Periarterial Sympathetic Nerves Research Articles

Eagle's syndrome, elongated styloid process and new evidence for pre-manipulative precautions for potential cervical arterial dysfunction

IntroductionSafety with upper cervical interventions is a frequently discussed and updated concern for physical therapists, chiropractors and osteopaths. IFOMPT developed the framework for safety assessment of the cervical spine, and this topic has been discussed in-depth with past masterclasses characterizing carotid artery dissection and cervical arterial dysfunction. Our masterclass will expand on this information with knowledge of specific anatomical anomalies found to produce Eagle's syndrome, and cause carotid artery dissection, stroke and even death.Eagle's syndrome is an underdiagnosed, multi-mechanism symptom assortment produced by provocation of the sensitive carotid space structures by styloid process anomalies. As the styloid traverses between the internal and external carotid arteries, provocation of the vessels and periarterial sympathetic nerve fibers can lead to various neural, vascular and autonomic symptoms. Eagle's syndrome commonly presents as neck, facial and jaw pain, headache and arm paresthesias; problems physical therapists frequently evaluate and treat. PurposeThis masterclass aims to outline the safety concerns, assessment and management of patients with Eagle's syndrome and styloid anomalies. By providing evidence of this common anomaly found in almost one-third of the population, hypothesis generation and clinical reasoning with patients presenting with head and neck symptoms can improve. ImplicationsIncluding styloid anomalies as potential hypotheses for patients with head and neck complaints can assist therapists in safe practice and expedite referral. The authors recommend updating the IFOMPT framework to incorporate Eagle's syndrome, a comprehensive autonomic assessment, and palpation of the stylohyoid complex to avoid potentially serious complications from conceivably hazardous interventions.

Simulated Microgravity Induces Regionally Distinct Neurovascular and Structural Remodeling of Skeletal Muscle and Cutaneous Arteries in the Rat.

Introduction: Mechanical forces and sympathetic influences are key determinants of vascular structure and function. This study tested the hypothesis that hindlimb unloading (HU) exerts diverse effects on forelimb and hindlimb small arteries of rats in functionally different regions of the skeletal muscle and skin. Methods: Male Wistar rats were subjected to HU for 2 weeks, then skeletal muscle arteries (deep brachial and sural) and skin arteries (median and saphenous) were examined in vitro using wire myography or isobaric perfusion and glyoxylic acid staining. Results: HU increased lumen diameter of both forelimb arteries but decreased diameter of the sural artery; the saphenous artery diameter was not affected. Following HU, maximal contractile responses to noradrenaline and serotonin increased in the forelimb but decreased in the hindlimb skeletal muscle feed arteries with no change in skin arteries; all region-specific alterations persisted after endothelium removal. HU increased the sensitivity to vasoconstrictors in the saphenous artery but not in the sural artery. In the saphenous artery, initially high sympathetic innervation density was reduced by HU, sparse innervation in the sural artery was not affected. Electrical stimulation of periarterial sympathetic nerves in isobarically perfused segments of the saphenous artery demonstrated a two-fold decrease of the contractile responses in HU rats compared to that of controls. Conclusion: HU induces contrasting structural and functional adaptations in forelimb and hindlimb skeletal muscle arteries. Additionally, HU had diverse effects in two hindlimb vascular regions. Hyper-sensitivity of the saphenous artery to vasoconstrictors appears to result from the shortage of trophic sympathetic influence. Importantly, HU impaired sympathetically induced arterial vasoconstriction, consistent with the decreased sympathetic constrictor response in humans following space flight.

Spinal cord injury alters purinergic neurotransmission to mesenteric arteries in rats.

Complications associated with spinal cord injury (SCI) result from unregulated reflexes below the lesion level. Understanding neurotransmission distal to the SCI could improve quality of life by mitigating complications. The long-term impact of SCI on neurovascular transmission is poorly understood, but reduced sympathetic activity below the site of SCI enhances arterial neurotransmission (1). We studied sympathetic neurovascular transmission using a rat model of long-term paraplegia (T2-3) and tetraplegia (C6-7). Sixteen weeks after SCI, T2-3 and C6-7 rats had lower blood pressure (BP) than sham rats (103 ± 2 and 97 ± 4 vs. 117 ± 6 mmHg, P < 0.05). T2-3 rats had tachycardia (410 ± 6 beats/min), and C6-7 rats had bradycardia (299 ± 10 beats/min) compared with intact rats (321 ± 4 beats/min, P < 0.05). Purinergic excitatory junction potentials (EJPs) were measured in mesenteric arteries (MA) using microlectrodes, and norepinephrine (NE) release was measured using amperometry. NE release was similar in all groups, while EJP frequency-response curves from T2-3 and C6-7 rats were left-shifted vs. sham rats. EJPs in T2-3 and C6-7 rats showed facilitation followed by run-down during stimulation trains (10 Hz, 50 stimuli). MA reactivity to exogenous NE and ATP was similar in all rats. In T2-3 and C6-7 rats, NE content was increased in left cardiac ventricles compared with intact rats, but was not changed in MA, kidney, or spleen. Our data indicate that peripheral purinergic, but not adrenergic, neurotransmission increases following SCI via enhanced ATP release from periarterial nerves. Sympathetic BP support is reduced after SCI, but improving neurotransmitter release might maintain cardiovascular stability in individuals living with SCI.NEW & NOTEWORTHY This study revealed increased purinergic, but not noradrenergic, neurotransmission to mesenteric arteries in rats with spinal cord injury (SCI). An increased releasable pool of ATP in periarterial sympathetic nerves may contribute to autonomic dysreflexia following SCI, suggesting that purinergic neurotransmission may be a therapeutic target for maintaining stable blood pressure in individuals living with SCI. The selective increase in ATP release suggests that ATP and norepinephrine may be stored in separate synaptic vesicles in periarterial sympathetic varicosities.

