Stimulator for the isolated phrenic nerve diaphragm preparation

A simple and inexpensive stimulator is described to deliver regular impulses of alternating characteristics suitable for direct and indirect stimulation of an isolated phrenic nerve diaphragm preparation. Output impedances of 5 and 10 ω provide independence of pulse size and shape from load characteristics.     

Substance P-like immunoreactivity in the phrenic nerve and diaphragm: axonal transport of substance P in the phrenic nerve

Substance P-like immunoreactivity was demonstrated in the phrenic nerve of rat in quantities similar to that found in vagus and sciatic nerves. In the diaphragm, the peptide was concentrated in the motor endplate zone. A surgical cut of the phrenic nerve was followed by disappearance of substance P from the motor endplate zone. In addition we could demonstrate axonal transport of substance P in the phrenic nerve after transection of the nerve.     

Decrease of the spontaneous non-quantal release of acetylcholine from the phrenic nerve in botulinum-poisoned rat diaphragm

Botulinum type A toxin (BoTx) has been found to diminish by 40% the spontaneous release of acetylcholine (ACh) from normal (acutely denervated) rat diaphragms incubated in the presence of 5 mM K⁺, while the release of ACh from chronically (4 days) denervated diaphragms was not affected during 2 h incubations. The toxin has been found to rapidly remove (within 10 min) the local depolarization of about 8 mV which developed in the end-plate zones of the diaphragms after the inhibition of cholinesterases; after the administration of BoTx, tubocurarine lost its ability to increase the resting membrane potential (H-response, Katz and Miledi 1977) in the end-plate area of anticholinesterasetreated muscles. It is concluded that BoTx inhibits the non-quantal release of ACh from the motor nerve fibres and that it probably acts directly on the nerve terminal surface membrane (without internalization). The H-response in the rat diaphragm reflects the non-quantal release of ACh from the nerve terminals and not from the muscle fibres.     

Transcutaneous,dual channel phrenic nerve stimulator for diaphragm pacing

An electrical stimulation system for bilateral diaphragm pacing was constructed. The stimulator instrument comprises a transmitter and two implantable receivers and nerve electrodes. An optimised transcutaneous link system is described which utilises a radio frequency coupling to transmit both power and stimulus information to the receiver. The receiver unit was constructed in thick-film hybrid technology using bipolar and c.m.o.s. circuits and was packaged in a hermetically sealed metal case. The construction of the transmitter allows a high reliability, low power consumption and flexibility of the stimulus parameter control. A complete, bilateral phrenic nerve pacing system was implanted into a patient suffering from tetraplegia. It is concluded that electrical phrenic nerve stimulation to pace the diaphragm is capable of being used in clinical therapy.     

Fatigue during continuous 20 Hz stimulation of the rat phrenic nerve diaphragm preparation

The site and mechanism of action of fatigue was investigated in the isolated rat phrenic nerve diaphrigm preparation, with indirect and direct stimulation at 20 Hz and recording of tension and EMG. An equal decay of the subtetanic tension during indirect and direct stimulation, and a parallel decay of tension and EMG, suggested a mechanism of fatigue localized to structures that were ‘seen’ by the EMG electrodes. A comparison of the responses to sub- and supra-maximum direct stimulation did not show increased fatigue at sub-maximum stimulation. Therefore, the fatigue was probably not caused by an increased threshold of the excitability of the sarcolemma. However, prolongation of the stimulus pulse during direct stimulation from 0.5 ms to 5 ms in the Citigued preparation caused a two-phasic recovery of tension. The initial phase, but not the slow phase, was inhibited by tetrodotoxin (TTX). Thus a recovery of sarcolemma action potentials could explain the initial phase. The slow phase was probably caused by a mechanism localized at more distal potential-dependent sites, probably in the T tubules.     

Spontaneous subminature end-plate potentials in mouse diaphragm muscle: evidence for synchronous release.

