Phrenic Afferents Research Articles

Diaphragm Pacing Elicits Ventilatory Plasticity in a Rodent Model of Spinal Cord Injury

Cervical spinal cord injury (cSCI) often results in respiratory impairments requiring the use of chronic mechanical ventilation (MV). Diaphragm pacing (DP) is an emerging clinical intervention designed to combat the deleterious effects of chronic MV by electrically stimulating the diaphragm muscle to maintain ventilation. Anecdotal clinical evidence suggests a rehabilitative benefit of DP; however, current clinical practice is limited by a lack of controlled investigations. Thus, the purpose of this ongoing study was to determine if daily DP could be used as a rehabilitative tool following cSCI. We hypothesized that following cSCI, DP would 1) induce a long-lasting increase in tidal volume 60 minutes after stimulation, and 2) result in sustained improvements in tidal volume after four days of daily DP in awake, behaving rats. To test these hypotheses, we implanted custom electromyography electrodes in the mid-costal region of the left and right hemidiaphragms of male Sprague-Dawley rats. One week later, all animals received a left C2 hemisection (C2Hx; n=14) and a subset also received a left C4-6 dorsal rhizotomy (n=3). One week after injury, rats received stimulation on the paralyzed hemidiaphragm (biphasic 30Hz, 0.8mA, 80 μs pulse width) in phase with breathing (Pacing n=5, Rhizotomy n=3), or served as controls (n=6). We delivered DP to animals within plethysmography chambers in an intermittent pattern (5 minutes on/5 minutes off) for 1 hour/day over 4 consecutive days. Using whole body plethysmography, we recorded ventilation before, during, and for 60 minutes after to examine the acute effects of DP. Additionally, we measured ventilation during normoxia (21% FiO2) and respiratory challenge (10.5% FiO2, 7% FiCO2) before and after C2Hx, and 24 hours after four days of DP to assess the rehabilitative potential of pacing. DP increased tidal volume (% change from pre-pacing) compared to control animals during stimulation (P =0.021). Animals receiving DP displayed an elevated tidal volume compared to controls for at least 60 minutes after DP (P=0.015). When compared to control animals 24 hours after the completion of the pacing protocol, DP animals displayed an increased tidal volume (mL/breath/100g) during both normoxia (P =0.006) and respiratory challenge (P =0.019). In contrast, animals pre-treated with rhizotomy showed a similar tidal volume (mL/breath/100g) compared to controls (normoxia: P=0.597; respiratory challenge: P=0.191), suggesting that DP-induced ventilatory plasticity requires phrenic afferents. Taken together, our data suggest that diaphragm pacing has rehabilitative potential and elicits functional respiratory improvements in rodents following C2Hx. Acknowledgements: This work was supported by funding from the National Institute of Health, grant numbers: R00 HL143207-01 (KAS), UL1TR001436 (TCH) and the APTA Acute Care Physical Therapy Seed Grant (TCH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intermittent activation of large diameter phrenic afferents induces phrenic motor plasticity

