repeating microvascular modules which markedly increase the surface available for neurohemal contact.

Facial flushing after thermocoagulation of the Gasserian ganglion.

The development of a facial flush during thermocoagulation of the Gasserian ganglion was monitored in 16 patients with pulse recording techniques and in a further 17 patients with thermography. There was a close association between the development of the facial flush in the distribution of one or more divisions of the trigeminal nerve and the subsequent demonstration of postoperative analgesia. In regions where significant changes took place, vascular pulsations increased 25-233% (mean 96%) and facial temperature rose 0.5-2.0 degrees C. The response persisted for up to an hour postoperatively, and was not diminished in patients with pre-operative analgesia from a previous procedure. Possible mechanisms for the facial flush, including stimulation of an active vasodilator system, the antidromic release of vasoactive substances from trigeminal nerve terminals and the release of tonic vasoconstriction are discussed. A practical application of the pulse recording technique used in the present investigation would be to monitor the distribution of vasodilatation at operation to avoid unwanted first division sensory loss.