1. Miniature end-plate potentials (min.e.p.p.s) were recorded from small muscle cells of mouse diaphragms. Min.e.p.p. amplitude histograms showed successive peaks which were integral multiples of the smallest peak. The smallest potentials (submin.e.p.p.s) averaged 0-3-0-6mV and the mean of the larger min.e.p.p.s averaged 3-7 mV, depending on the muscle cell diameter. There was a positive correlation between time-to-peak and min.e.p.p. amplitude. Time-to-peak of the submin.e.p.p.s fell slightly below the regression line through the larger min.e.p.p.s. 2. Sometimes min.e.p.p. amplitude distributions changed spontaneously such that the mean of the major mode min.e.p.p.s decreased twofold during which time the mean of the submin.e.p.p.s did not change. Spontaneous decreases were most pronounced during low frequencies of release (10/min) achieved at 32 degrees C. 3. Small changes in temperature (2 degrees C steps in the range 32-40 degrees C) greatly altered the number of peaks of min.e.p.p. amplitude histograms without noticeably changing the position of the submin.e.p.p. peak. At 32 degrees C submin.e.p.p.s composed 5-20% of the histograms and the amplitude of the major mode peak was twelve to fifteen-times that of the submin.e.p.p.s. Over-all bell-shaped distributions were obtained at 37 degrees C which showed up to eight peaks with the major peak at the fourth to sixth peak. Temperatures slightly above 37 degrees C gave a flat distribution with the mean amplitude at the third peak. Min.e.p.p. amplitude histograms were initially skewed (mostly small min.e.p.p.s) after a 40 degrees C heat challenge. 4. Two to eight-times the normal concentration of Ca2+ in the saline reversibly increased the min.e.p.p. frequency and also decreased the mean of the major mode min.e.p.p.s (two to nine-times) without noticeably changing the mean of the submin.e.p.p.s. 5. Botulinum toxin A, 10(5) X intraperitoneal median lethal dose (10(5)I.P.LD50)/ml., almost abolished min.e.p.p.s in 30-90 min. The relative proportion of submin.e.p.p.s increased and the mean of the major mode min.e.p.p.s usually did not change during the initial decrease in frequency. Major mode min.e.p.p.s essentially ceased after 200-1000 were generated and remaining min.e.p.p.s of some cells showed skewed distributions with three small peaks that were integral multiples of the submin.e.p.p. peak. Smaller min.e.p.p.s were more resistant to block than the larger min.e.p.p.s and, although frequencies were low, small min.e.p.p.s were recorded after 4 hr of botulinum toxin incubation. 6. Colchicine (5 X 10(-4)M) within minutes reduced the major mode min.e.p.p.s by half (mean of major peak reduced to sixth or seventh peak). Additional colchicine (10(-3)M reduced the major mode min.e.p.p. amplitude to a fifth of that of control (mean of major mode min.e.p.p.s at the third peak) with no change in position of the submin.e.p.p. peak. Min.e.p.p. amplitudes slowly recovered to half control values after washing. 7...     

Effects of hexamethonium on transmitter release from the rat phrenic nerve.

Hexamethonium (HEX) was applied to isolated transected diaphragm-phrenic nerve preparations of the rat in order to further elucidate the functional role of the presynaptic nicotinic autoreceptors. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the neuromuscular junctions in the presence and absence of HEX to determine the relative effect of this nicotinic antagonist on end-plate sensitivity and evoked release. In this study we show that HEX enhances transmitter release for the first few stimuli, but this action is not maintained during a train-of-six stimulation. While these results support the hypothesis that transmitter released from the nerve terminal normally has a negative feedback effect by depressing transmitter release it is proposed that HEX also has secondary actions on the neuromuscular junction that are unrelated to autoreceptor blockage. The results with HEX suggests that the presynaptic receptors may differ pharmacologically from the postsynaptic receptors.     

