Anatomy of blood microcirculation in the pig epicardial ganglionated nerve plexus.

Embolization of coronary arteries and their terminal arterioles causes ischemia of all tissues distributed within a cardiac wall including the intrinsic cardiac ganglionated nerve plexus (ICGP). The disturbed blood supply to the ICGP causes chronic sympathetic activation with succeeding atrial and ventricular arrhythmias. This study analyses the anatomy of microcirculation of epicardial nerves and ganglia using the hearts of 11 domestic pigs. Our findings demonstrate that thicker epicardial nerves are normally supplied with blood via 12 epineural arterioles penetrating the endoneurium regularly along a nerve, and forming an endoneurial capillary network, which drains the blood into the myocardial blood flow. The mean diameter of intraneural capillaries was 7.2 ± 0.2 µm, while the diameters of arterioles were 25.8 ± 0.7 µm and involved 45 endothelial cells accompanied by circular smooth muscle cells. Usually, two or three arterioles with a mean diameter of 28.9 ± 1.7 µm supplied blood to any epicardial ganglion, in which arterioles proceeded into a network of capillaries with a mean diameter of 6.9 ± 0.3 µm. Both the epicardial nerves and the ganglia distributed near the porta venarum of the heart had tiny arterioles that anastomosed blood vessels from the right and the left coronary arteries. The density of blood vessels in the epicardial nerves was significantly lesser compared with the ganglia. Our electron microscopic observations provided evidence that blood vessels of the pig epicardial nerves and ganglia may be considered as either arterioles or capillaries that have quantitative and qualitative differences comparing to the corresponding blood vessels in humans and, therefore, a pig should not be considered as an animal model of the first choice for further heart functional studies seeking to improve the treatment of cardiac arrhythmias via trans-coronary cardiac neuroablation. STRUCTURED ABSTRACT: This study details the anatomy of microcirculation of epicardial nerves and ganglia, from which intracardiac nerves and bundles of nerve fibers extend into all layers of the atrial and ventricular walls in the most popular animal model of experimental cardiology and cardiac surgery - the domestic pig. Our findings provided evidence that blood vessels of the pig epicardial nerves and ganglia may be considered as either arterioles or capillaries that have quantitative and qualitative differences comparing to the corresponding blood vessels in humans and, therefore, a pig should not be considered as an animal model of the first choice for further heart functional studies seeking to improve the treatment of cardiac arrhythmias via trans-coronary cardiac neuroablation.

Vasculo-Neuronal Coupling: Retrograde Vascular Communication to Brain Neurons.

Continuous cerebral blood flow is essential for neuronal survival, but whether vascular tone influences resting neuronal function is not known. Using a multidisciplinary approach in both rat and mice brain slices, we determined whether flow/pressure-evoked increases or decreases in parenchymal arteriole vascular tone, which result in arteriole constriction and dilation, respectively, altered resting cortical pyramidal neuron activity. We present evidence for intercellular communication in the brain involving a flow of information from vessel to astrocyte to neuron, a direction opposite to that of classic neurovascular coupling and referred to here as vasculo-neuronal coupling (VNC). Flow/pressure increases within parenchymal arterioles increased vascular tone and simultaneously decreased resting pyramidal neuron firing activity. On the other hand, flow/pressure decreases evoke parenchymal arteriole dilation and increased resting pyramidal neuron firing activity. In GLAST-CreERT2; R26-lsl-GCaMP3 mice, we demonstrate that increased parenchymal arteriole tone significantly increased intracellular calcium in perivascular astrocyte processes, the onset of astrocyte calcium changes preceded the inhibition of cortical pyramidal neuronal firing activity. During increases in parenchymal arteriole tone, the pyramidal neuron response was unaffected by blockers of nitric oxide, GABAA, glutamate, or ecto-ATPase. However, VNC was abrogated by TRPV4 channel, GABAB, as well as an adenosine A1 receptor blocker. Differently to pyramidal neuron responses, increases in flow/pressure within parenchymal arterioles increased the firing activity of a subtype of interneuron. Together, these data suggest that VNC is a complex constitutive active process that enables neurons to efficiently adjust their resting activity according to brain perfusion levels, thus safeguarding cellular homeostasis by preventing mismatches between energy supply and demand. SIGNIFICANCE STATEMENT: We present evidence for vessel-to-neuron communication in the brain slice defined here as vasculo-neuronal coupling. We showed that, in response to increases in parenchymal arteriole tone, astrocyte intracellular Ca2+ increased and cortical neuronal activity decreased. On the other hand, decreasing parenchymal arteriole tone increased resting cortical pyramidal neuron activity. Vasculo-neuronal coupling was partly mediated by TRPV4 channels as genetic ablation, or pharmacological blockade impaired increased flow/pressure-evoked neuronal inhibition. Increased flow/pressure-evoked neuronal inhibition was blocked in the presence of adenosine A1 receptor and GABAB receptor blockade. Results provide evidence for the concept of vasculo-neuronal coupling and highlight the importance of understanding the interplay between basal CBF and resting neuronal activity.