We recently showed that in anesthetized rats, 30 minutes of electrical stimulation applied to the diaphragm muscle induces phrenic afferent dependent plasticity characterized by a long-lasting increase in diaphragm electromyography amplitude. Our previous data also indicate that large diameter phrenic afferents may be most effective at eliciting plasticity. Here we directly test the hypothesis that intermittent activation of large diameter phrenic afferents induces phrenic motor plasticity. In urethane anesthetized and mechanically ventilated male Sprague-Dawley rats, we isolated the left and right phrenic nerves. We applied inspiratory-triggered electrical stimulation (biphasic; 40Hz; 160mA) to one phrenic nerve in a sustained (n= 5, 25 minutes) or intermittent (n=5; 5 X 5-minute episodes) pattern, and recorded contralateral motor output before, during, and for 60 minutes after stimulation. We used a narrow pulse width (0.1ms) to preferentially activate large diameter afferents. At 60 minutes, rats receiving sustained stimulation showed no difference in contralateral phrenic motor amplitude compared to time controls not receiving stimulation (n=5; P=0.1320). In contrast, rats receiving intermittent stimulation had an increase at 60 minutes compared to time controls (P=0.0005) and sustained stimulation (P=0.0024). This suggests that intermittent, but not sustained, phrenic nerve stimulation induces contralateral phrenic motor plasticity. To test the hypothesis that phrenic afferents are necessary for intermittent induced plasticity, separate animals received either a left, C3-C6 dorsal rhizotomy (n=3) or laminectomy (n=4) prior to intermittent stimulation. Laminectomy controls expressed a long-lasting increase in contralateral phrenic motor amplitude compared to time controls (n=5) at 60 minutes after stimulation (P=0.0112). Animals receiving a dorsal rhizotomy were not different than time controls at 60 minutes (P=0.4058), indicating phrenic afferents are necessary for the induction of plasticity. These data show for the first time that intermittent activation of large diameter phrenic afferents induces phrenic motor plasticity. A detailed understanding of afferent mediated plasticity will be beneficial in the development of therapeutic techniques utilizing electrical stimulation of the diaphragm muscle (i.e., diaphragm pacing). This work was supported by funding from the National Institute of Health, grant numbers: R00 HL143207-01 (KAS). The authors declare no competing financial interests. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Potentiated respiratory metaboreflex in older males and females: afferent or efferent mechanisms?

Potentiated respiratory metaboreflex in older males and females: afferent or efferent mechanisms?

Diaphragm stimulation elicits phrenic afferent-induced neuromuscular plasticity

We hypothesized that activation of phrenic afferents induces diaphragm motor plasticity. In anesthetized and spontaneously breathing rats we delivered 40 Hz, low threshold (twitch and 1.5X twitch threshold), inspiratory-triggered stimulation to the left hemidiaphragm for 30 min to activate ipsilateral phrenic afferents. Diaphragm amplitude ipsilateral and contralateral to stimulation were increased for 60 min following both currents compared to time controls not receiving stimulation. Diaphragm stimulation was repeated in laminectomy controls or following a unilateral C3-C6 dorsal rhizotomy to eliminate phrenic afferent volleys. Laminectomy controls expressed neuromuscular plasticity post-stimulation. In contrast, ipsilateral and contralateral diaphragm amplitude following dorsal rhizotomy was lower than laminectomy controls and no different than time controls, suggesting diaphragm motor plasticity was not induced post-rhizotomy. Our results indicate that diaphragm stimulation induces a novel form of plasticity in the phrenic motor system which requires phrenic afferent activation. Respiratory motor plasticity elicited by diaphragm stimulation may have value as a therapeutic strategy to improve diaphragm output in neuromuscular conditions.

Acute Diaphragm Stimulation Elicits Phrenic Afferent Induced Neuromuscular Plasticity

Activation of phrenic afferents increases phrenic motor output and induces short‐term plasticity. Here we tested the hypothesis that activation of phrenic afferents via acute diaphragm stimulation induces neuromuscular plasticity within the phrenic motor system. Bilateral EMG activity was recorded from the mid‐costal diaphragm of anesthetized and spontaneously breathing rats. Inspiratory‐triggered stimulation was delivered to the left diaphragm for 30 minutes at twitch threshold (n=7; 0.6 ± 0 mA) or 1.5X twitch threshold (n=7; 1.0 ± 0.12 mA), and output was recorded for 60 minutes post‐stimulation. Arterial blood gas samples revealed that PaCO2 was no different than baseline during or after stimulation (P=0.2204) or across experimental groups (P=0.3534). Diaphragm amplitude ipsilateral and contralateral to stimulation was increased 60 minutes following threshold (P=0.0021 and P=0.0096) and 1.5X threshold (P=0.0003 and P=0.0448) stimulation compared to time controls (n=7). Separate groups of rats received 1.5X threshold stimulation and either a C3‐C6 laminectomy (n=6; 0.8 ± 0.06mA) or a left C3‐C6 dorsal rhizotomy (n=6; 0.7 ± 0.10mA) to prevent afferent input from reaching the spinal cord. Laminectomy controls continued to express neuromuscular plasticity ipsilateral (P=0.0017) and contralateral (P=0.0076) to stimulation compared to time controls. By contrast, following unilateral dorsal rhizotomy, ipsilateral and contralateral diaphragm amplitude 60 minutes post‐stimulation was lower than laminectomy controls (P=0.0107 and P=0.0115) and no different than time controls (P=0.5692 and P=0.4784), suggesting neuromuscular plasticity was not induced. Our results indicate that acute diaphragm stimulation induces a novel form of neuromuscular plasticity which requires activation of phrenic afferents. We hypothesize that phrenic afferent induced neuromuscular plasticity may be valuable as a therapeutic strategy following spinal cord injury.