The Preferential Inhibitory Effect of Olmesartan, a New Angiotensin II Type 1 Antagonist, on Sympathetic Nerve Terminals in Isolated Canine Splenic Artery

Effects of olmesartan (RNH-6270: (5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxy-4-(1-hydroxy-1-methylethyl)-2-propyl{4-[2-(tetrazol-5-yl)-phenyl]phenyl}methylimidazol-5-carboxylase, an active form of olmesartan medoxomil (CS-866)) was investigated in isolated, perfused canine splenic arterial preparations. Neither exogenous noradrenaline- nor ATP-induced vasoconstrictor responses were modified by treatment with the used concentrations of olmesartan (1 – 100 nM). A high concentration of 10 nM angiotensin II caused a potentiation of either noradrenaline- and ATP-induced constrictions, although 1 nM angiotensin II did not induce any potentiating effects for these responses. These potentiations were inhibited by olmesartan in a concentration-related manner. Periarterial nerve electrical stimulation (PNS) readily induced a biphasic constriction consisting of an initial P2X purinoceptor-mediated vasoconstriction followed by a prolonged mainly α1-adrenoceptor-mediated response. PNS-induced 1st and 2nd peaked responses were significantly inhibited by olmesartan in a concentration-related manner. With a low concentration of 1 nM angiotensin II, which did not induce any vascular effects by itself, PNS-induced responses were markedly enhanced. The enhanced responses were inhibited by olmesartan. It is concluded that endogenous angiotensin II exerts its stimulating action on the releases of ATP and noradrenaline from the periarterial sympathetic nerve terminal, and olmesartan has an inhibitory property on angiotensin II-induced potentiation of endogenous ATP- and noradrenaline-induced responses.

Sympathetic Neurotransmission in Resistance Mesenteric Arteries in Obesity‐Related Hypertension

Hyperactivity of sympathetic nerves has an important role in the pathogenesis of obesity‐related hypertension. The mechanisms linking obesity to hypertension are unclear. Small mesenteric arteries (MA, &lt;300 mm dia.) are resistance blood vessels that are densely innervated by sympathetic nerves. Nerve varicosities release norepinephrine (NE) and ATP which cause arterial constriction. Impaired control of NE and/or ATP release and clearance would cause hypertension. Alterations in sympathetic nerves supplying MAs contribute to hypertension in several animal models. This study examined whether alterations of sympathetic neurotransmission in the resistance MAs contribute to high blood pressure in high fat‐fed (HF) Dahl salt‐sensitive rats. Sympathetic nerves supplying MAs maintained in vitro were stimulated focally. Electrically‐evoked NE and ATP release were detected by using amperometry with carbon fiber electrodes, and intracellular recording of excitatory junction potentials (EJPs) from arterial smooth muscle cells, respectively. Data were obtained from male and female rats at 10, 17, and 24 weeks of control and HF diet. Peak amplitudes of NE oxidation current and EJPs were assessed. NE oxidation current from all groups were blocked by a Na+ channel blocker, tetrodotoxin (TTX, 300 nM), suggesting that NE release in neurogenic. EJPs from all groups were abolished by TTX, and PPADS (10 μM), a P2X receptor antagonist, indicating that EJPs were neurogenic and purinergic. Frequency (1–30 Hz, 50 pulses) response curves using amperometric measurement of NE release showed similar responses in MAs from control and HF fed male and female rats. EJP frequency (0.5–10Hz, 5 pulses) response curves were also similar in MA from control and HF fed male and female rats. We investigated prejunctional α2‐adrenergic receptor function using the α2 receptor agonist (UK14,304) and antagonist (yohimbine). UK14,304 (0.0001–10 μM) inhibited NE and ATP release equally well and in a concentration‐dependent manner in MAs from control and HF rats. In the presence of 1 μM yohimbine, NE and ATP release was increased equally in MA from all rats. We conclude that adrenergic and purinergic neurotransmission by periarterial sympathetic nerves are not affected in HF Dahl salt sensitive rats. Elevated blood pressure in this animal model is not dependent on altered sympathetic neurotransmission to the mesenteric vasculature.Support or Funding InformationNIH 2P01HL070687 (For JJG), and Post‐doctoral training scholarship, Faculty of Medicine, Thammasat University, Thailand (For SS).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Micro-anatomic distribution of peri-arterial renal sympathetic nerves