Excessive peptidergic sensory innervation of cutaneous arteriole-venule shunts (AVS) in the palmar glabrous skin of fibromyalgia patients: implications for widespread deep tissue pain and fatigue.

OBJECTIVE: To determine if peripheral neuropathology exists among the innervation of cutaneous arterioles and arteriole-venule shunts (AVS) in fibromyalgia (FM) patients. SETTING: Cutaneous arterioles and AVS receive a convergence of vasoconstrictive sympathetic innervation, and vasodilatory small-fiber sensory innervation. Given our previous findings of peripheral pathologies in chronic pain conditions, we hypothesized that this vascular location may be a potential site of pathology and/or serotonergic and norepinephrine reuptake inhibitors (SNRI) drug action. SUBJECTS: Twenty-four female FM patients and nine female healthy control subjects were enrolled for study, with 14 additional female control subjects included from previous studies. AVS were identified in hypothenar skin biopsies from 18/24 FM patient and 14/23 control subjects. METHODS: Multimolecular immunocytochemistry to assess different types of cutaneous innervation in 3 mm skin biopsies from glabrous hypothenar and trapezius regions. RESULTS: AVS had significantly increased innervation among FM patients. The excessive innervation consisted of a greater proportion of vasodilatory sensory fibers, compared with vasoconstrictive sympathetic fibers. In contrast, sensory and sympathetic innervation to arterioles remained normal. Importantly, the sensory fibers express α2C receptors, indicating that the sympathetic innervation exerts an inhibitory modulation of sensory activity. CONCLUSIONS: The excessive sensory innervation to the glabrous skin AVS is a likely source of severe pain and tenderness in the hands of FM patients. Importantly, glabrous AVS regulate blood flow to the skin in humans for thermoregulation and to other tissues such as skeletal muscle during periods of increased metabolic demand. Therefore, blood flow dysregulation as a result of excessive innervation to AVS would likely contribute to the widespread deep pain and fatigue of FM. SNRI compounds may provide partial therapeutic benefit by enhancing the impact of sympathetically mediated inhibitory modulation of the excess sensory innervation.

Influence of estradiol supplementation on neuropeptide Y neurotransmission in skeletal muscle arterioles of F344 rats.

The effects of estradiol on neuropeptide Y (NPY) neurotransmission in skeletal muscle resistance vessels have not been described. The purpose of this study was to determine the effects of long-term estradiol supplementation on NPY overflow, degradation, and vasoconstriction in gastrocnemius first-order arterioles of adult female rats. Female rats (4 mo; n = 34) were ovariectomized (OVX) with a subset (n = 17) receiving an estradiol pellet (OVE; 17ß-estradiol, 4 µg/day). After conclusion of the treatment phase (8 wk), arterioles were excised, placed in a physiological saline solution (PSS) bath, and cannulated with micropipettes connected to albumin reservoirs. NPY-mediated vasoconstriction via a Y(1)-agonist [Leu31Pro34]NPY decreased vessel diameter 44.54 ± 3.95% compared with baseline; however, there were no group differences in EC(50) (OVE: -8.75 ± 0.18; OVX: -8.63 ± 0.10 log M [Leu31Pro34]NPY) or slope (OVE: -1.11 ± 0.25; OVX: -1.65 ± 0.34% baseline/log M [Leu31Pro34]NPY). NPY did not potentiate norepinephrine-mediated vasoconstriction. NPY overflow experienced a slight increase following field stimulation and significantly increased (P < 0.05) over control conditions in the presence of a DPPIV inhibitor (diprotin A). Estradiol status did not affect DPPIV activity. These data suggest that NPY can induce a moderate decrease in vessel diameter in skeletal muscle first-order arterioles, and DPPIV is active in mitigating NPY overflow in young adult female rats. Long-term estradiol supplementation did not influence NPY vasoconstriction, overflow, or its enzymatic breakdown in skeletal muscle first-order arterioles.

