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Journal of Visualized Experiments : JoVE Feb 2018Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating...
Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.
Topics: Animals; Mice; Motor Neurons; Phrenic Nerve
PubMed: 29553523
DOI: 10.3791/56758 -
American Journal of Respiratory and... Oct 2022
Topics: Diaphragm; Electric Stimulation; Electric Stimulation Therapy; Humans; Phrenic Nerve; Sleep Apnea, Central
PubMed: 35772121
DOI: 10.1164/rccm.202206-1024LE -
The Journal of Neuroscience : the... Jul 2017Gonadal steroids modulate CNS plasticity, including phrenic long-term facilitation (pLTF), a form of spinal respiratory neuroplasticity resulting in increased phrenic...
Gonadal steroids modulate CNS plasticity, including phrenic long-term facilitation (pLTF), a form of spinal respiratory neuroplasticity resulting in increased phrenic nerve motor output following exposure to acute intermittent hypoxia (aIH; three 5 min episodes, 10.5% O). Despite the importance of respiratory system neuroplasticity, and its dependence on estrogen in males, little is known about pLTF expression or mechanisms of estrogen signaling in females. Here, we tested the hypotheses that (1) pLTF expression in young, gonadally intact female rats would be expressed during estrous cycle stages in which 17β-estradiol (E2) is naturally high (e.g., proestrus vs estrus), (2) pLTF would be absent in ovariectomized (OVX) rats and in physiological conditions in which serum progesterone, but not E2, is elevated (e.g., lactating rats, 3-10 d postpartum), and (3) acute E2 administration would be sufficient to restore pLTF in OVX rats. Recordings of phrenic nerve activity in female Sprague Dawley rats (3-4 months) revealed a direct correlation between serum E2 levels and pLTF expression in cycling female rats. pLTF was abolished with OVX, but was re-established by acute E2 replacement (3 h, intraperitoneal). To identify underlying E2 signaling mechanisms, we intrathecally applied BSA-conjugated E2 over the spinal phrenic motor nucleus and found that pLTF expression was restored within 15 min, suggesting nongenomic E2 effects at membrane estrogen receptors. These data are the first to investigate the role of ovarian E2 in young cycling females, and to identify a role for nongenomic estrogen signaling in any form of respiratory system neuroplasticity. Exposure to acute intermittent hypoxia induces phrenic long-term facilitation (pLTF), a form of spinal respiratory motor plasticity that improves breathing in models of spinal cord injury. Although pathways leading to pLTF are well studied in males and estradiol (E2) is known to be required, it has seldom been investigated in females, and underlying mechanisms of E2 signaling are unknown in either sex. We found that while ovariectomy abolished pLTF, it could be restored by acute systemic E2, or by intraspinal application of the membrane-impermeable E2 (BSA-conjugated E2; 15 min). The ability of nongenomic estrogen signaling within the cervical spinal cord to recover respiratory neuroplasticity in disorders of respiratory insufficiency suggests that membrane estrogen receptors may represent novel therapeutic targets to restore breathing in both sexes.
Topics: Animals; Estradiol; Estrogens; Estrous Cycle; Female; Long-Term Potentiation; Neuronal Plasticity; Ovariectomy; Phrenic Nerve; Rats; Rats, Sprague-Dawley; Respiratory Mechanics
PubMed: 28592693
DOI: 10.1523/JNEUROSCI.0433-17.2017 -
Experimental Neurology Jan 2022Intrapleural injections of cholera toxin B conjugated to saporin (CTB-SAP) selectively eliminates respiratory (e.g., phrenic) motor neurons, and mimics motor neuron...
Nonsteroidal anti-inflammatory drug (ketoprofen) delivery differentially impacts phrenic long-term facilitation in rats with motor neuron death induced by intrapleural CTB-SAP injections.
