Etude du nerf phrenique chez le cobaye (Cavia porcellus, L., 1756)

The aim of the present study to ascertain the mode of origin of the phrenic nerve and to provide a morphological basis for experimental studies of this nerve in the guinea pig. In sketches made of the dissections, in 10 male and 10 female guinea pigs adults, the modes of origin of the phrenic roots were demonstrated to arise from the fourth to the seventh cervical nerves. Four types of origin could be distinguished. The phrenic nerve of the guinea pig has three or four roots.

Ramificação e distribuição dos nervos frênicos no músculo diafragma do gato doméstico

The ramification and the distribution of the phrenic nerves right and left had been studied in 30 muscles diaphragms of unknown breed adult domestic cats, 7 males and 23 females, and were fixed in 10% buffered formalin solution. After fixation and dissection, it was observed that the phrenic nerves ramified for the respective carnous parts of the muscle diaphragm, pars lumbalis, costalis and sternalis, and had finished in higher frequency in dorsolateral trunk and ventral branch (63.33%). We observed the following arrangements: dorsal, ventral and lateral branches (25.0%); dorsal branch and ventrolateral trunk (6.66%); dorsolateral and ventrolateral trunks (3.33%); dorsolateral trunk, lateral and ventral branches (1.66%). The phrenic nerves had distributed symmetrically in 11 samples (36.66%), only showing the termination in dorsolateral trunk and ventral branch. The dorsal branches supplied pars lumbalis (73.33% to right and 56.66% to the left) and pars costalis (13.33% to right and 10.0% to the left). The right dorsal branch supplied the crus mediale dexter of the right pillar (100.0%) and the left dorsal branch supplied the crus mediale sinister of the right pillar and the left pillar (100.0%). The lateral branches supply pars lumbalis (23.33% to right and 33.33% to the left), pars costalis (96.66% to right and 100.0% to the left) and pars sternalis (3.33% only to the right). The ventral branches supplied the ventral region of pars costalis (46.66 % to right and 43.33% to the left) and pars sternalis (96.66% to right and 100.0% to the left). Four female animals (13.33%) had shown fibers crossing proceeding from the left ventral branch for right antimere had been that in one of these samples (3.33%) occurred connection between the left ventral branch and the right.

Diaphragmatic pacing stimulation in spinal cord injury: anesthetic and perioperative management

OBJECTIVE: The standard therapy for patients with high-level spinal cord injury is long-term mechanical ventilation through a tracheostomy. However, in some cases, this approach results in death or disability. The aim of this study is to highlight the anesthetics and perioperative aspects of patients undergoing insertion of a diaphragmatic pacemaker. METHODS: Five patients with quadriplegia following high cervical traumatic spinal cord injury and ventilator-dependent chronic respiratory failure were implanted with a laparoscopic diaphragmatic pacemaker after preoperative assessments of their phrenic nerve function and diaphragm contractility through transcutaneous nerve stimulation. ClinicalTrials.gov:NCT01385384. RESULTS: The diaphragmatic pacemaker placement was successful in all of the patients. Two patients presented with capnothorax during the perioperative period, which resolved without consequences. After six months, three patients achieved continuous use of the diaphragm pacing system, and one patient could be removed from mechanical ventilation for more than 4 hours per day. CONCLUSIONS: The implantation of a diaphragmatic phrenic system is a new and safe technique with potential to improve the quality of life of patients who are dependent on mechanical ventilation because of spinal cord injuries. Appropriate indication and adequate perioperative care are fundamental to achieving better results.

Sural nerve involvement in experimental hypertension: morphology and morphometry in male and female normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)

Background: The sural nerve has been widely investigated in experimental models of neuropathies but information about its involvement in hypertension was not yet explored. The aim of the present study was to compare the morphological and morphometric aspects of different segments of the sural nerve in male and female spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Rats aged 20 weeks (N = 6 in each group) were investigated. After arterial pressure and heart rate recordings in anesthetized animals, right and left sural nerves were removed and prepared for epoxy resin embedding and light microscopy. Morphometric analysis was performed with the aid of computer software, and took into consideration the fascicle area and diameter, as well as myelinated fiber number, density, area and diameter. Results: Significant differences were observed for the myelinated fiber number and density, comparing different genders of WKY and SHR. Also, significant differences for the morphological (thickening of the endoneural blood vessel walls and lumen reduction) and morphometric (myelinated fibers diameter and G ratio) parameters of myelinated fibers were identified. Morphological exam of the myelinated fibers suggested the presence of a neuropathy due to hypertension in both SHR genders. Conclusions: These results indicate that hypertension altered important morphometric parameters related to nerve conduction of sural nerve in hypertensive animals. Moreover the comparison between males and females of WKY and SHR allows the conclusion that the morphological and morphometric parameters of sural nerve are not gender related. The morphometric approach confirmed the presence of neuropathy, mainly associated to the small myelinated fibers. In conclusion, the present study collected evidences that the high blood pressure in SHR is affecting the sural nerve myelinated fibers.