Interactions between adenosine and K+ channel-related pathways in the coupling of somatosensory activation and pial arteriolar dilation.

Multiple, perhaps interactive, mechanisms participate in the linkage between increased neural activity and cerebral vasodilation. In the present study, we assessed whether neural activation-related pial arteriolar dilation (PAD) involved interactions among adenosine (Ado) A(2) receptors (A(2)Rs), large-conductance Ca(2+)-operated K(+) (BK(Ca)) channels, and inward rectifier K(+) (K(ir)) channels. In rats with closed cranial windows, we monitored sciatic nerve stimulation (SNS)-induced PAD in the absence or presence of pharmacological blockade of A(2)Rs (ZM-241385), ecto-5'-nucleotidase (α,ß-methylene-adenosine diphosphate), BK(Ca) channels (paxilline), and K(ir) channels (BaCl(2)). Individually, these interventions led to 53-66% reductions in SNS-induced PADs. Combined applications of these blockers led to little or no further repression of SNS-induced PADs, suggesting interactions among A(2)Rs and K(+) channels. In the absence of SNS, BaCl(2) blockade of K(ir) channels produced 52-80% reductions in Ado and NS-1619 (BK(Ca) channel activator)-induced PADs. In contrast, paxilline blockade of BK(Ca) channels was without effect on dilations elicited by KCl (K(ir) channel activator) and Ado suffusions, indicating that Ado- and NS-1619-associated PADs involved K(ir) channels. In addition, targeted ablation of the superficial glia limitans was associated with a selective 60-80% loss of NS-1619 responses, suggesting that the BK(Ca) channel participation (and paxilline sensitivity) derived largely from channels within the glia limitans. Additionally, blockade of either PKA or adenylyl cyclase caused markedly attenuated pial arteriolar responses to SNS and, in the absence of SNS, responses to Ado, KCl, and NS-1619. These findings suggested a key, possibly permissive, role for A(2)R-linked cAMP generation and PKA-induced K(+) channel phosphorylation in somatosensory activation-evoked PAD.

ATP overflow in skeletal muscle 1A arterioles.

The purpose of this study was to investigate the sources of ATP in the 1A arteriole, and to investigate age-related changes in ATP overflow. Arterioles (1A) from the red portion of the gastrocnemius muscle were isolated, cannulated and pressurized in a microvessel chamber with field stimulation electrodes. ATP overflow was determined using probes specific for ATP and null probes that were constructed similar to the ATP probes, but did not contain the enzyme coating. ATP concentrations were determined using a normal curve (0.78 to 25 micromol l(-1) ATP). ATP overflow occurred in two phases. Phase one began in the first 20 s following stimulation and phase two started 35 s after field stimulation. Tetrodotoxin, a potent neurotoxin that blocks action potential generation in nerves, abolished both phases of ATP overflow. alpha1-Receptor blockade resulted in a small decrease in ATP overflow in phase two, but endothelial removal resulted in an increase in ATP overflow. ATP overflow was lowest in 6-month-old rats and highest in 12- and 2-month-old rats (P<0.05). ATP overflow measured via biosensors was of neural origin with a small contribution from the vascular smooth muscle. The endothelium seems to play an important role in attenuating ATP overflow in 1A arterioles.

Presynaptic inhibition of neural vasodilator pathways to submucosal arterioles by release of purines from sympathetic nerves.