Intrapleural injections of cholera toxin B conjugated to saporin (CTB-SAP) selectively eliminates respiratory (e.g., phrenic) motor neurons, and mimics motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. Additionally, microglial density increases in the phrenic motor nucleus following CTB-SAP. This CTB-SAP rodent model allows us to study the impact of motor neuron death on the output of surviving phrenic motor neurons, and the underlying mechanisms that contribute to enhancing or constraining their output at 7 days (d) or 28d post-CTB-SAP injection. 7d CTB-SAP rats elicit enhanced phrenic long-term facilitation (pLTF) through the Gs-pathway (inflammation-resistant in naïve rats), while pLTF is elicited though the Gq-pathway (inflammation-sensitive in naïve rats) in control and 28d CTB-SAP rats. In 7d and 28d male CTB-SAP rats and controls, we evaluated the effect of cyclooxygenase-1/2 enzymes on pLTF by delivery of the nonsteroidal anti-inflammatory drug, ketoprofen (IP), and we hypothesized that pLTF would be unaffected by ketoprofen in 7d CTB-SAP rats, but pLTF would be enhanced in 28d CTB-SAP rats. In anesthetized, paralyzed and ventilated rats, pLTF was surprisingly attenuated in 7d CTB-SAP rats and enhanced in 28d CTB-SAP rats (both p < 0.05) following ketoprofen delivery. Additionally in CTB-SAP rats: 1) microglia were more amoeboid in the phrenic motor nucleus; and 2) cervical spinal inflammatory-associated factor expression (TNF-α, BDNF, and IL-10) was increased vs. controls in the absence of ketoprofen (p < 0.05). Following ketoprofen delivery, TNF-α and IL-10 expression was decreased back to control levels, while BDNF expression was differentially affected over the course of motor neuron death in CTB-SAP rats. This study furthers our understanding of factors (e.g., cyclooxygenase-1/2-induced inflammation) that contribute to enhancing or constraining pLTF and its implications for breathing following respiratory motor neuron death.
Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Death; Cholera Toxin; Ketoprofen; Long-Term Potentiation; Male; Microglia; Motor Neurons; Neuromuscular Diseases; Phrenic Nerve; Rats; Rats, Sprague-Dawley; Saporins
PubMed: 34634309
DOI: 10.1016/j.expneurol.2021.113892 -
Medicine Jul 2023The aim of this study is to utilize ultrasound to evaluate the normal cross-sectional area (CSA) of the phrenic nerve (PN), at the level of the anterior scalene muscle....
The aim of this study is to utilize ultrasound to evaluate the normal cross-sectional area (CSA) of the phrenic nerve (PN), at the level of the anterior scalene muscle. The study included 62 PNs in 31 healthy subjects (13 men, 18 women); mean age, 36.6 years; mean height, 161.1 cm; mean weight, 69.6 kg; and mean body mass index 25.8 kg/m2. High-resolution ultrasound images of the bilateral PNs were obtained by a radiologist with 15 years of experience in neuromusculoskeletal ultrasound. Three separate CSA measurements for the bilateral PNs bilaterally were obtained. Images were also reviewed by an experienced neurologist to evaluate for inter-rater variability. The mean CSA of the right PN was 0.54 mm2 ± 0.16. The mean CSA of the left PN was 0.53 mm2 ± 0.18. We believe that the reference values for the normal CSA of the PNs obtained in our study could help in the sonographic evaluation of PN enlargement, as it relates to the diagnosis of various diseases affecting the PN. Furthermore, knowledge of its location and size, at the level of the scalene muscle, could help prevent PN-related complications during interventional procedures in that area. Additionally, for each participant, demographic information of age and gender as well as body mass index, weight, and height were documented.
Topics: Male; Humans; Female; Adult; Phrenic Nerve; Ultrasonography; Neck Muscles; Reference Values; Healthy Volunteers
PubMed: 37505169
DOI: 10.1097/MD.0000000000034181 -
Journal of Physiology and Pharmacology... Aug 2013The aim was to investigate whether intravenous infusion of remifentanil would depress phrenic long term facilitation (pLTF) evoked by acute intermittent hypoxia (AIH) in...