Risultati funzionali a lungo termine dopo sutura o plicatura del diaframma

Obbiettivo. Analizzare la funzionalità polmonare e diaframmatica dopo interventi di plicatura del diaframma con rete di rinforzo peri-costale eseguiti per relaxatio e riparazione di ernia transdiaframmatica cronica mediante riduzione e sutura diretta. Metodi. Dal 1996 al 2010, 10 pazienti con relaxatio unilaterale del diaframma e 6 pazienti con ernia transdiaframmatica cronica misconosciuta sono stati sottoposti a chirurgia elettiva. Gli accertamenti preoperatori e al follow-up di 12 mesi includevano prove di funzionalità respiratoria, misura della pressione massimale inspiratoria alla bocca in clino e ortostatismo, emogasanlisi, TC del torace e dispnea score. Risultati. I pazienti dei due gruppi non differivano in termini di funzionalità respiratoria preoperatoria nè di complicanze postoperatorie; al follow-up a 12 mesi il gruppo Eventrazione mostrava un significativo aumento del FEV1% (+18,2 – p<0.001), FVC% (+12,8 – p<0.001), DLCO% (+6,84 – p=0,04) e pO2 (+9,8 mmHg – p<0.001). Al contrario nrl gruppo Ernia solo il miglioramento della pO2 era significativo (+8.3 – p=0.04). Sebbene la massima pressione inspiratoria (PImax) fosse aumentata in entrambi i gruppi al follow-up, i pazienti operati per ernia mostravano un miglioramento limitato con persistente caduta significativa della PImax dall’ortostatismo al clinostatismo (p<0.001). Il Transitional dyspnoea score è stato concordante con tali miglioramenti pur senza differenze significative tra i due gruppi. La TC del torace ha evidenziato una sopraelevazione dell’emidiaframma suturato, senza recidiva di ernia, mentre i pazienti sottoposti a plicatura hanno mantenuto l’ipercorrezione. Conclusioni. L’utilizzo di un rinforzo protesico è sicuro e sembra assicurare risultati funzionali migliori a distanza in termini di flussi respiratori e di movimento paradosso del diaframma (valutato mediante PImax). Lacerazioni estese del diaframma coinvolgenti le branche principali di suddivisione del nervo frenico si associano verosimilmente a una relaxatio che può quindi ridurre il guadagno funzionale a lungo termine se non adeguatamente trattata mediante l’utilizzo di un rinforzo protesico.

Role of Rab3 GDP/GTP Exchange Protein in Synaptic Vesicle Trafficking at the Mouse Neuromuscular Junction

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca2+-dependent neurotransmitter release. These small G proteins are activated by Rab3 GDP/GTP exchange protein (Rab3 GEP). To determine the function of Rab3 GEP during neurotransmitter release, we have knocked out Rab3 GEP in mice. Rab3 GEP−/− mice developed normally but died immediately after birth. Embryos at E18.5 showed no evoked action potentials of the diaphragm and gastrocnemius muscles in response to electrical stimulation of the phrenic and sciatic nerves, respectively. In contrast, axonal conduction of the spinal cord and the phrenic nerve was not impaired. Total numbers of synaptic vesicles, especially those docked at the presynaptic plasma membrane, were reduced at the neuromuscular junction ∼10-fold compared with controls, whereas postsynaptic structures and functions appeared normal. Thus, Rab3 GEP is essential for neurotransmitter release and probably for formation and trafficking of the synaptic vesicles.

Restriction of intramuscular neurite branching and extension is lost in agrin and rapsyn-deficient mice