Capsaicin-sensitive extrinsic sensory nerves and submucosal vasodilator neurons provide important vasodilator input to submucosal arterioles, but relatively little is known about the signaling between these populations and the sympathetic vasoconstrictor innervation. This study examined whether release of sympathetic purines can modulate dilator nerves. In vitro submucosal preparations from guinea pig ileum were modified to leave the parent mesenteric artery intact so that perivascular sympathetic and extrinsic afferent nerves could be activated by a bipolar stimulating electrode placed on the parent artery, and submucosal vasodilator neurons were activated using focal electrodes placed on submucosal ganglia. The outside diameter of submucosal arterioles was monitored using videomicroscopy, and dilator responses were examined after preconstricting vessels 80-95% with prostaglandin F(2alpha) (400 nM). Mesenteric nerve stimulation evoked a frequency-dependent dilation, with suramin (100 microM) present throughout to inhibit P(2X) receptor-mediated vasoconstrictions. In the presence of guanethidine (10 microM) to inhibit sympathetic purine release, superfusion of ATP (200 nM-6 microM) caused a concentration-dependent inhibition of nerve-evoked dilations. Vasodilations to substance P (10 nM) were not inhibited by ATP in the presence of guanethidine, implicating a presynaptic effect of ATP on neurotransmitter release. The inhibitory effect of ATP was blocked by the adenosine receptor antagonist 8-phenyltheophylline (8-PT; 10 microM). In addition, 8-PT increased the amplitude of nerve-evoked dilations, suggesting a tonic inhibitory effect of adenosine receptors on vasodilator release. Dilations evoked by electrical stimulation of submucosal ganglia were also inhibited almost 50% by ATP (2 microM) and its nonhydrolyzable analog, alpha,beta-methylene-ATP (10 microM). These data suggest that sympathetic varicosities release ATP or a related purine that can act at presynaptic adenosine receptors on extrinsic sensory and submucosal vasodilator neurons to inhibit neurotransmitter release.

Identification of neuron types in the submucosal ganglia of the mouse ileum.

The continuing and even expanding use of genetically modified mice to investigate the normal physiology and development of the enteric nervous system and for the study of pathophysiology in mouse models emphasises the need to identify all the neuron types and their functional roles in mice. An investigation that chemically and morphologically defined all the major neuron types with cell bodies in myenteric ganglia of the mouse small intestine was recently completed. The present study was aimed at the submucosal ganglia, with the purpose of similarly identifying the major neuron types with cell bodies in these ganglia. We found that the submucosal neurons could be divided into three major groups: neurons with vasoactive intestinal peptide (VIP) immunoreactivity (51% of neurons), neurons with choline acetyltransferase (ChAT) immunoreactivity (41% of neurons) and neurons that expressed neither of these markers. Most VIP neurons contained neuropeptide Y (NPY) and about 40% were immunoreactive for tyrosine hydroxylase (TH); 22% of all submucosal neurons were TH/VIP. VIP-immunoreactive nerve terminals in the mucosa were weakly immunoreactive for TH but separate populations of TH- and VIP-immunoreactive axons innervated the arterioles in the submucosa. Of the ChAT neurons, about half were immunoreactive for both somatostatin and calcitonin gene-related peptide (CGRP). Calretinin immunoreactivity occurred in over 90% of neurons, including the VIP neurons. The submucosal ganglia and submucosal arterioles were innervated by sympathetic noradrenergic neurons that were immunoreactive for TH and NPY; no VIP and few calretinin fibres innervated submucosal neurons. We conclude that the submucosal ganglia contain cell bodies of VIP/NPY/TH/calretinin non-cholinergic secretomotor neurons, VIP/NPY/calretinin vasodilator neurons, ChAT/CGRP/somatostatin/calretinin cholinergic secretomotor neurons and small populations of cholinergic and non-cholinergic neurons whose targets have yet to be identified. No evidence for the presence of type-II putative intrinsic primary afferent neurons was found.

Sympathetic vasoconstrictor regulation of mouse colonic submucosal arterioles is altered in experimental colitis.

Recent studies suggest that altered neural regulation of the gastrointestinal microvasculature contributes to the pathogenesis of inflammatory bowel disease. Therefore, we employed video microscopy techniques to monitor nerve-evoked vasoconstrictor responses in mouse colonic submucosal arterioles in vitro and examined the effect of 2,4,6-trinitrobenzene sulphonic acid (TNBS) colitis. Nerve stimulation (2-20 Hz) caused frequency-dependent vasoconstrictor responses that were abolished by tetrodotoxin (300 nm) and guanethidine (10 microm). The P2 receptor antagonist suramin (100 microm) or the alpha(1)-adrenoceptor antagonist prazosin (100 nm) reduced the vasoconstriction and the combination of suramin and prazosin completely abolished responses. Nerve-evoked constrictions of submucosal arterioles from mice with TNBS colitis were inhibited by prazosin but not suramin. Superfusion of ATP (10 microm) resulted in large vasoconstrictions in control mice but had no effect in mice with colitis whereas constrictions to phenylephrine (3 microm) were unaffected. P2X(1) receptor immunohistochemistry did not suggest any alteration in receptor expression following colitis. However, Western blotting revealed that submucosal P2X(1) receptor expression was increased during colitis. In contrast to ATP, alphabeta-methylene-ATP (1 microm), which is resistant to catabolism by nucleotidases, constricted control and TNBS arterioles. This indicates that reduced purinergic transmission to submucosal arterioles may be due to increased degradation of ATP during colitis. These data comprise the first description of the neural regulation of mouse submucosal arterioles and identify a defect in sympathetic regulation of the GI vasculature during colitis due to reduced purinergic neurotransmission.