Phrenic afferent activation modulates cardiorespiratory output in the adult rat.

Phrenic afferents project to brainstem areas responsible for cardiorespiratory control and the mid-cervical spinal cord containing the phrenic motor nucleus. Our purpose was to quantify the impact of small- and large-diameter phrenic afferent activation on phrenic motor output. Anesthetized and ventilated rats received unilateral phrenic nerve stimulation while contralateral phrenic motor output and blood pressure were recorded. Twelve currents of 40-Hz inspiratory-triggered stimulation were delivered (20 s on, 5 min off) to establish current response curves. Stimulation pulse width was varied to preferentially activate large-diameter phrenic afferents (narrow pulse width) and recruit small-diameter fibers (wide pulse width). Contralateral phrenic amplitude was elevated immediately poststimulation at currents above 35 µA for wide and 70 µA for narrow pulse stimulation when compared with animals not receiving stimulation (time controls). Wide pulse width stimulation also increased phrenic burst frequency at currents ≥35 µA, caused a transient decrease in mean arterial blood pressure at currents ≥50 µA, and resulted in a small change in heart rate at 300 µA. Unilateral dorsal rhizotomy attenuated stimulation-induced cardiorespiratory responses indicating that phrenic afferent activation is required. Additional analyses compared phrenic motor amplitude with output before stimulation and showed that episodic activation of phrenic afferents with narrow pulse stimulation can induce short-term plasticity. We conclude that the activation of phrenic afferents 1) enhances contralateral phrenic motor amplitude when large-diameter afferents are activated, and 2) when small-diameter fibers are recruited, the amplitude response is associated with changes in burst frequency and cardiovascular parameters.NEW & NOTEWORTHY Acute, inspiratory-triggered stimulation of phrenic afferents increases contralateral phrenic motor amplitude in adult rats. When small-diameter afferents are recruited, the amplitude response is accompanied by an increase in phrenic burst frequency, a transient decrease in mean arterial blood pressure, and a slight increase in heart rate. Repeated episodes of large-diameter phrenic afferent activation may also be capable of inducing short-term plasticity.

Electrical activation of phrenic afferent neurons evokes phrenic motor plasticity following cervical spinal cord injury