The phrenic nerve enters the diaphragm at approximately embryonic day 12.5 (E12.5) in the mouse. The secondary nerve trunk advances along the centre of the diaphragm muscle and extends tertiary branches primarily towards the lateral side during normal embryonic development. In the present study we quantified the intramuscular neurite branching in the most ventral region of the diaphragm at E15.5 and E18.5 in wild-type mice, agrin knock-out mice (KOAG) and rapsyn knock-out mice (KORAP). KOAG and KORAP have decreased muscle contraction due to their inability to maintain/form acetylcholine receptor (AChR) clusters during embryonic development. Heterozygote mothers were anaesthetised via an overdose of Nembutal (30 mg; Boeringer Ingelheim, Ridgefield, CT, USA) and killed via cervical dislocation. There were increases in the number of branches exiting the medial side of the phrenic nerve trunk in KOAG and KORAP compared to wild-type mice, but not on the lateral side at E15.5 and E18.5. However, the number of bifurcations in the periphery significantly increased on both the medial and lateral sides of the diaphragm at E15.5 and E18.5 in KOAG and KORAP compared to control mice. Furthermore, neurites extended further on both the medial and lateral sides of the diaphragm at E15.5 and E18.5 in KOAG and KORAP compared to wild-type mice. Together these results show that the restriction of neurite extension and bifurcations from the secondary nerve trunk is lost in both KOAG and KORAP allowing us the opportunity to investigate the factors that restrict motoneuron behaviour in mammalian muscles.

The motor neurite terminal determines where neuromuscular synapses form in the absence of agrin

Agrin is a proteoglycan secreted by motor neurite terminals that functions to initiate and maintain AChR clusters at the nerve terminal. This led to the theory that neurite terminals decide where neuromuscular synapses form by secreting agrin. However, initiation of AChR clustering occurs in the absence of the innervating motoneuron and in the absence of agrin. In this instance, the muscle, not the nerve, is deciding the location of neuromuscular synapses by drawing neurite terminals towards pre-existing AChR clusters. If this were true, one would expect the initial innervation patterns to be the same in agrin-deficient mice and wild-type mice. To test this we quantified the intramuscular axonal branching and synapse formation in the diaphragm at E14.5 in agrin-deficient mice and wild-type mice. Heterozygote mothers were anaesthetised with Nembutal (30 mg) and killed via cervical dislocation. In the diaphragm, the nerve trunk runs down the centre of the muscle and extends branches primarily toward the lateral side. In agrin-deficient mice however, we found significantly more branches exited the phrenic nerve trunk, branched in the periphery and extended further on the medial side. Moreover, we found that the percentage α-bungarotoxin/synaptophysin colocalisations, markers of pre- and postsynaptic differentiation, respectively, was the same in agrin-deficient mice and wild-type mice. These results show that initial innervation patterns are not the same in agrin-deficient mice and wild-type mice indicating neurite terminals, not muscle, decide the placement of neuromuscular synapses in the absence of agrin.

Central chemoreceptors and neural mechanisms of cardiorespiratory control

The arterial partial pressure (P CO2) of carbon dioxide is virtually constant because of the close match between the metabolic production of this gas and its excretion via breathing. Blood gas homeostasis does not rely solely on changes in lung ventilation, but also to a considerable extent on circulatory adjustments that regulate the transport of CO2 from its sites of production to the lungs. The neural mechanisms that coordinate circulatory and ventilatory changes to achieve blood gas homeostasis are the subject of this review. Emphasis will be placed on the control of sympathetic outflow by central chemoreceptors. High levels of CO2 exert an excitatory effect on sympathetic outflow that is mediated by specialized chemoreceptors such as the neurons located in the retrotrapezoid region. In addition, high CO2 causes an aversive awareness in conscious animals, activating wake-promoting pathways such as the noradrenergic neurons. These neuronal groups, which may also be directly activated by brain acidification, have projections that contribute to the CO2-induced rise in breathing and sympathetic outflow. However, since the level of activity of the retrotrapezoid nucleus is regulated by converging inputs from wake-promoting systems, behavior-specific inputs from higher centers and by chemical drive, the main focus of the present manuscript is to review the contribution of central chemoreceptors to the control of autonomic and respiratory mechanisms.

Are l-glutamate and ATP cotransmitters of the peripheral chemoreflex in the rat nucleus tractus solitarius ?

