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.

Granular Layer Neurons Control Cerebellar Neurovascular Coupling Through an NMDA Receptor/NO-Dependent System.

Neurovascular coupling (NVC) is the process whereby neuronal activity controls blood vessel diameter. In the cerebellum, the molecular layer is regarded as the main NVC determinant. However, the granular layer is a region with variable metabolic demand caused by large activity fluctuations that shows a prominent expression of NMDA receptors (NMDARs) and nitric oxide synthase (NOS) and is therefore much more suitable for effective NVC. Here, we show, in the granular layer of acute rat cerebellar slices, that capillary diameter changes rapidly after mossy fiber stimulation. Vasodilation required neuronal NMDARs and NOS stimulation and subsequent guanylyl cyclase activation that probably occurred in pericytes. Vasoconstriction required metabotropic glutamate receptors and CYP ω-hydroxylase, the enzyme regulating 20-hydroxyeicosatetraenoic acid production. Therefore, granular layer capillaries are controlled by the balance between vasodilating and vasoconstricting systems that could finely tune local blood flow depending on neuronal activity changes at the cerebellar input stage. SIGNIFICANCE STATEMENT: The neuronal circuitry and the biochemical pathways that control local blood flow supply in the cerebellum are unclear. This is surprising given the emerging role played by this brain structure, not only in motor behavior, but also in cognitive functions. Although previous studies focused on the molecular layer, here, we shift attention onto the mossy fiber granule cell (GrC) relay. We demonstrate that GrC activity causes a robust vasodilation in nearby capillaries via the NMDA receptors-neuronal nitric oxide synthase signaling pathway. At the same time, metabotropic glutamate receptors mediate 20-hydroxyeicosatetraenoic acid-dependent vasoconstriction. These results reveal a complex signaling network that hints for the first time at the granular layer as a major determinant of cerebellar blood-oxygen-level-dependent signals.

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.

Spatiotemporal distribution of neurovascular alignment in remodeling adult rat mesentery microvascular networks.

An emerging area of microvascular research focuses on the links between neural and vascular patterning. However, the functional dependence between vascular and neural growth in adult tissues remains underinvestigated. The objective of this study was to determine the spatial and temporal coordination between vascular and neural networks over a time course of adult microvascular growth. Mesentery tissues from adult male Wistar rats were harvested prior to stimulation, and 2, 10 and 30 days after angiogenesis stimulated by mast cell degranulation. Tissues were immunolabeled for PECAM (endothelial cell marker) and class III ß-tubulin (peripheral nerve marker). Neurovascular alignment was quantified per vessel category: arterioles (>20 µm), pre-capillary arterioles (10-20 µm), post-capillary venules (10-20 µm), venules (>20 µm), capillaries (<10 µm) and capillary sprouts. Neurovascular alignment along pre-capillary arterioles, capillaries, post-capillary venules and venules was decreased compared to unstimulated levels on days 2 and 10. These decreases inversely correlated with increases in vessel density per vessel category. By day 30, alignment either returned to unstimulated levels or was increased compared to day 10. These results suggest that neurovascular alignment arises after microvascular network growth and is present along arterioles, venules and even capillaries.

Oscillating neuro-capillary coupling during cortical spreading depression as observed by tracking of FITC-labeled RBCs in single capillaries.