Objective To investigate the distribution of sympathetic nerves around human renal arteries. Methods We collected 25 renal arteries from the patients who had a retroperitoneal laparoscopic nephrectomy and bilateral renal arteries from 7 human autopsy subjects. Each artery was equally divided into segments, which were made into paraffin sections. We examined the distribution of nerves around renal arteries and manually counted nerves by using optical microscope. Results A total of 3 065 nerves were identified from 258 sections. About 98% of all nerves existed within the renal arterial adventitia and the outside tissue. The mean number of nerves between the ventral, doirsal, superior, and inferior region of surgical specimens was 4.7±1.3, 1.4±0.7, 3.1±1.3, and 2.1±1.2, respectively. The mean number of nerves in the ventral region was greater compared with the doirsal region (P=0.000), the mean number of nerves in the superior region was also greater compared with the inferior region (P=0.005). The mean number of nerves in the middle segments was obviously greater compared with the distal segments (14.5±4.4 and 8.3±3.0, P=0.000). The mean number of nerves between the ventral, doirsal, superior, and inferior region of autopsy specimens was 5.4±1.4, 1.5±0.7, 3.1±0.8, and 2.8±1.0, respectively. The mean number of nerves in the ventral region was greater compared with the doirsal region(P=0.000), whereas the mean number of nerves was similar between the superior and inferior region (P=0.440). The mean number of nerves among the proximal, middle, and distal segments was 11.2±4.4, 17.6±4.4, and 8.5±2.2. It was obvious that the mean number of nerves in the middle segments was greater compared with the proximal and diatal segments (P=0.000). The distribution of sympathetic nerves around the left renal arteries was not considerably different compared with the right renal arteries (Psurgical=0.100, Pautopsy=0.916). Conclusion The mean number of nerves in the ventral region is greatest and least in the doirsal region. Along the renal artery, the distribution of nerves is relatively greater in the middle segments. Nerve anatomic distribution around the left renal arteries was not considerably different compared with the right renal arteries. Key words: Renal sympathetic denervation; Resistant hypertension; Renal artery; Sympathetic nerves; Anatomic distribution

Macrophage depletion lowers blood pressure and restores sympathetic nerve α2-adrenergic receptor function in mesenteric arteries of DOCA-salt hypertensive rats.

We tested the hypothesis that vascular macrophage infiltration and O2 (-) release impairs sympathetic nerve α2-adrenergic autoreceptor (α2AR) function in mesenteric arteries (MAs) of DOCA-salt hypertensive rats. Male rats were uninephrectomized or sham operated (sham). DOCA pellets were implanted subcutaneously in uninephrectomized rats who were provided high-salt drinking water or high-salt water with apocynin. Sham rats received tap water. Blood pressure was measured using radiotelemetry. Treatment of sham and DOCA-salt rats with liposome-encapsulated clodronate was used to deplete macrophages. After 3-5, 10-13, and 18-21 days of DOCA-salt treatment, MAs and peritoneal fluid were harvested from euthanized rats. Norepinephrine (NE) release from periarterial sympathetic nerves was measured in vitro using amperometry with microelectrodes. Macrophage infiltration into MAs as well as TNF-α and p22(phox) were measured using immunohistochemistry. Peritoneal macrophage activation was measured by flow cytometry. O2 (-) was measured using dihydroethidium staining. Hypertension developed over 28 days, and apocynin reduced blood pressure on days 18-21. O2 (-) and macrophage infiltration were greater in DOCA-salt MAs compared with sham MAs after day 10. Peritoneal macrophage activation occurred after day 10 in DOCA-salt rats. Macrophages expressing TNF-α and p22(phox) were localized near sympathetic nerves. Impaired α2AR function and increased NE release from sympathetic nerves occurred in MAs from DOCA-salt rats after day 18. Macrophage depletion reduced blood pressure and vascular O2 (-) while restoring α2AR function in DOCA-salt rats. Macrophage infiltration into the vascular adventitia contributes to increased blood pressure in DOCA-salt rats by releasing O2 (-), which disrupts α2AR function, causing enhanced NE release from sympathetic nerves.