Peripheral chemoreflex activation in awake rats or in the working heart-brainstem preparation (WHBP) produces sympathoexcitation, bradycardia and an increase in the frequency of phrenic nerve activity. Our focus is the neurotransmission of the sympathoexcitatory component of the chemoreflex within the nucleus of the tractus solitarius (NTS), and recently we verified that the simultaneous antagonism of ionotropic glutamate and purinergic P(2) receptors in the NTS blocked the pressor response and increased thoracic sympathetic activity in awake rats and WHBP, respectively, in response to peripheral chemoreflex activation. These previous data suggested the involvement of ATP and L-glutamate in the NTS in the processing of the sympathoexcitatory component of the chemoreflex by unknown mechanisms. For a better understanding of these mechanisms, here we used a patch-clamp approach in brainstem slices to evaluate the characteristics of the synaptic transmission of NTS neurons sending projections to the ventral medulla, which include the premotor neurons involved in the generation of the sympathetic outflow. The NTS neurons sending projections to the ventral medulla were identified by previous microinjection of the membrane tracer dye, 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate (DiI), in the ventral medulla and the spontaneous (sEPSCs) and tractus solitarius (TS)-evoked excitatory postsynaptic current (TS-eEPSCs) were recorded using patch clamp. With this approach, we made the following observations on NTS neurons projecting to the ventral medulla: (i) the sEPSCs and TS-eEPSCs of DiI-labelled NTS neurons were completely abolished by 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), an antagonist of ionotropic non-NMDA glutamatergic receptors, showing that they are mediated by L-glutamate; (ii) application of ATP increased the frequency of appearance of spontaneous glutamatergic currents, reflecting an increased exocytosis of glutamatergic vesicles; and (iii) ATP decreased the peak of TS-evoked glutamatergic currents. We conclude that L-glutamate is the main neurotransmitter of spontaneous and TS-evoked synaptic activities in the NTS neurons projecting to the ventral medulla and that ATP has a dual modulatory role on this excitatory transmission, facilitating the spontaneous glutamatergic transmission and inhibiting the TS-evoked glutamatergic transmission. These data also suggest that ATP is not acting as a cotransmitter with L-glutamate, at least at the level of this subpopulation of NTS neurons studied.

Glutamatergic and purinergic mechanisms on respiratory modulation in the caudal NTS of awake rats

In the present study we evaluated the role of ionotropic glutamate receptors and purinergic P2 receptors in the caudal commissural NTS (cNTS) on the modulation of the baseline respiratory frequency (fR), and on the tachypneic response to chemoreflex activation in awake rats. The selective antagonism of ionotropic glutamate receptors with kynurenic acid (2 nmol/50 nl) in the cNTS produced a significant increase in the baseline fR but no changes in the tachypneic response to chemoreflex activation. The selective antagonism of purinergic P2 receptors by PPADS (0.25 nmol/50 nl) in the cNTS produced no changes in the baseline fR or in the tachypneic response to chemoreflex activation. The data indicate that glutamate acting on ionotropic receptors in the cNTS plays a inhibitory role on the modulation of the baseline fR but had no effect on the tachypneic response to chemoreflex activation, while ATP acting on P2 receptors in the cNTS plays no major role in the modulation of the baseline fR or in the tachypneic response to chemoreflex activation. We suggest that neurotransmitters other than L-glutamate and ATP are involved in the processing of the tachypneic response of the chemoreflex at the cNTS level. (C) 2008 Elsevier B.V. All rights reserved.

Reproducibility of twitch mouth pressure, sniff nasal inspiratory pressure, and maximal inspiratory pressure.

Twitch mouth pressure (Pmo,tw) during magnetic phrenic nerve stimulation and sniff nasal inspiratory pressure (SNIP) were recently proposed as alternative noninvasive methods for assessing inspiratory muscle strength. This study aimed to compare their reproducibility with maximal inspiratory pressure (MIP) in normal subjects. Ten healthy subjects were studied at functional residual capacity in semirecumbent position. Cervical magnetic phrenic nerve stimulation was performed during gentle expiration against an occlusion incorporating a small leak. Constancy of stimulation was controlled by recording diaphragmatic electromyogram. Within and between-session reproducibility of pressure were studied for Pmo,tw, SNIP, and MIP. The subjects were studied during a session of 10 manoeuvres repeated after 1 day and 1 month. The mean values were 16 cmH2O for Pmo,tw, 118 cmH2O for SNIP, and 115 cmH2O for MIP. For the three tests, the within subject variation was small in relation to between-subject variation, with the intraclass correlation coefficient ranging 0.79-0.90 for Pmo,tw, 0.85-0.92 for SNIP, and 0.88-0.92 for MIP. At 1 day interval, the coefficient of repeatability (2 SD of differences) was 3.6 cmH2O for Pmo,tw, 32 cmH2O for SNIP and 28 cmH2O for MIP. At 1 month interval, the coefficient of repeatability was 5.8 cmH2O for Pmo,tw, 23 cmH2O for SNIP and 21 cmH2O for MIP. We conclude that the within session reproducibility of the new tests twitch mouth pressure and sniff nasal inspiratory pressure is sufficient to be clinically useful. For sniff nasal inspiratory pressure, the between session reproducibility established after 1 day was maintained after 1 month. For twitch mouth pressure, the between session reproducibility declined slightly after 1 month. These characteristics should be considered when using these methods to follow an individual patient over time.