Postischemic augmentation of conducted dilation in cerebral arterioles.

BACKGROUND AND PURPOSE: Conducted vasomotor responses likely play an important role in cerebrovascular regulation, but it is unclear how these responses may be affected by ischemia. The purpose of this study was to evaluate the hypothesis that cerebral ischemia and reperfusion (I/R) alters vascular conduction in cerebral arterioles. METHODS: Middle cerebral artery occlusion (MCAO) was induced by an intraluminal filament technique in 4 groups of rats: (A) 2-hour MCAO/24-hour reperfusion (n=14); (B) 2-hour MCAO/1-hour reperfusion (n=7); (C) 1-hour MCAO/24-hour reperfusion (n=6); and (D) 1-hour MCAO/1-hour reperfusion (n=5). Neurological status and infarction (2,3,5-triphenyltetrazolium chloride staining) were evaluated after I/R. Conducted vasomotor responses were assessed in intracerebral branches of the MCA, by following the longitudinal spread of vasodilation or vasoconstriction to localized microapplication of ATP or adenosine. RESULTS: Local microapplication of ATP evoked a biphasic constriction (17+/-3%) and dilation (7+/-2%) response, whereas adenosine elicited only dilation (11+/-2%). These local responses spread longitudinally along sham-control arterioles (1 mm conduction distance) with rapid spatial decay. Ischemia followed by 24-hour reperfusion (groups A and C) led to a marked potentiation of conducted dilation responses: dilation to ATP conducted with virtually no decay in I/R arterioles. Augmentation of conductivity was not observed in the 1-hour reperfusion groups (B and D). Moreover, I/R did not alter conducted constriction. CONCLUSIONS: Ischemia-reperfusion led to a specific augmentation of conducted vasodilation in cerebral arterioles. Presumably, enhanced conductivity may improve cerebral perfusion after ischemia.

Changes in vasoconstrictor and vasodilator neurotransmitters in nerves supplying arterioles in developing colorectal polyps.

OBJECTIVE: To examine the changes that occur in the immunohistochemistry of vasoconstrictor and vasodilator transmitters in nerves supplying early and advanced colorectal polyps. SUBJECTS AND METHODS: We studied the perivascular innervation of submucosal arterioles of colorectal polyps (n = 18) and the innervation of the epithelial layer of polyps compared to normal controls (n=8), using immunohistochemical markers for the neurotransmitters; noradrenaline (NA) (marker used; tyrosine hydroxylase (TH)), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), and calcitonin gene-related polypeptide (CGRP). (Advanced polyps; villous adenomas>1.5 cm, polyps with severe dysplasia or partial carcinoma). RESULTS: In submucosal arterioles there was a progressive decrease from controls through early polyps to advanced polyps in TH and NPY perivascular immunoreactivity (P<0.015 for both). VIP and SP immunoreactivity was higher in early polyps compared to controls, but markedly reduced in advanced polyps (P<0.05 for VIP). Sparse CGRP immunoreactivity was present in polyps only. Neural VIP and SP immunoreactivity in the lamina propria of polyp mucosa was more intense than in controls. CONCLUSION: There is a decrease in vasoconstrictor neurotransmitters NPY and NA (shown by TH) around submucosal arterioles of both early and advanced polyps, but an increase in the vasodilator neurotransmitters, particularly VIP, in early colorectal polyps. These results suggest a predominantly vasodilatory neural influence in early polyps, perhaps indicating a mechanism that maintains polyp growth.

The role of the glia limitans in ADP-induced pial arteriolar relaxation in intact and ovariectomized female rats.