5-Hydroxytryptamine uptake and release in relation to aggregation of rabbit platelets

1. The aggregation of blood platelets was measured in vitro at different time intervals after the addition of 5-hydroxytryptamine (5-HT) or of reserpine to platelet-rich plasma of untreated rabbits and of rabbits injected with reserpine and 5-HT, i.e. while the platelets were taking up 5-HT or releasing it under the influence of reserpine.2. When 5-HT was added to stirred platelet-rich plasma the platelets aggregated reversibly within 1 min. The velocity of aggregation increased with 5-HT concentrations of 0.1-30 muM and decreased with higher concentrations.3. (-)-Adrenaline, which alone did not produce aggregation, markedly accelerated the aggregation caused by 5-HT. The acceleration was greatest when 5-HT and adrenaline were added simultaneously.4. 5-HT added to the platelet-rich plasma in amounts that exceeded the 5-HT capacity of the platelets progressively diminished the velocity of aggregation produced by 5-HT plus adrenaline until aggregation was completely inhibited. Smaller amounts of 5-HT produced a transient inhibition of aggregation.5. The aggregation of platelets from reserpinized rabbits was inhibited by less 5-HT than the aggregation of platelets from normal rabbits.6. (-)-Adrenaline aggregated platelets of untreated rabbits but not those of reserpinized rabbits or of rabbits injected with 5-HT when reserpine was added in vitro 1-30 min previously.7. Platelets obtained from rabbits treated first with reserpine and subsequently injected with 5-HT were not aggregated by 5-HT plus adrenaline. During incubation in vitro these platelets progressively recovered their aggregability but this recovery was delayed by the monoamine oxidase inhibitor Pargyline.8. Imipramine in concentrations which did not influence platelet aggregation by adenosine diphosphate (ADP) abolished aggregation produced by 5-HT or by 5-HT plus adrenaline.9. The inhibitory effect of adenosine on platelet aggregation was concentration-dependent and similar whether aggregation was produced by ADP, 5-HT or 5-HT plus adrenaline.10. It is proposed that aggregation brought about by 5-HT is connected with its active uptake into the platelets and is caused by ADP formed from ATP during the active uptake of the amine. When 5-HT is no longer actively taken up, it also ceases to cause or to potentiate aggregation.     

Left phrenic nerve anatomy relative to the coronary venous system: Implications for phrenic nerve stimulation during cardiac resynchronization therapy

The objective of this study was to quantitatively characterize anatomy of the human phrenic nerve in relation to the coronary venous system, to reduce undesired phrenic nerve stimulation during left‐sided lead implantations. We obtained CT scans while injecting contrast into coronary veins of 15 perfusion‐fixed human heart‐lung blocs. A radiopaque wire was glued to the phrenic nerve under CT, then we created three‐dimensional models of anatomy and measured anatomical parameters. The left phrenic nerve typically coursed over the basal region of the anterior interventricular vein, mid region of left marginal veins, and apical region of inferior and middle cardiac veins. There was large variation associated with the average angle between nerve and veins. Average angle across all coronary sinus tributaries was fairly consistent (101.3°–111.1°). The phrenic nerve coursed closest to the middle cardiac vein and left marginal veins. The phrenic nerve overlapped a left marginal vein in >50% of specimens. Clin. Anat. 28:621–626, 2015. © 2015 Wiley Periodicals, Inc.     

An anatomical study of the full-length phrenic nerve and its blood supply: clinical implications for endoscopic dissection