The aim was to investigate whether intravenous infusion of remifentanil would depress phrenic long term facilitation (pLTF) evoked by acute intermittent hypoxia (AIH) in adult, male, urethane anaesthetized Sprague-Dawley rats, bilaterally vagotomized, paralyzed and mechanically ventilated. The experimental group received a remifentanil infusion (0.5 μg/kg/min i.v., n=12), whereas the control group (n=6) received saline. Rats were exposed to AIH protocol. Phrenic nerve amplitude (PNA), burst frequency (f) and breathing rhythm parameters (Ti, Te, Ttot) were analyzed during 5 hypoxias and at 15, 30, and 60 minutes after the final hypoxia, and compared to baseline values. At the end of the experiment, the infusion of remifentanil was stopped and phrenic nerve activity was compared to baseline values prior to remifentanil infusion. In the control group, peak phrenic nerve activity (pPNA) significantly increased at 60 min (T60, increase by 138.8±28.3%, p=0.006) after the last hypoxic episode compared to baseline values, i.e. pLTF was induced. In remifentanil treated rats, there were no significant changes in peak phrenic nerve activity at T60 compared to baseline values (decrease by 5.3±16.5%, p>0.05), i.e. pLTF was abolished. Fifteen minutes following cessation of remifentanil infusion, pPNA increased by 93.2±40.2% (p<0.05) and remained increased compared to pre-remifentanil-infusion values for more than 30 minutes, i.e. pLTF could be observed after cessation of the remifentanil infusion. In conclusion, the short acting μ-opioid receptor agonist, remifentanil, reversibly abolished phrenic long term facilitation in urethane anesthetized rats.
Topics: Analgesics, Opioid; Animals; Hypnotics and Sedatives; Hypoxia; Long-Term Potentiation; Male; Phrenic Nerve; Piperidines; Rats; Rats, Sprague-Dawley; Remifentanil
PubMed: 24101395
DOI: No ID Found -
Toxicon : Official Journal of the... Mar 2017Phospholipases A (PLAs) constitute a class of extensively studied toxins, isolated from snake venoms. Basic PLA isoforms mediate various toxicological effects, while the...
Exploring and understanding the functional role, and biochemical and structural characteristics of an acidic phospholipase A AplTx-I, purified from Agkistrodon piscivorus leucostoma snake venom.
Phospholipases A (PLAs) constitute a class of extensively studied toxins, isolated from snake venoms. Basic PLA isoforms mediate various toxicological effects, while the acidic isoforms generally have higher enzymatic activities, but do not promote evident toxic effects. The functions of these acidic isoforms in snake venoms are still not completely understood and more studies are needed to characterize the biological functions and diversification of acidic toxins in order to justify their abundant presence in these secretions. Recently, Lomonte and collaborators demonstrated, in a proteomic and toxicological study, high concentrations of PLAs in the venom of Agkistrodon piscivorus leucostoma. We have, herein, purified and characterized an acidic PLA from this snake venom, denominated AplTx-I, in order to better understand its biochemical and structural characteristics, as well as its biological effects. AplTx-I was purified using two chromatographic steps, in association with enzymatic and biological assays. The acidic toxin was found to be one of the most abundant proteins in the venom of A. p. leucostoma; the protein was monomeric with a molecular mass of 13,885.8 Da, as identified by mass spectrometry ESI-TOF and electrophoresis. The toxin has similar primary and tridimensional structures to those of other acidic PLAs, a theoretical and experimental isoelectric point of ≈5.12, and a calcium-dependent enzyme activity of 25.8985 nM/min/mg, with maximum values at 37 °C and pH 8.0. Despite its high enzymatic activity on synthetic substrate, AplTx-I did not induce high or significant myotoxic, coagulant, anticoagulant, edema, neuromuscular toxicity in mouse phrenic nerve-diaphragm preparations or antibacterial activities. Interestingly, AplTx-I triggered a high and selective neuromuscular toxicity in chick biventer cervicis preparations. These findings are relevant to provide a deeper understanding of the pharmacology, role and diversification of acidic phospholipase A isoforms in snake venoms.