Anatomic Assessment of Sympathetic Peri-Arterial Renal Nerves in Man

BackgroundAlthough renal sympathetic denervation therapy has shown promising results in patients with resistant hypertension, the human anatomy of peri-arterial renal nerves is poorly understood. ObjectivesThe aim of our study was to investigate the anatomic distribution of peri-arterial sympathetic nerves around human renal arteries. MethodsBilateral renal arteries were collected from human autopsy subjects, and peri-arterial renal nerve anatomy was examined by using morphometric software. The ratio of afferent to efferent nerve fibers was investigated by dual immunofluorescence staining using antibodies targeted for anti–tyrosine hydroxylase and anti–calcitonin gene–related peptide. ResultsA total of 10,329 nerves were identified from 20 (12 hypertensive and 8 nonhypertensive) patients. The mean individual number of nerves in the proximal and middle segments was similar (39.6 ± 16.7 per section and 39.9 ± 1 3.9 per section), whereas the distal segment showed fewer nerves (33.6 ± 13.1 per section) (p = 0.01). Mean subject-specific nerve distance to arterial lumen was greatest in proximal segments (3.40 ± 0.78 mm), followed by middle segments (3.10 ± 0.69 mm), and least in distal segments (2.60 ± 0.77 mm) (p < 0.001). The mean number of nerves in the ventral region (11.0 ± 3.5 per section) was greater compared with the dorsal region (6.2 ± 3.0 per section) (p < 0.001). Efferent nerve fibers were predominant (tyrosine hydroxylase/calcitonin gene–related peptide ratio 25.1 ± 33.4; p < 0.0001). Nerve anatomy in hypertensive patients was not considerably different compared with nonhypertensive patients. ConclusionsThe density of peri-arterial renal sympathetic nerve fibers is lower in distal segments and dorsal locations. There is a clear predominance of efferent nerve fibers, with decreasing prevalence of afferent nerves from proximal to distal peri-arterial and renal parenchyma. Understanding these anatomic patterns is important for refinement of renal denervation procedures.

MR-guided high-focused ultrasound for renal sympathetic denervation-a feasibility study in pigs.

BackgroundRenal sympathetic denervation has recently gained clinical relevance for the treatment of therapy-resistant hypertension. Denervation is currently mainly performed using catheter-based transarterial radiofrequency ablation of periarterial sympathetic nerve fibers. Since this approach has numerous limitations, we conducted a study to evaluate the feasibility, safety, and efficacy of magnetic resonance-guided high-focused ultrasound (MRgHiFUS) for renal sympathetic denervation in pigs as an alternative to catheter-based ablation.MethodsRenal periarterial MRgHiFUS was performed under general anesthesia in ten pigs. Blood pressure measurements and magnetic resonance imaging (MRI) of the kidneys, renal arteries, and surrounding structures were obtained immediately before and after the interventions and after 4 weeks. Histological examinations of periarterial tissues and determination of renal norepinephrine (NE) concentration were performed to assess treatment efficacy.Results and discussionIn each pig, 9.8 ± 2.6 sonications with a mean energy deposition of 2,670 ± 486 J were performed. The procedure was well tolerated by all pigs. No major complications occurred. MRgHiFUS induced periarterial edema in three pigs, but only one pig showed corresponding histological changes. The NE level of the treated kidney was lower in five pigs (-8% to -38%) compared to the untreated side. Overall, there was no significant difference between the NE values of both kidneys in any of the treated pigs. Postinterventional MRI indicated absorption of ultrasound energy at the transverse process and fascia.ConclusionMRgHiFUS had some thermal periarterial effects but failed to induce renal denervation. Insufficient energy deposition is most likely attributable to a small acoustic window with beam path impediment in the porcine model. Since HiFUS treatment in humans is expected to be easier to perform due to better access to renal sympathetic nerves, further studies of this method are desirable to investigate the potential of MRgHiFUS as an alternative for patients not suitable for catheter-based renal sympathicolysis.

Reduction of vasoconstriction by removing periarterial nerve in human gastroepiploic artery imply prevention of vasospasm

BackgroundThe right gastroepiploic artery (GEA) is used as an in‐situ arterial graft for coronary artery bypass grafting (CABG). However, the GEA is more likely to cause vasospasms compared to the internal thoracic artery. We hypothesized that the significant cause of GEA spasms is triggered by periarterial sympathetic nerve, since the GEA is classified as a muscular artery.Materials and MethodsUnused parts of the GEA were obtained from patients who underwent CABG. The vessel was assigned to control (N+) and denervation (N−) group. For N− group, periarterial nerve was microscopically removed. These vessels were investigated by hematoxylin‐eosin (H‐E) and immunohistochemical staining. Then, each vessel was exposed to electrical field stimulation (EFS).ResultsHistological analysis revealed that the periarterial connective tissues including neuropeptide Y (NPY) were removed from lateral layer to external elastic membrane in the N−, whereas they were preserved in N+. The vasoconstriction by EFS with serial frequency was consistently lower in N− than N+ (p&lt;0.05 at 20 and 50Hz; n=8 each). These vasoconstrictions were inhibited by tetrodotoxin. Moreover, endothelium dependent relaxation and vasoconstriction of the smooth muscle were similar in both groups.ConclusionsThe removal of periarterial nerves from human GEA can prevent graft spasms without disturbance of endothelial and smooth muscle functions.