Anatomical data from our laboratory indicate that phrenic afferent fibers can project across the spinal midline and neurophysiological data indicate that electrical activation of phrenic afferents can increase contralateral phrenic motor output. We have also observed that following chronic unilateral C2 hemisection (C2Hx) phrenic nerve afferent fibers undergo significant anatomical remodeling. Here we tested the hypothesis that following chronic C2Hx, activation of ipsilateral (same side as injury) phrenic afferent neurons would induce contralateral (opposite to injury) phrenic motor plasticity. Twelve weeks after unilateral C2Hx (n=9) or C2 laminectomy (n=8), adult Sprague‐Dawley rats were anesthetized, vagotomized, and ventilated to enable phrenic motor output to be studied under controlled conditions. The phrenic nerve ipsilateral to C2Hx or laminectomy was electrically stimulated using a bipolar electrode while contralateral phrenic motor output was recorded. Rats received 30 minutes of inspiratory triggered stimulation (90μA, 40pps, 1ms pulse width) and contralateral phrenic motor output was recorded for one hour. Analysis of contralateral burst amplitude using a two‐way repeated measures ANOVA revealed a significant effect of time (F3,45=9.505, P<0.0001), but not treatment (C2Hx vs. laminectomy, F1,15=0.1577, P=0.6969). Thus, phrenic burst amplitude was significantly changing over the 60 minute post‐stimulation, and both experimental groups had increased phrenic burst amplitude during this period (P<0.05 vs. pre‐stimulation baseline). These results indicate that electrical activation of phrenic nerve afferent fibers can induce a sustained increased (facilitation) of contralateral phrenic motor output after spinal cord injury. The response was not statistically different in the laminectomy group, which indicates that the phrenic afferent‐induced facilitation does not require conditions induced by the spinal cord injury. The ability of phrenic afferent neurons to evoke phrenic motor plasticity after spinal cord injury suggests that these afferent pathways could be important to rehabilitation of the diaphragm motor system post‐injury.Support or Funding InformationK99HL143207‐01 (KS), F32NS095620‐01 (KS), T32HL134621‐01A1 (LBW), SPARC OT2 OD023854 (DDF)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phrenic afferent projections to the cervical spinal cord in spinal intact and spinal injured rats

The phrenic nerve contains both myelinated and unmyelinated afferent projections. Relatively little is known, however, regarding the spinal distribution of these sensory projections. Accordingly, our first goal was to create a map of phrenic afferent projections throughout the cervical spinal cord. Afferents were traced via prolonged phrenic nerve soak with Cascade Blue (CB) in adult Sprague‐Dawley rats. Spinal tissues were harvested, sectioned (40μm) from C2–C7, and processed with routine immunochemical methods. Extensive labeling of afferent projections was observed throughout the ipsilateral dorsal horn (laminae II and III) and intermediate grey region (laminae IV and V) in segments C2–C6. Phrenic afferent projections were crossed the spinal midline in 13% of the tissue sections, and were occasionally observed to reach the contralateral dorsal horn (laminae III). Robust phrenic motoneuron (PhrMN) labeling was observed from C3–C5, and PhrMN dendrites projected across the spinal midline in 30% of tissue sections from C3–C5. Our second objective was to determine if high cervical spinal cord injury (SCI) induced anatomical plasticity in the phrenic sensorimotor system. The phrenic nerve was traced using the same CB method in Sprague‐Dawley rats at 12 weeks following lateral hemisection of the C2 spinal cord. Preliminary analysis indicates that following SCI, phrenic afferents and PhrMN dendrites project across the spinal midline at approximately the same frequency as in spinal‐intact rats (p>0.36). However, we noted that the likelihood of observing phrenic afferent projections extending to the region of the ipsilateral phrenic motor nucleus (laminae VIII‐IX) was increased after SCI (p<0.05). By combining CB labeling with Choline acetyltransferase (ChAT) immunostaining, we verified the presence of laminae VIII‐IX phrenic afferents by observing CB‐positive and ChAT negative fibers. We conclude that phrenic sensory afferent projections 1) are prominent in the ipsilateral C3–C5 dorsal horn and intermediate grey region, 2) can project to the contralateral C3–C5 dorsal horn, and 3) undergo remodeling after high cervical SCI.Support or Funding InformationSPARC OT2 OD023854 (DDF), 1 R01 NS080180‐01A1 (DDF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phrenic afferent stimulation modulates cardiorespiratory output in the adult rat