For surgeries aimed at the dissection of full-length phrenic nerve, a full appreciation of its trajectory, blood supply and correlation with adjacent anatomical structures is necessary, especially for endoscopic manipulations. A fresh cadaver study was conducted with the purpose of avoiding surgical complications and ensuring further efficacy and efficiency of endoscopic manipulations. Ten fresh adult cadavers were dissected. Special attention was paid to the topography of the origin, the trajectory of the phrenic nerve, and its anatomic communication with the surrounding vessels and organs. In the second side of the cadavers, thoracic endoscopic manipulations and observations were also performed. The full length of the phrenic nerve was 24.6 ± 1.7 and 30.6 ± 1.8 cm on the right and left side, respectively; the blood supply of the phrenic nerve in the thoracic cavity came exclusively from the pericardiacophrenic artery; the distance between the origin of the pericardiacophrenic artery and that of the internal thoracic artery ranged from 0.5 to 5.2 cm on the right side, and from 1.4 to 5.6 cm on the left; most of the pericardiacophrenic veins intermingled with small vessels of pericardium and pleura, forming a venous network and joining the innominate vein. Endoscopic dissection of the thoracic phrenic nerve together with the accompanying pericardiacophrenic artery can be performed. Extreme attention should be paid during surgery to a section of about 6 cm in length of the artery originating from the internal thoracic artery, while the accompanying veins do not require to be spared.     

The accessory phrenic nerve in the rat

Summary Reinnervation studies of the diaphragm led us to reinvestigate the normal anatomy of the phrenic nerve of the rat. The phrenic nerve originates from the cervical nerve roots C₄ and C₅. In 16 out of 19 normal rats an accessory phrenic nerve was observed receiving its segmental fibres from C₆. The number of myelinated axons of the accessory phrenic nerve varied from 41 to 101 (mean: 64.3, i.e. about 15% of the average number of axons in the common phrenic nerve). The accessory phrenic nerve innervates the dorsal part of the costal and the lateral part of the crural region, whereas the remaining parts of the hemidiaphragm are supplied by the segments C₄ and C₅. There is no evidence for any additional contribution to the motor innervation of the diaphragm from intercostal nerves.Direktor: Prof. Dr. W. ZenkerThis study was supported by the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich.     

家兔膈神经放电实验中常见问题的解决方法以及与膈肌放电、呼吸运动的同步记录

针对机能实验课膈神经放电实验中常见的问题,提出了切实有效的解决方法以及观察呼吸运动调节的综合实验。     

Abolishment of non-quantal release of acetylcholine from the mouse phrenic nerve endings by toosendanin

The hyperpolarizing effect (H-effect) of d-tubocurarine on the end-plate of the isolated diaphragm pretreated with an anticholinesterase was irreversibly abolished by toosendanin (1 X 10(-5) g/ml), indicating the blockade of spontaneous non-quantal release of acetylcholine (ACh). The H-effect was also inhibited, but temporarily, when toosendanin (a dose of 0.6 LD50) was subcutaneously injected into the mouse and the diaphragm was isolated 40-120 min after injection. During such an inhibitory period, however, spontaneous release of ACh remained facilitated. It is concluded that the effect of toosendanin on non-quantal release of ACh was different from its effect on quantal release not only at the direction but also at the time course.     

Immunohistochemical evidence for the existence of angiotensin II-containing nerve terminals in the brain and spinal cord in the rat

Using the indirect immunohistochemical method it has been possible to demonstrate angiotensin II or angiotensin II-like immunoreactivity in probable axons and nerve terminals in the brain and spinal cord of normal rat, resistant to bilateral nephrectomy. A high density of angiotensin II-cotaining nerve terminals has been found in the substantia gelatinosa of the spinal cord and the medulla oblongata, suggesting a role of this peptide in the mediation of sensory information to the CNS. A high density was found in the sympathetic lateral column, which supports the view that this peptide is involved in central blood pressure control. A plexus of angiotensin II nerve terminals in the locus coeruleus may control activity in the coerulocortical noradrenergic pathways. The existence of a substantial innervation of the medial external layer of the median eminence may suggest a role of angiotensin II in anterior pituitary control as, for example, a hypothalamic hormone.     

Electrical stimulation of the phrenic nerve in dogs

N. N. Burdenko Institute of Neurosurgery, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. N. Konovalov.) Translated from Byullenten' Éksperimental'noi Biologii i Meditsiny, Vol. 106, No. 7, pp. 19–21, July, 1988.