Denervation of gastroepiploic artery graft can reduce vasospasm

The right gastroepiploic artery is useful as an in situ arterial graft for coronary artery bypass grafting. However, the gastroepiploic artery is more likely to cause vasospasms compared with the internal thoracic artery. We hypothesized that the cause of the spasms is the stimulation of the periarterial sympathetic nerve, because the gastroepiploic artery is classified as a muscular artery. In this study, we examined whether the spasm is reduced by removing the periarterial sympathetic nerve. Unused parts of the gastroepiploic artery were obtained from patients who underwent coronary artery bypass grafting. The vessel was cut into 2 segments, and they were assigned to control (N+) and denervation (N-) groups. The periarterial nerve was microscopically removed from the vessels of the N- group. The vessels in both groups were investigated by hematoxylin-eosin or immunohistochemical staining, and they were stimulated by electrical field stimulation with serial frequency for isometric tension measurement. Histologic analyses revealed that periarterial connective tissues including neuropeptide Y were removed to expose the external elastic membrane in the N- vessel, whereas they were preserved in N+. The mean contraction by electrical field stimulation with serial frequency was consistently lower in N- than in N+ (P<.05 at 20 and 50 Hz; n=8 each). Endothelium-dependent relaxation and contractile function of the smooth muscle were similar in both groups. The removal of the periarterial sympathetic nerve from the human gastroepiploic artery reduced vascular contraction, elicited by peripheral nerve stimulation, without disturbing endothelial and smooth muscle contractile functions. This reduction may contribute to the prevention of vasospasms.

Antioxidant treatment restores prejunctional regulation of purinergic transmission in mesenteric arteries of deoxycorticosterone acetate-salt hypertensive rats

Norepinephrine (NE) and ATP are co-released by periarterial sympathetic nerves. In mesenteric arteries (MA) from deoxycorticosterone-acetate (DOCA)-salt hypertensive rats, ATP, but not norepinephrine, release is impaired suggesting that their release may be regulated differently. We tested the hypothesis that different calcium channels contribute to ATP and norepinephrine release from sympathetic nerves in vitro in MA from normotensive and DOCA-salt hypertensive rats and that oxidative stress disrupts prejunctional regulation of co-transmission. Excitatory junction potentials (EJPs) were used to measure ATP release. Norepinephrine release was measured amperometrically with carbon-fiber microelectrodes. CdCl 2 (30 μM) inhibited norepinephrine release in sham and DOCA-salt arteries by 78% and 85%, respectively. The N-type calcium channel antagonist, ω-conotoxin GVIA (CTX, 0.1 μM) inhibited norepinephrine release by 50% and 67% in normotensive and DOCA-salt arteries, respectively while CTX blocked EJPs. The P/Q-type calcium channel antagonist ω-agatoxin IVA (ATX; 0.03 μM) reduced norepinephrine release in sham but not DOCA-salt arteries and increased EJPs in sham but not DOCA-salt arteries. ATX did not increase EJPs in sham arteries in the presence of the α 2-adrenergic receptor antagonist, yohimbine (1 μM). α 2-Autoreceptor-sensitive EJP facilitation is impaired in DOCA-salt hypertension but this response is restored in DOCA-salt rats treated chronically with the antioxidant, apocynin. Apocynin restored α 2-autoreceptor regulation of norepinephrine release. We conclude that ATP released from periarterial sympathetic nerves is controlled directly by N-type calcium channels. Norepinephrine release is controlled by N and P/Q type calcium channels. Norepinephrine release controlled by P/Q channels acts at α 2-adrenergic receptors to inhibit norepinephrine release suggesting that there may be multiple pools of norepinephrine in periarterial sympathetic nerves. Regulation of norepinephrine release by α 2-autoreceptors and P/Q-type channels is impaired in DOCA-salt hypertension and α 2-autoreceptor function is disrupted by oxidative stress.

