The flat slabs allow proper orientation of the nerve tissue in the mold before the resin hardens. Selected sections were stained with Toluidine Blue. Ten images from each nerve specimen were analyzed using the freely available Neurocounter morphometric software 1 by two researchers blind to nerve identity to count the number of myelinated axons. This software recognizes myelinized axons as objects and automatically evaluates their number as well as several morphometric parameters.
The animals were anesthetized by means of intraperitoneal injection of a combination anesthetic containing ketamine and xylazine at a total dose of 1. After laparotomy, the liver was gently pushed away to expose the diaphragm muscle.
Bipolar stainless steel electrodes in the form of small parallel hooks distance of 3 mm were inserted into the diaphragm muscle. Signals were recorded from both the contralateral and ispilateral hemidiaphragms. Similar results were achieved using WB analysis Figure 1C. Figure 1. Image of C4 spinal cord segment showing expression of retrogradely labeled phrenic motoneurons in contralateral phrenic nucleus PN 8 days after PhN ligation and bilateral injection of retrograde tracer Fluorogold FG into diaphragm A.
Immunolabeling with SMI antibody a specific marker for neurofilaments was performed to visualize axons in the phrenic nerve. Figure 2. We next examined anterograde degeneration of the phrenic nerve below the ligation point Figures 3A,B. Axons of the contralateral phrenic nerve showed the typical morphological characteristics of intact myelinated axons. In the ipsilateral nerve, evident axonal loss was detected, and the tissue contained numerous disintegrated myelin structures typical for Wallerian degeneration.
Axonal counts indicated massive loss of nerve fibers in the ipsilateral nerve Figure 3. Eight days continuous phrenic nerve PhN ligation caused axonal degeneration in the ipsilateral PhN and abnormalities at neuromuscular junctions NMJs in the ipsilateral hemidiaphragm.
Noticeable axonal degeneration in PhN below ligation point ipsilateral A. Quantitative analysis revealed significantly lower number of intact axons in ipsilateral than in contralateral PhN B. Numbers of intact, partially denervated and fully denervated arrowhead NMJs in ipsilateral vs.
Figure 4. Representative confocal images showing morphological changes at diaphragm NMJs after unilateral PhN ligation. Ipsilateral hemidiaphragm shows NMJ abnormalities represented by partial B,E , arrows or complete denervation F, arrows. Marked preterminal axon thinning is visible in ipsilateral diaphragm B, asterisk.
This was further supported by pathological alterations at the diaphragm NMJs. As shown in Figure 3C , differences in numbers of NMJs intact, partially or fully denervated between the contra- and ipsilateral hemidiaphragm were significant. The number of intact NMJs was three-fold lower in the ipsilateral than the contralateral hemidiaphragm. These alterations were identified as partial Figures 4B,E , arrows or full denervation Figure 4F , arrow. However, the decrease was nine — times stronger in the ipsilateral than the contralateral hemidiaphragm 0.
While there was no difference in breathing frequency on both sides of the diaphragm, the overall muscle activity differed dramatically.
PhN ligation caused rapid and pronounced reduction in EMG activity amplitude on the ipsilateral side, while the amplitude of EMG on the contralateral side slightly increased Figure 5B. Eight days after continuous PhN ligation, EMG on both sides of the diaphragm muscles changed markedly: on the ipsilateral side the residual activity disappeared, whereas on the contralateral side there was several-fold increase in EMG amplitude while breathing frequency remained unchanged Figure 5C.
This increase in EMG activity was due to an enormous increase in muscle fibers activity, but not to prolongation of that activity, since the duration of the rhythmic contractions did not change significantly. Figure 5. Phrenic nerve injury is a well-established model, since the nerve ligation or transection deactivates descending excitatory drive from the phrenic motoneurons to the diaphragm. Experimental and clinical data have shown that PhN injury can have significant negative effects on diaphragm and lung, affecting their function.
Recently published data revealed that the complete paralysis of the unilateral diaphragm could influence the loss of vital capacity and total lung capacity in the aged rats Ding et al.