Topics: Agkistrodon; Animals; Chickens; Crotalid Venoms; Mice; Molecular Weight; Neuromuscular Junction; Phospholipases A2; Phrenic Nerve; Protein Isoforms; Rats, Wistar
PubMed: 28063838
DOI: 10.1016/j.toxicon.2017.01.002 -
Journal of Neurology, Neurosurgery, and... Feb 1996Respiratory failure is a common and potentially life threatening complication in patients with Guillain-Barré syndrome. The incidence of phrenic nerve involvement and...
Respiratory failure is a common and potentially life threatening complication in patients with Guillain-Barré syndrome. The incidence of phrenic nerve involvement and the predictive value of phrenic nerve conduction and diaphragmatic needle EMG were studied in 40 patients with Guillain-Barré syndrome within the first three days of admission to hospital. The negative peak onset latency of the diaphragmatic compound muscle action potential (CMAP), and its amplitude, duration, and area were abnormal in 83%. The need for ventilation was correlated with diaphragmatic CMAP amplitude (P = 0.005), and area (P = 0.001), but not with latency or duration. Abnormalities in diaphragmatic needle EMG were found in 45%, mainly a decreased number of motor unit potentials. The abnormalities correlated with the need for ventilation (P = 0.013). Of the 40% who required ventilation, all had either abnormal phrenic conduction, abnormal diaphragmatic needle EMG, or both. Eighty one per cent of the ventilated patients had abnormal forced vital capacity on the day of the electrophysiological examination. The results indicate that phrenic nerve conduction studies and diaphragmatic EMG are useful in detecting respiratory involvement in patients with Guillain-Barré syndrome and in identifying those at risk of respiratory failure.
Topics: Adolescent; Adult; Aged; Electromyography; Female; Humans; Male; Median Nerve; Middle Aged; Neural Conduction; Phrenic Nerve; Polyradiculoneuropathy; Respiration
PubMed: 8708652
DOI: 10.1136/jnnp.60.2.191 -
Thorax Mar 1957
Topics: Diaphragm; Humans; Muscular Diseases; Phrenic Nerve; Respiration Disorders; Thoracic Diseases
PubMed: 13422397
DOI: 10.1136/thx.12.1.50 -
The Journal of Physiology Jun 1985Comparisons were made of the activities of the dorsal buccal branch (d.b.b.) of the facial nerve which innervates the alae nasi and of the alar muscles themselves (alae...
Comparisons were made of the activities of the dorsal buccal branch (d.b.b.) of the facial nerve which innervates the alae nasi and of the alar muscles themselves (alae nasi) with the activity of the phrenic nerve in eight anaesthetized, spontaneously breathing dogs during hypercapnia, end-expiratory airway occlusion, and chest wall compression, before and after vagotomy. Nerve and muscle activities were recorded from bipolar cuff or wire electrodes respectively and processed by a moving average technique. Peak facial d.b.b., alae nasi, and phrenic activity all increased linearly with hypercapnia. Airway occlusion prolonged the duration of phrenic as well as facial d.b.b. and alae nasi activity. A comparison of electrical activity during unoccluded and occluded inspirations indicated a facilitation of phrenic activity but an inhibition of both facial d.b.b. and alae nasi activities associated with volume feed-back. These volume-related feed-back effects were abolished by vagotomy. After vagotomy, peak facial d.b.b., alae nasi and phrenic activities increased during lower chest wall compression and decreased with upper chest wall compression when compared to the preceding control breath. Alae nasi and facial nerve activities, like that of the phrenic nerve, respond to respiratory chemical and reflex influences.
Topics: Anesthesia, Intravenous; Animals; Carbon Dioxide; Dogs; Electromyography; Facial Muscles; Facial Nerve; Nose; Phrenic Nerve; Respiration; Ribs; Tidal Volume; Vagotomy
PubMed: 3926993
DOI: 10.1113/jphysiol.1985.sp015715