Sympathetic neurogenic Ca2+ signalling in rat arteries: ATP, noradrenaline and neuropeptide Y

The sympathetic nervous system (SNS) plays an essential role in the control of total peripheral vascular resistance by controlling the contraction of small arteries. The SNS also exerts long-term trophic influences in health and disease; SNS hyperactivity accompanies most forms of human essential hypertension, obesity and heart failure. At their junctions with smooth muscle cells, the peri-arterial sympathetic nerves release ATP, noradrenaline (NA) and neuropeptide Y (NPY) onto smooth muscle cells. Confocal Ca(2+) imaging studies reveal that ATP and NA each produce unique types of postjunctional Ca(2+) signals and consequent smooth muscle cell contractions. Neurally released ATP activates postjunctional P2X(1) receptors to produce local, non-propagating Ca(2+) transients, termed 'junctional Ca(2+) transients', or 'jCaTs'. Neurally released NA binds to alpha(1)-adrenoceptors and can activate Ca(2+) waves or more uniform global changes in [Ca(2+)]. Neurally released NPY does not appear to produce Ca(2+) transients directly, but significantly modulates NA-induced Ca(2+) signalling. The neural release of ATP and NA, as judged by postjunctional Ca(2+) signals, electrical recording of excitatory junction potentials and carbon fibre amperometry to measure NA, varies markedly with the pattern of nerve activity. This probably reflects both pre- and postjunctional mechanisms, which are not yet fully understood. These phenomena, together with different temporal patterns of sympathetic nerve activity in different regional circulations, are probably an important mechanistic basis of the important selective regulation of regional vascular resistance and blood flow by the sympathetic nervous system.

Phase Resetting of Arterial Vasomotion by Burst Stimulation of Perivascular Nerves

Arteries display cyclic diameter variations, vasomotion. In vivo, these rhythmic contractions are modulated by the influence of sympathetic nerves. In this study, we investigated the effect of burst stimulation of intramural nerves in vitro on the vasomotion of rat mesenteric small arteries. Vessels were mounted for isometric force measurement. After initiation of vasomotion with noradrenaline (0.5–2 µM), periarterial sympathetic nerves were stimulated electrically (10 impulses at 20 Hz) at approximately half-minute intervals. With a delay of 2–3 s, a neurogenic burst caused a brief contraction of the vascular smooth muscle and altered the period of the current vasomotion cycle. The effect on amplitude decayed rapidly and was practically not apparent in the next vasomotion cycle after the burst. With respect to period, stimulation at increasing intervals from the trough in force of vasomotion caused gradual prolongation of the cycle until a critical interval was reached, after which cycle duration was reduced instead. Since subsequent cycles were not affected, a change in phase remained. When two segments of oscillating arteries were mounted in a two-vessel myograph, simultaneously applied bursts of impulses synchronized their oscillation. The data suggest that changes in neural activity are able to make different vessels oscillate in phase, thereby coordinating vasomotion in different parts of the vascular tree, possibly explaining the synchronicity of vasomotion in different vascular beds that can be observed in vivo.

Chronic in ovo hypoxia decreases pulmonary arterial contractile reactivity and induces biventricular cardiac enlargement in the chicken embryo.

Although chronic prenatal hypoxia is considered a major cause of persistent pulmonary hypertension of the newborn, experimental studies have failed to consistently find pulmonary hypertensive changes after chronic intrauterine hypoxia. We hypothesized that chronic prenatal hypoxia induces changes in the pulmonary vasculature of the chicken embryo. We analyzed pulmonary arterial reactivity and structure and heart morphology of chicken embryos maintained from days 6 to 19 of the 21-day incubation period under normoxic (21% O(2)) or hypoxic (15% O(2)) conditions. Hypoxia increased mortality (0.46 vs. 0.14; P < 0.01) and reduced the body mass of the surviving 19-day embryos (22.4 +/- 0.5 vs. 26.6 +/- 0.7 g; P < 0.01). A decrease in the response of the pulmonary artery to KCl was observed in the 19-day hypoxic embryos. The contractile responses to endothelin-1, the thromboxane A(2) mimetic U-46619, norepinephrine, and electrical-field stimulation were also reduced in a proportion similar to that observed for KCl-induced contractions. In contrast, no hypoxia-induced decrease of response to vasoconstrictors was observed in externally pipped 21-day embryos (incubated under normoxia for the last 2 days). Relaxations induced by ACh, sodium nitroprusside, or forskolin were unaffected by chronic hypoxia in the pulmonary artery, but femoral artery segments of 19-day hypoxic embryos were significantly less sensitive to ACh than arteries of control embryos [pD(2) (= -log EC(50)): 6.51 +/- 0.1 vs. 7.05 +/- 0.1, P < 0.01]. Pulmonary vessel density, percent wall area, and periarterial sympathetic nerve density were not different between control and hypoxic embryos. In contrast, hypoxic hearts showed an increase in right and left ventricular wall area and thickness. We conclude that, in the chicken embryo, chronic moderate hypoxia during incubation transiently reduced pulmonary arterial contractile reactivity, impaired endothelium-dependent relaxation of femoral but not pulmonary arteries, and induced biventricular cardiac hypertrophy.

Chronic moderate hypoxia during in ovo development alters arterial reactivity in chickens.