In the present study we speculated, whether changes in the ipsilateral hemidiaphragm e. To answer this fundamental question, immunohistochemical and histological staining, Western blotting, Elisa and electrophysiology were used.
However, its functional importance in the respiratory pathway brainstem-phrenic motoneurons — phrenic nerve — diaphragm still remains unclear.
We have previously reported that the premotor bulbospinal respiratory pathway connecting the bulbar respiratory centers with the motoneurons of the phrenic nucleus in dog and rat is nitrergic Marsala et al. The phrenic nerves are generally considered as motor nerves whose primary function is to supply motor innervation to the diaphragm Mantilla and Sieck, By means of a retrograde tracing technique we provide data showing direct projection from the hemidiaphragm to the contralateral FG-labeled phrenic motoneurons of the phrenic nucleus.
Recently published data indicate that 2 weeks of tetrodoxin TTX phrenic nerve blockade significantly increased both the total phrenic motoneuron surface area and the dendritic surface area Mantilla et al.
These authors suggest that ipsilateral phrenic motoneuron morphological adaptations are consistent with normalization of motoneuron excitability following prolonged alterations in motoneuron activity.
Phrenic motoneuron structural plasticity is probably more dependent on motoneuron activity or descending input than muscle fiber activity. Furthermore, motoneurons have high metabolic activity associated with neurotransmission Agar and Durham, Murphy et al. As reported in previous studies Saji and Miura, ; Marsala et al. The ability of motoneurons to handle calcium may also be important in determining their response to stimulation Chao et al.
Previous studies from our and other labs Lips and Keller, ; Palecek et al. Phrenic motor neuron loss and subsequent diaphragm NMJ abnormalities are well described in various experimental models Prakash et al. Bashan et al. The pattern of diaphragm denervation has been studied in the central region of the diaphragm, particularly innervated by phrenic motor neurons located at the C5 level of the spinal cord Laskowski and Sanes, Although we found marked reduction in cGMP levels in both the contra- and ipsilateral hemidiaphragm, the decrease was significantly greater nine times on the side of the ligation.
Currently, the functional effects of unilateral 8-day continuous PhN ligation are unknown but significant effects can be inferred from recently published experimental study Ding et al. Reid et al. Abraham et al.
Our results support sGC-dependent cGMP signaling in the lower respiratory pathway, but the precise target of cGMP action in the diaphragm muscle still remains unclear. Bartsch and Ivanov investigated the network of interactions between the brain, cardiac and respiratory system, and demonstrated that a network approach to physiological interactions is necessary to understand how modulations in the regulatory mechanism of individual systems translate into reorganization of physiological interactions across the organism.
It is known that the function of inspiratory muscles is very important in the breathing process, because they are required to contract repetitively without interruption throughout life. Our results show remarkable unilateral silencing of EMG activity immediately after PhN ligation, and the failure of recovery of EMG activity in the ipsilateral diaphragm muscle 8 days after pathological continuous nerve ligation.
We also show spontaneous and increased EMG activity in the contralateral hemidiaphragm. Such enhancement, recorded 8 days after unilateral PhN ligation, seems to be the result of increased muscle fiber activity. Mantilla and Sieck observed the absence of EMG activity in the ipsilateral diaphragm immediately and 3 days after C2 hemisection using diaphragm electrodes.
They recorded progressive increase in the proportion of spontaneous recovery of EMG activity ispilaterally to C2 hemisection over time. These findings are inconsistent with previously reported spontaneous ipsilateral increase in EMG activity. Vinit et al. These authors also indicated that ipsilateral phrenic nerve reactivation was greater at 3 months compared with 7 days post-SCI, and that it was enhanced after contralateral phrenicotomy.
There was gradual recovery of rhythmic diaphragm muscle activity ipsilaterally to cervical spinal cord injury over time, consistent with neuroplasticity and strengthening of spared, contralateral descending premotor input to the phrenic motoneurons Mantilla et al.