Coupling between capillary red blood cell (RBC) movements and neuronal dysfunction during cortical spreading depression (CSD) was examined in rats by employing a high-speed camera laser-scanning confocal fluorescence microscope system in conjunction with our Matlab domain software (KEIO-IS2). Following microinjection of K(+) onto the surface of the brain, changes in electroencephalogram (EEG), DC potential and tissue optical density were all compatible with the occurrence of a transient spreading neuronal depression. RBC flow in single capillaries was not stationary. Unpredictable redistribution of RBCs at branches of capillaries was commonly observed, even though no change in diameter was apparent at the reported site of the capillary sphincter and no change of arteriolar-venule pressure difference was detected. There appeared to be a slow morphological change of astroglial endfeet. When local neurons were stunned transiently by K(+) injection, the velocity and oscillation frequency of RBCs flowing in nearby capillaries started to decrease. The flow in such capillaries was rectified, losing oscillatory components. Sluggish floating movements of RBCs in pertinent capillaries were visualized, with occasional full stops. When CSD subsided, RBC movements recovered to the original state. We postulate that neuronal depolarization blocks oscillatory signaling to local capillaries via low-shear plasma viscosity increases in the capillary channels, and a complex interaction between the RBC surface and the buffy coat on the capillary wall surface increases the capillary flow resistance. Then, when CSD subsides and oscillatory neuronal function is recovered, the normal physiological conditions are restored.

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.

[Decreased numerical density of pericapillary oligodendrocytes in the cortex in schizophrenia].

Autopsy samples from the prefrontal cortex were obtained from 22 schizophrenic cases and 22 normal controls. Capillaries and oligodendrocytes were viewed in paraffin sections stained with a Luxol-fast blue + cresyl violet. Electron microscopy was applied to study the ultrastructure of oligodendrocytes. Numerical density of pericapillary oligodendrocytes was measured in layer V of Broadmann's area 10. Subjects with schizophrenia had a significantly lower (-20,8%) numerical density of pericapillary oligodendrocytes compared to controls. The analysis for schizophrenic subgroups with predominantly positive or predominantly negative symptoms revealed a significant decrease in the numerical density of pericapillary oligodendrocytes only in the latter one (-29,4%). The numerical density of pericapillary oligodendrocytes in cases with predominantly positive symptoms also decreased (-8,3%) but not differed significantly from the controls. Electron microscopy revealed the prominent ultrastructural dystrophic and degenerative alterations of pericapillary oligodendrocytes in the prefrontal cortex in schizophrenic brains. The present study points out that the population of pericapillary oligodendrocytes is involved in the pathogenesis of schizophrenia. Damage and loss of pericapillary oligodendrocytes might lead to disruption of the blood-brain barrier and as a result to dysregulation of metabolic trafficking across the blood-brain barrier in the prefrontal cortex in schizophrenia.

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.

Adrenergic and cholinergic innervation of pulmonary tissue in the pig.

Both the adrenergic and the cholinergic components of the autonomic nervous system (ANS) have been found to be an important source of nerve fibres supplying the lungs. On the other hand, data regarding the innervation of the pulmonary tissue in breeding animals are surprisingly scarce. Therefore, in the present study noradrenergic and acetylcholinesterase-positive (AChE-positive) innervation of the lungs of sexually immature pigs was studied using histochemical methods. Studies were performed on six juvenile female pigs (aged 9 weeks, body weight 15-20 kg). Samples of the tissue were collected from the caudal lobe of the right lung. 70 microm cryostat sections were processed for the sucrose-potassium phosphate-glyoxylic acid technique to determine the occurrence and distribution of noradrenergic nerve fibres, while AChE-positive nerves were detected according to the acetylcholinesterase histochemistry. The present results revealed a dense network of noradrenergic nerve fibres localised mainly in the muscular membrane surrounding the epithelium of the bronchuli while AChE-positive nerve terminals supplied functional capillary vessels localised in the inter-alveolar septum and mucous membrane of the bronchi and bronchuli. The results of the present study confirm those of physiological experiments reporting the influence of the adrenergic and cholinergic components of the autonomic nervous system on the lung functions of pigs.

Cholinergic regulation of pericyte-containing retinal microvessels.