Central chemoreceptors and neural mechanisms of cardiorespiratory control

The arterial partial pressure (P CO2) of carbon dioxide is virtually constant because of the close match between the metabolic production of this gas and its excretion via breathing. Blood gas homeostasis does not rely solely on changes in lung ventilation, but also to a considerable extent on circulatory adjustments that regulate the transport of CO2 from its sites of production to the lungs. The neural mechanisms that coordinate circulatory and ventilatory changes to achieve blood gas homeostasis are the subject of this review. Emphasis will be placed on the control of sympathetic outflow by central chemoreceptors. High levels of CO2 exert an excitatory effect on sympathetic outflow that is mediated by specialized chemoreceptors such as the neurons located in the retrotrapezoid region. In addition, high CO2 causes an aversive awareness in conscious animals, activating wake-promoting pathways such as the noradrenergic neurons. These neuronal groups, which may also be directly activated by brain acidification, have projections that contribute to the CO2-induced rise in breathing and sympathetic outflow. However, since the level of activity of the retrotrapezoid nucleus is regulated by converging inputs from wake-promoting systems, behavior-specific inputs from higher centers and by chemical drive, the main focus of the present manuscript is to review the contribution of central chemoreceptors to the control of autonomic and respiratory mechanisms.

INHIBITION OF THE CAUDAL PRESSOR AREA REDUCES CARDIORESPIRATORY CHEMOREFLEX RESPONSES

The caudal pressor area (CPA) is a brainstem area located close to the spinal cord. The activation of the CPA increases sympathetic activity and mean arterial pressure (MAP) by mechanisms dependent on the commissural nucleus of the solitary tract (commNTS) and rostroventrolateral medulla, however, the signals that activate the CPA to produce these responses are still unknown. Therefore, in the present study, we investigated the activity of glutamatergic and GABAergic mechanisms from the CPA and commNTS in rats exposed to hypoxia and the effects of the inhibition of CPA neurons on cardiorespiratory responses to peripheral chemoreceptor activation with i.v. sodium cyanide (NaCN). Male Sprague-Dawley rats (250-280 g, n=5-8/group) were used. In conscious rats, most of the commNTS neurons (66 +/- 11%) and part of the CPA neurons (36 +/- 7%) activated by hypoxia (8% O2) were glutamatergic (contained VGLUT2mRNA). Small part of the neurons activated during hypoxia was GABAergic (contained GAD-67mRNA) in the commNTS (9 +/- 4%) or the CPA (6 +/- 2%). In urethane anesthetized rats, the inhibition of CPA neurons with bilateral injections of muscimol (GABA-A agonist, 2 mM) reduced baseline MAP, splanchnic sympathetic nerve discharge (SND) and phrenic nerve discharge (PND). Muscimol into the CPA also reduced by around 50% the pressor and sympathoexcitatory responses and the increase in PND to peripheral chemoreceptor activation with NaCN (50 mu g/kg i.v.), without changing sympathetic baroreflex responses. These data suggest that CPA mechanisms facilitate cardiorespiratory responses to peripheral chemoreflex activation. Immunohistochemistry results also suggest that at least part of the CPA mechanisms activated by hypoxia is glutamatergic. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Ventrolateral medulla mechanisms involved in cardiorespiratory responses to central chemoreceptor activation in rats

A rise in arterial PCO(2) stimulates breathing and sympathetic activity to the heart and blood vessels. In the present study, we investigated the involvement of the retrotrapezoid nucleus (RTN) and glutamatergic mechanisms in the Botzinger/C1 region (Botz/C1) in these responses. Splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane-anesthetized, sino-aortic-denervated, vagotomized, and artificially ventilated rats subjected to hypercapnia (end-expiratory CO(2) from 5% to 10%). Phrenic activity was absent at end-expiratory CO(2) of 4%, and strongly increased when end-expiratory CO(2) reached 10%. Hypercapnia also increased sSND by 103 +/- 7%. Bilateral injections of the GABA-A agonist muscimol (2 mM) into the RTN eliminated the PND and blunted the sSND activation (Delta = +56 +8%) elicited by hypercapnia. Injections of NMDA receptor antagonist AP-5 (100 mM), non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 100 mM) or metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 100 mM) bilaterally into the Botz/C1 reduced PND (Delta = +43 +/- 7%, +52 +/- 6% or +56 +/- 11%, respectively). MCPG also reduced sSND (Delta = +41 +/- 7%), whereas AP-5 and DNQX had no effect. In conclusion, the increase in sSND caused by hypercapnia depends on increased activity of the RTN and on metabotropic receptors in the Botz/C1, whereas PND depends on increased RTN activity and both ionotropic and metabotropic receptors in the Botz/C1.