We previously observed arterial sympathetic hyperinnervation and endothelial dysfunction in the chicken embryo after exposure to chronic hypoxia. We now investigate whether changes in arterial properties could also be observed at 14-15 weeks of life. Eggs of White Leghorn chicken were incubated under normoxic or moderately hypoxic (15% O2 from days 6-19 of a 21-day incubation) conditions. Experiments were performed at 14-15 weeks of life under standard conditions (Hm: males exposed to hypoxia; Hf: females exposed to hypoxia; Nm: males exposed to normoxia; Nf: females exposed to normoxia). Body weight at hatching and at 14-15 weeks was not affected by in ovo exposure to hypoxia. Mean arterial pressure and heart rate were not significantly altered by chronic in ovo hypoxia. However, isolated femoral arteries were more sensitive to electrical stimulation (frequency in Hz of half-maximal contraction, Hm: 1.62+/-0.33, Hf: 1.92+/-0.88, Nm: 2.49+/-0.49, Nf: 2.83+/-0.31) and pharmacological stimulation of peri-arterial sympathetic nerves (contraction in N/m in response to tyramine: Hm: 5.27+/-0.85, Hf: 4.10+/-0.9, Nm: 2.26+/-0.67, Nf: 3.65+/-0.51, p=0.07) after in ovo hypoxia. In side branches of the femoral artery, the effect of NO synthase blockade with L-NAME on contraction (in N/m) in response to high K+ (Hm: 0.35+/-0.91, Hf: 1.29+/-0.36, Nm: 2.88+/-0.19, Nf: 2.79+/-0.58) and on the sensitivity to acetylcholine (DeltapD2, H: 0.32+/-0.11, N: 0.62+/-0.05) was reduced after in ovo hypoxia. The present study shows that exposure to chronic moderate hypoxia during development affects the contractile and relaxing arterial responses of 14- to 15-week-old chickens. Although hypoxia did not lead to changes in blood pressure at this age, the observed effects on arterial sympathetic and endothelial function may represent early signs of future cardiovascular abnormalities.

Chronic hypoxia stimulates periarterial sympathetic nerve development in chicken embryo.

Epidemiological findings suggest an association between low-for-age birth weight and the risk to develop coronary heart diseases in adulthood. During pregnancy, an imbalance between fetal demands and supply may result in permanent alterations of neuroendocrine development in the fetus. We evaluated whether chronic prenatal hypoxia increases arterial sympathetic innervation. Chicken embryos were maintained from 0.3 to 0.9 of the 21-day incubation period under normoxic (21% O(2)) or hypoxic conditions (15% O(2)). At 0.9 incubation, the degree of sympathetic innervation of the embryonic femoral artery was determined by biochemical, histological, and functional (in vitro contractile reactivity) techniques. Chronic hypoxia increased embryonic mortality (32% versus 13%), reduced body weight (21.9+/-0.4 versus 25.4+/-0.6 g), increased femoral artery norepinephrine (NE) content (78.4+/-9.4 versus 57.5+/-5.0 pg/mm vessel length), and increased the density of periarterial sympathetic nerve fibers (14.4+/-0.7 versus 12.5+/-0.6 counts/10(4) microm(2)). Arteries from hypoxic embryos were less sensitive to NE (pD(2), 5.99+/-0.04 versus 6. 21+/-0.10). In the presence of cocaine, however, differences in sensitivity were no longer present. In the embryonic heart, NE content (156.9+/-11.0 versus 108.1+/-14.7 pg/mg wet wt) was also increased after chronic hypoxia. In the chicken embryo, chronic moderate hypoxia leads to sympathetic hyperinnervation of the arterial system. In humans, an analogous mechanism may increase the risk for cardiovascular disease in adult life.

INHIBITION OF SYMPATHETIC NORADRENERGIC TRANSMISSION BY GUANABENZ AND GUANETHIDINE IN RAT ISOLATED MESENTERIC ARTERY: INVOLVEMENT OF NEURONAL POTASSIUM CHANNELS