We examined whether the glia limitans (GL) influences pial arteriolar relaxation elicited in vivo by the purinergic (P(2)Y(1) receptor) agonist ADP in female rats, and whether that influence is altered in ovariectomized (Ovx) females. A validated model for GL injury was used, topical application of the gliotoxin L-alpha-aminoadipic acid (L-alphaAAA), 24 h before the study. In both intact and Ovx females, L-alphaAAA had no effect on responses to the NO donor, S-nitroso-N-acetyl penicillamine, but ADP-induced pial arteriolar dilations were significantly reduced (by 33-90%), compared with vehicle-treated controls. When N(G)-nitro-L-arginine (L-NNA) was administered to L-alphaAAA-treated rats, the ADP response was virtually lost in intact females, but no further reductions were observed in the Ovx rats. On the other hand, in L-alphaAAA-treated Ovx females, when the gap junction blocker, Gap 27, was subsequently added to the suffusate, ADP reactivity fell to very low levels. In vehicle-treated control rats, L-NNA and Gap 27 reduced ADP reactivity by approximately 50% in intact and Ovx females, respectively. An earlier study indicated that the endothelium was a key site of influence for L-NNA (intact) and Gap 27 (Ovx). Thus present and previous results imply that the ADP response in pial arterioles represents the additive actions of an endothelial and a GL component. That supposition was confirmed in the present study by the finding that combining endothelial and GL injury produced an essentially complete loss of ADP reactivity in both intact and Ovx females. Finally, topical application of the selective P(2)Y(1) antagonist, MRS-2179, was associated with a nearly complete suppression of the ADP response in both intact and Ovx females. These results suggest that 1) ADP-induced pial arteriolar dilation involves additive contributions from P(2)Y(1) receptors present in both vascular endothelium and the GL; 2) the influence of the GL component is not altered by ovariectomy; and 3) the gap junction-dependent component of the ADP response in Ovx females is unlikely to include the GL and probably resides in the vessels themselves.

Estrogen selectively increases sensory nociceptor innervation of arterioles in the female rat.

Differences exist in vascular function and disease susceptibility in males and females, and estrogen is apparently a primary factor. One mechanism by which estrogen may influence vascular function is by affecting vasomotor innervation. We have shown previously that estrogen increases calcitonin gene-related peptide (CGRP)-immunoreactive sensory innervation of the rat mammary gland, but it is not known if this occurs in other tissues. The objective of this study was to determine if estrogen modulates CGRP-immunoreactive innervation of vascular and non-vascular tissues. Ovariectomized adult virgin female rats were implanted with pellets containing 17beta-estradiol or placebo. After 7 days, innervation was examined in the external ear, jejunal mesenteric arterioles, superficial epigastric, femoral, and uterine arteries, and foot skin. Immunofluorescence microscopy of the external ear pinna revealed increased CGRP-immunoreactive sensory innervation in estrogen-treated rats, and this was attributable specifically to increased innervation of arterioles. Tyrosine hydroxylase-immunoreactive innervation was unchanged. Total nerve density, revealed by the pan-neuronal marker PGP 9.5, was also greater after estrogen treatment, implying structural proliferation of nociceptor vasodilator fibers. Mesenteric arteriolar CGRP-immunoreactive nerve density was also selectively increased by estrogen treatment. However, estrogen did not affect CGRP-immunoreactive nerve density of superficial epigastric, femoral, or uterine arteries, or foot skin. Therefore, estrogen increases sensory innervation of arterioles, but not of large arteries or skin. We conclude that sensory nociceptor vasodilatory innervation of arterioles is selectively enriched by estrogen, which may influence cardiovascular function in health and disease.

Distribution and organization of the nerve fiber and ganglion cells of the human choroid.

Antibodies to the 68, 160 and 200 kD of the neurofilament triplets were used to study the distribution and organization of neuronal structures in the human choroid. Choroidal axons were observed in the suprachoroid and vascular laminae but absent from the choriocapillary layer. Most axons were situated in the suprachoroid. In this layer, there were band-like bundles. The two thickest band-like bundles could constitute the long ciliary nerve, while the rest could constitute short ciliary nerves. These bundles ran through the suprachoroid, branching out on the suprachoroid and the vascular laminae until they reached the ciliary body. In the submacular area of the suprachoroid, the branches of the band-like bundles were so intermingled that they looked like a meshwork. In the vascular layer, the large vessels and their primary branches were accompanied by paravascular axons. Some paravascular axons penetrated the medium-caliber vessel layer and in the submacular area interwove to form a network parallel to the arteriole walls. In addition, perivascular axons were revealed by antibodies to neuropeptides. Choroidal ganglion cells were more numerous in the central choroid, specifically in a circumferential area corresponding to the entrance of the short posterior ciliary arteries and their primary branches, and in the vicinity of the submacular area. These cells presented bipolar and multipolar morphology. The high concentration of innervation in the central human choroid could be necessary to maintain strict blood flow in this zone; thus if required, these neuron structures could induce early vasodilation reflexes at the entrance of the choroidal blood vessels to increase the blood flow.