This suggests that spontaneous recovery of the respiratory pathway may depend on post-lesional time. The datasets generated for this study are available on request to the corresponding author. LH and NL wrote the manuscript. All authors contributed to the manuscript revision, read and approved the submitted version. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Abraham, R. Such information is important for nerve stimulation, because myelinated nerve fibres have a much lower amplitude-duration threshold upon nerve stimulation than non-myelinated fibres 41 , Typical stimulation protocols for transvenous phrenic nerve stimulation can vary up to a hundred-fold in intensity 0. If applied for central sleep apnoea, the stimulation should target the myelinated fibres and should, therefore, be accomplished with the lowest possible amplitude-duration thresholds that result in the intended rhythmic activation of the diaphragm.
This is necessary to prevent any undesired stimulation of the nonmyelinated catecholaminergic fibres that are also present within the phrenic nerve.
This concern is relevant, because direct electric stimulation of the right subclavian ansa did elevate noradrenaline and cAMP concentrations in plasma harvested in the coronary sinus of dogs Such a catecholaminergic stimulation of the heart may, therefore, further increase the chronic upregulation of sympathetic activity that is already present in patients with central sleep apnoea 44 , 45 and that is associated with increased mortality these patients 15 , 16 , Galen, C.
Translated from the Greek with an introduction and commentary by Margaret Tallmadge May. Mendelsohn, A. Cervical variations of the phrenic nerve. Article PubMed Google Scholar. Loukas, M. Surgical anatomy of the accessory phrenic nerve. Luschka, H. Der nervus phrenicus des menschen; eine monographie. Laupp, Mitchell, G. The innervation of the heart. Br Heart J 15 , — Than, M. Variations in the formation of the cardiac plexus—a study in human foetuses.
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Phrenic nerve stimulation for the treatment of central sleep apnea. Zhang, X. Safety and feasibility of chronic transvenous phrenic nerve stimulation for treatment of central sleep apnea in heart failure patients.
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Presence of functional sarcoplasmic reticulum in the developing heart and its confinement to chamber myocardium. Hikspoors, J. Development of the human infrahepatic inferior caval and azygos venous systems. Verlinden, T.
Morphology of the human cervical vagus nerve: implications for vagus nerve stimulation treatment. Kumar, P. Peripheral chemoreceptors: function and plasticity of the carotid body.
Canella, C. Anatomical study of phrenic nerve using ultrasound. Song, A. Development of the rat phrenic nerve and the terminal distribution of phrenic afferents in the cervical cord. Anat Embryol Berl , — Muller Botha, G. The anatomy of phrenic nerve termination and the motor innervation of the diaphragm.
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Am J Obstet Gynecol , — Paintal, A. A study of right and left atrial receptors. Luchner, A. Interactions between the sympathetic nervous system and the cardiac natriuretic peptide system. Harrison, R. Observations on the venous system in certain Pinnipedia and Cetacea.
Proceedings of the Zoological Society of London , — Ya, J. Expression of the smooth-muscle proteins alpha-smooth-muscle actin and calponin, and of the intermediate filament protein desmin are parameters of cardiomyocyte maturation in the prenatal rat heart. Anat Rec , — Roux, N. Faithful innervation of the diaphragm is essential for normal muscular function, but our understanding of this process is less clear than it is for other muscular systems. Here p. The authors use a genetic approach to study the involvement of the Sema3—Npn-1 signalling pathway in phrenic nerve targeting and fasciculated growth during establishment of the diaphragm in mice.
The authors show that systemic ablation of Npn-1 does not impact the initial branch formation and guidance of the phrenic nerves. However, Sema3—Npn-1 signalling does govern phrenic nerve fasciculation and branching during innervation of the costal muscles of the diaphragm in a cell autonomous manner. Interestingly, phrenic axons misproject into the central tendon region of the diaphragm and innervate ectopic muscles in the absence of Sema3—Npn-1 signalling, leading the authors to hypothesise that additional factors released by misprojecting growth cones cause ectopic myocyte fusion.
Next, the authors demonstrate that Slit2 and Robo1, members of an axon-guidance-cue family, are expressed in phrenic motor neurons and migrating myoblasts, respectively, during diaphragm innervation. Further, Slit1 and Slit2 exert an attractive effect on primary myoblasts. This paper provides important new insights into both phrenic nerve development and myocyte fusion.
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