Anatomical work from our laboratory indicates that small and large diameter phrenic nerve afferent fibers localize to the dorsal laminae, project throughout the ipsilateral spinal gray matter and occasionally reach the contralateral dorsal horn. In ongoing experiments, we are testing the hypothesis that activation of phrenic afferent neurons modulates contralateral phrenic motor output and arterial blood pressure in the adult rat. In anesthetized and ventilated adult Sprague‐Dawley rats, one phrenic nerve was electrically stimulated using a bipolar electrode, while respiratory bursting in the contralateral phrenic nerve and arterial blood pressure were recorded. Inspiratory triggered phrenic nerve stimulation was delivered for 20 seconds (approximately 18 breaths) at 40pps using a narrow (0.1 ms) pulse width to target activation of large diameter myelinated axons, or a wide (1.0 ms) pulse width to recruit small diameter myelinated and unmyelinated afferents. The experimental protocol consisted of eight currents (10, 25, 50, 70, 90, 120, 160, 220μA) delivered in a random order, each separated by a 5 minute recovery period (i.e. no stimulation). Each stimulus current caused a progressive increase in contralateral phrenic amplitude during the stimulation period. Phrenic amplitude remained above baseline for at least 1 minute after the cessation of stimulation. At lower currents (<50μA), the wide pulse stimulation was more effective at increasing contralateral phrenic output compared to the narrow pulse stimulation, while higher currents (>50μA) elicited a similar increase in contralateral amplitude regardless of pulse width. In addition, phrenic nerve stimulation was associated with a brief decrease, followed by an increase in mean arterial blood pressure during stimulation. In separate rats, a unilateral dorsal root rhizotomy (C3, C4, C5) was performed prior to phrenic nerve stimulation to eliminate afferent input to the spinal cord. This treatment prevented both the increase in contralateral phrenic amplitude and blood pressure at all currents and both pulse widths, indicating that the observed responses are due to activation of phrenic afferents. These preliminary results confirm that activation of phrenic afferents can modulate the activity of the contralateral phrenic motor pool, and suggest that phrenic afferents are capable of initiating phrenic motor plasticity.Support or Funding Information1F32NS095620‐01 (KS), T32‐ND043730 (MS), SPARC OT2 OD023854 (DDF), 1 R01 NS080180‐01A1 (DDF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Anatomy and physiology of phrenic afferent neurons.

Large-diameter myelinated phrenic afferents discharge in phase with diaphragm contraction, and smaller diameter fibers discharge across the respiratory cycle. In this article, we review the phrenic afferent literature and highlight areas in need of further study. We conclude that 1) activation of both myelinated and nonmyelinated phrenic sensory afferents can influence respiratory motor output on a breath-by-breath basis; 2) the relative impact of phrenic afferents substantially increases with diaphragm work and fatigue; 3) activation of phrenic afferents has a powerful impact on sympathetic motor outflow, and 4) phrenic afferents contribute to diaphragm somatosensation and the conscious perception of breathing. Much remains to be learned regarding the spinal and supraspinal distribution and synaptic contacts of myelinated and nonmyelinated phrenic afferents. Similarly, very little is known regarding the potential role of phrenic afferent neurons in triggering or modulating expression of respiratory neuroplasticity.

Anatomical Characterization of Phrenic Afferent Projections Following Cervical Spinal Cord Injury