Cholinergic innervation of pial arteries in senescent rats: an immunohistochemical study.

Perivascular acetylcholine (ACh)-immunoreactive nerve fibres were demonstrated in basilar and middle cerebral arteries, in pial arteries and arterioles and in intracerebral arteries of male Fisher 344 rats of 6 months (young), 15 months (adult) and 22 months (senescent). Analysis included whole mounts of basilar and middle cerebral arteries, of pial arteries and sections of brain including pia-arachnoid membrane to demonstrate the localization of nerve fibres throughout the wall of pial and of intracerebral arteries. ACh-immunoreactive nerve fibres were demonstrated by indirect immunohistochemistry using a polyclonal anti-ACh antibody and their relative density was quantified. Perivascular ACh-immunoreactive nerve fibres were located in basilar and middle cerebral arteries, in pial arteries and arterioles and in intracerebral arteries. These fibres were found in the adventitia and adventitia-media border with a higher density in pial rather than in intracerebral arteries. A decrease of ACh-immunoreactive nerve fibres was observed both in pial and intracerebral arteries of adult or senescent rats compared to younger cohorts. The direct demonstration of ACh-immunoreactive nerve fibres in the cerebrovascular tree may contribute to evaluate the influence of experimental and pathological conditions on cerebrovascular cholinergic neuroeffector mechanisms, including a role of cholinergic innervation in the pathophysiology of cerebrovascular disease of the elderly.

Reduced PO(2) and adenosine formation preserve arteriolar nitric oxide synthesis during sympathetic constriction in the rat intestine.

Previous reports by this laboratory have indicated that a flow-dependent fall in arteriolar wall PO(2 )may be a stimulus for the sustained release of endothelial nitric oxide (NO) during sympathetic vasoconstriction in the superfused rat intestine. In this study, we tested the hypothesis that locally formed adenosine serves as the link between the fall in local PO(2) and NO synthesis under these conditions. Adenosine applied via pressurized micropipettes directly onto the wall or at a distance of 25 microm from the wall of first-order arterioles (resting diameter = 54 +/- 1 microm) elicited dose-dependent dilations of 15-46% that were significantly reduced by the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA, 10(-4) M). Arteriolar responses to sympathetic nerve stimulation were enhanced by 57-66% in the presence of L-NMMA or when tissue PO(2) was prevented from falling under a high O(2) superfusate. Adenosine deaminase (2.0 U/ml) or the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (4 x 10(-4) M) completely blocked the enhancing effect of L-NMMA on sympathetic constriction. These results are consistent with the hypothesis that the fall in arteriolar wall and/or tissue PO(2) that accompanies sympathetic arteriolar constriction in the rat intestine can lead to local adenosine production, which in turn preserves endothelial NO release.

Effects of near-infrared low-level laser irradiation on microcirculation.

BACKGROUND AND OBJECTIVE: Recently, there has been an increase in the clinical application of low-level laser irradiation (LLLI) in various fields. The present study was conducted to explore the effects of LLLI on microcirculation. STUDY DESIGN/MATERIAL AND METHODS: We investigated the effects of LLLI on rat mesenteric microcirculation in vivo, and on cytosolic calcium concentration ([Ca2+]i) in rat vascular smooth muscle cells (VSMCs) in vitro. RESULTS: LLLI caused potent dilation in the laser-irradiated arteriole, which led to marked increases in the arteriolar blood flow. The changes were partly attenuated in the initial phase by the superfusion of 15 microM L-NAME, but they were not affected by local denervation. Furthermore, LLLI caused a power-dependent decrease in [Ca2+]i in VSMCs. CONCLUSION: The circulatory changes observed seemed to be mediated largely by LLLI-induced reduction of [Ca2+]i in VSMCs, in addition to the involvement of NO in the initial phase.