The present study investigated the effects of the α 2-adrenoceptor agonist guanabenz and the adrenergic neurone blocking drug guanethidine on the resting and stimulation-induced (S-I) effluxes of radioactivity from rat isolated mesenteric artery preparations in which the noradrenergic transmitter stores had been radiolabelled with [ 3H]-noradrenaline. The efflux of radioactivity evoked by electrical field stimulation of periarterial sympathetic nerves (60s trains of 1ms pulses, 2Hz, 12V) was taken as an index of transmitter noradrenaline release. Guanabenz (0.1–10μ M) decreased, in a concentration-dependent manner, both the resting and S-I effluxes of radioactivity. Guanethidine (0.1 and 1μ M) also decreased S-I efflux but increased resting efflux, both effects being concentration dependent. The inhibitory effects of guanabenz on both resting and S-I effluxes were reduced by blockade of the neuronal amine carrier with desipramine (1μ M). The inhibitory effect of guanabenz on resting efflux was prevented by inhibition of monoamine oxidase with pargyline (100μ M). The inhibitory effect of guanabenz on S-I efflux was not due to activation of prejunctional α 2-adrenoceptors since the inhibition was not blocked by the selective α 2-adrenoceptor antagonist idazoxan (0.1μ M). However, the inhibitory effect of guanabenz and guanethidine on S-I efflux was reduced by the inhibitor of Ca 2+-activated potassium channels apamin (0.1μ M). The findings suggest that guanabenz, like guanethidine, enters noradrenergic nerve terminals by the neuronal amine carrier. The inhibition of resting efflux produced by guanabenz may be due to inhibition of neuronal monoamine oxidase. The enhancement of resting efflux produced by guanethidine is attributable to its indirect sympathomimetic action. Finally, guanabenz and guanethidine may inhibit transmitter noradrenaline release by activating potassium channels on sympathetic noradrenergic nerve terminals. These findings may be relevant to the mechanism of adrenergic neurone blockade.

Prejunctional effects of cromakalim, nicorandil and pinacidil on noradrenergic transmission in rat isolated mesenteric artery

The present study investigated the effects of cromakalim, nicorandil and pinacidil on resting and stimulation-induced (S-I) effluxes of radioactivity from rat isolated mesenteric artery preparations in which the noradrenergic transmitter stores had been radiolabelled with [3H]-noradrenaline. The efflux of radioactivity evoked by field stimulation of peri-arterial sympathetic nerves (pulses at 2 Hz frequency in trains of 60 s duration) was taken as an index of transmitter noradrenaline release. Cromakalim (1-100 microM) and nicorandil (1-1000 microM) produced minor effects on resting and S-I effluxes of radioactivity, but these did not exhibit concentration-dependency. Pinacidil (1-1000 microM) produced concentration-dependent increases, in both resting and S-I effluxes of radioactivity. With 1000 microM pinacidil, resting and S-I effluxes were increased to approximately 348% and 358% of their respective control values. The effects of pinacidil on resting and S-I effluxes were unaltered when the neuronal amine transport system was inhibited by desipramine (1 microM). Inhibition of monoamine oxidase with pargyline (100 microM) treatment markedly reduced the enhancement of resting efflux by 1000 microM pinacidil but did not alter its effect on S-I efflux. It is proposed that the enhanced resting efflux produced by pinacidil without pargyline treatment consists of deaminated [3H]-noradrenaline metabolites formed from [3H]-noradrenaline displaced from transmitter storage vesicles by pinacidil. The enhancement of S-I efflux by pinacidil does not appear to involve disruption of alpha 2-adrenoceptor auto-inhibition of transmitter release since equi-effective concentrations of phentolamine (1 microM) and pinacidil (1000 microM) produced additive effects on S-I efflux, whereas increasing the concentration of phentolamine from 1 to 2M produced no further increases in S-I efflux. In conclusion, this study has provided no evidence of a prejunctional inhibitory effect of the potassium channel openers cromakalim, nicorandil and pinacidil on transmitter noradrenaline release. However, the findings with pinacidil suggest that, in high concentrations, pinacidil displaces noradrenaline from transmitter stores, such that deaminated noradrenaline metabolites are released from the nerve terminals. Furthermore, pinacidil enhances S-I transmitter noradrenaline release, possibly by blocking neuronal potassium channels.

Effects of the venom of the Theridiid spider, Steatoda capensis Hann, on autonomic transmission in rat isolated atria and caudal artery

The possibility of α-latrotoxin-like activity in the crude venom gland extract (VGE) of a related Theridiid spider, Steatoda capensis Hann, was investigated. The VGE from female S. capensis Hann spiders produced vasoconstriction in isolated segments of rat caudal artery but was without effect in artery segments obtained from rats that had been pretreated with reserpine (2.5 mg/kg) 24 hr prior to experimentation, indicating that the vasoconstriction was due to the release of noradrenaline from periarterial sympathetic nerves. Steatoda capensis Hann VGE also increased the rate of beating of rat isolated atrial preparations. The positive chronotropic action of the VGE was partly due to the release of noradrenaline from atrial sympathetic nerves since it was reduced by the β-adrenoceptor antagonist propranolol, and smaller increases in rate were observed in atria taken from rats pretreated with reserpine. The positive chronotropic effect of the VGE was enhanced by atropine, suggesting that the VGE also releases acetylcholine from atrial parasympathetic nerves. The VGE evoked release of radioactivity from rat atria in which the transmitter stores of the atrial intramural noradrenergic nerves had been labelled with [ 3H]noradrenaline. There appeared to be two components of the release, one involving ω-conotoxin GVIA-sensitive Ca 2+ channels, and the other independent of extracellular Ca 2+.