The aim of this study was to test the hypothesis that the neurotransmitter acetylcholine regulates the function of pericyte-containing retinal microvessels. A vasoactive role for acetylcholine is suggested by the presence of muscarinic receptors on pericytes, which are abluminally positioned contractile cells that may regulate capillary perfusion. However, little is known about the response of retinal microvessels to this neurotransmitter. Here we assessed the effects of cholinergic agonists on microvessels freshly isolated from the adult rat retina. Ionic currents were monitored via perforated patch pipettes; intracellular Ca(2+) levels were quantified with the use of fura 2, and microvascular contractions were visualized with the aid of time-lapse photography. We found that activation of muscarinic receptors elevated pericyte calcium levels, increased depolarizing Ca(2+)-activated chloride currents and caused pericytes to contract in a Ca(2+)-dependent manner. Most contracting pericytes were near capillary bifurcations. Contraction of a pericyte caused the adjacent capillary lumen to constrict. Thus acetylcholine may serve as a vasoactive signal by regulating pericyte contractility and thereby capillary perfusion in the retina.

Cutaneous blood flow and thermoregulation in Prader-Willi syndrome patients.

We examined adrenergic controlled cutaneous blood flow and temperature regulation in Prader-Willi syndrome. A body mass index was calculated for each participant. Thermal and laser Doppler finger probes were applied for continuous simultaneous surface temperature and capillary blood flow recording. Analysis with respect to group, age, body mass index, and genetic cause were performed. There were 32 patients (mean = 17.5 years of age) and five control subjects (mean = 15.6 years of age). There were no significant differences in mean ages or sex between groups. There was no significant difference in averaged blood flow measures with respect to group (P = 0.81), age (P = 0.16), body mass index (P = 0.54), or genetic identification (P = 0.81). There was no significant difference in average temperature measures as a function of group (P = 0.95), body mass index (P = 0.82), or genetic identification (P = 0.95). There was a significant difference in average temperature (P = 0.008) and trend in temperature change over time (P = 0.07) with respect to age for both patients and control subjects. Younger participants had higher average temperatures (30.6 degrees C vs 28.4 degrees C) in both study groups. We conclude that the central regulation and adrenergic control of cutaneous temperature and blood flow regulation in Prader-Willi syndrome at rest is not different from control subjects. These observations strengthen prior observations that a primary disturbance in parasympathetic autonomic regulation exists in Prader-Willi syndrome.

Functional immunohistochemistry of neuropeptides and nitric oxide synthase in the nerve fibers of the supratentorial dura mater in an experimental migraine model.

The supratentorial cerebral dura of the albino rat is equipped with a rich sensory innervation both in the connective tissue and around blood vessels, which includes nociceptive axons and their terminals; these display intense calcitonin gene-related peptide (CGRP) immunoreactivity. Stereotactic electrical stimulation of the trigeminal (Gasserian) ganglion, regarded as an experimental migraine model, caused marked increase and disintegration of club-like perivascular CGRP-immunopositive nerve endings in the dura mater and induced an apparent increase in the lengths of CGRP-immunoreactive axons. Intravenous administration of sumatriptan or eletriptan, prior to electrical stimulation, prevented disintegration of perivascular terminals and induced accumulation of CGRP in terminal and preterminal portions of peripheral sensory axons. Consequently, immunopositive terminals and varicosities increased in size; accumulation of axoplasmic organelles resulted in the "hollow" appearence of numerous varicosities. Since triptans exert their anti-migraine effect by virtue of agonist action on 5-HT(1D/B) receptors, we suggest that these drugs prevent the release of CGRP from perivascular nerve terminals in the dura mater by an action at 5-HT(1D/B) receptors. Nitroglycerine (NitroPOHL), given subcutaneously to rats, induces increased beading of nitric oxide synthase (NOS)-immunoreactive nerve fibers in the supratentorial cerebral dura mater, and an apparent increase in the number of NOS-immunoreactive nerve fibers in the dural areas supplied by the anterior and middle meningeal arteries, and the sinus sagittalis superior. Structural alterations of nitroxidergic axons innervating blood vessels of the dura mater support the idea that nitric oxide (NO) is involved in the induction of headache, a well-known side effect of coronary dilator agents.

[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.

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.

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.