The phrenic nerve has a rich component of sensory afferents, and there is some evidence that activation of these afferents can affect neuroplasticity in the phrenic motor system after cervical spinal cord injury (SCI). We are presently examining the neuroanatomical substrate that could enable phrenic afferent modulation of the spinal phrenic circuitry following high cervical SCI. Cholera toxin‐β (CT‐β) was applied to the cut end of the phrenic nerve to label spinal phrenic afferent projections in spinal‐intact adult Sprague‐Dawley rats and at 2‐8 wks‐ or 1 yr after lateralized C2 spinal hemisection (C2Hx). Cervical segments (C3‐C5) were also stained for C‐fiber projections using calcitonin gene related peptide (CGRP) and isolectin‐IB4. In spinal‐intact animals, CT‐β ‐labeled phrenic afferent projections terminated primarily in the ipsilateral mid‐cervical dorsal horn (laminae II‐III) and the intermediate grey matter (laminae IV‐V). CGRP and IB4 showed positive labeling in laminae I‐II. Coupling CT‐β labeling with transynaptic labeling of the phrenic motor circuit (via pseudorabies virus) demonstrated co‐labeling of afferent projections and spinal interneurons that are connected to the phrenic circuit. Contralaterally projecting phrenic afferents were not observed in spinal intact rats; however, axonal extensions across the midline were observed one year after C2HX. These early findings demonstrate primary afferent relationships with the spinal phrenic circuit that may play a role in post‐SCI respiratory neuroplasticity.Funding: NIH 1R01NS080180 (DDF), State of Florida Brain and Spinal Cord Injury Research Program

Autonomic adjustments to exercise in humans.

Autonomic nervous system adjustments to the heart and blood vessels are necessary for mediating the cardiovascular responses required to meet the metabolic demands of working skeletal muscle during exercise. These demands are met by precise exercise intensity-dependent alterations in sympathetic and parasympathetic nerve activity. The purpose of this review is to examine the contributions of the sympathetic and parasympathetic nervous systems in mediating specific cardiovascular and hemodynamic responses to exercise. These changes in autonomic outflow are regulated by several neural mechanisms working in concert, including central command (a feed forward mechanism originating from higher brain centers), the exercise pressor reflex (a feed-back mechanism originating from skeletal muscle), the arterial baroreflex (a negative feed-back mechanism originating from the carotid sinus and aortic arch), and cardiopulmonary baroreceptors (a feed-back mechanism from stretch receptors located in the heart and lungs). In addition, arterial chemoreceptors and phrenic afferents from respiratory muscles (i.e., respiratory metaboreflex) are also capable of modulating the autonomic responses to exercise. Our goal is to provide a detailed review of the parasympathetic and sympathetic changes that occur with exercise distinguishing between the onset of exercise and steady-state conditions, when appropriate. In addition, studies demonstrating the contributions of each of the aforementioned neural mechanisms to the autonomic changes and ensuing cardiac and/or vascular responses will be covered.

Functional and histopathological identification of the respiratory failure in a DMSXL transgenic mouse model of Myotonic Dystrophy

SUMMARYAcute and chronic respiratory failure is one of the major and potentially life-threatening features in individuals with myotonic dystrophy type 1 (DM1). Despite several clinical demonstrations showing respiratory problems in DM1 patients, the mechanisms are still not completely understood. This study was designed to investigate whether the DMSXL transgenic mouse model for DM1 exhibits respiratory disorders and, if so, to identify the pathological changes underlying these respiratory problems. Using pressure plethysmography, we assessed the breathing function in control mice and DMSXL mice generated after large expansions of the CTG repeat in successive generations of DM1 transgenic mice. Statistical analysis of breathing function measurements revealed a significant decrease in the most relevant respiratory parameters in DMSXL mice, indicating impaired respiratory function. Histological and morphometric analysis showed pathological changes in diaphragmatic muscle of DMSXL mice, characterized by an increase in the percentage of type I muscle fibers, the presence of central nuclei, partial denervation of end-plates (EPs) and a significant reduction in their size, shape complexity and density of acetylcholine receptors, all of which reflect a possible breakdown in communication between the diaphragmatic muscles fibers and the nerve terminals. Diaphragm muscle abnormalities were accompanied by an accumulation of mutant DMPK RNA foci in muscle fiber nuclei. Moreover, in DMSXL mice, the unmyelinated phrenic afferents are significantly lower. Also in these mice, significant neuronopathy was not detected in either cervical phrenic motor neurons or brainstem respiratory neurons. Because EPs are involved in the transmission of action potentials and the unmyelinated phrenic afferents exert a modulating influence on the respiratory drive, the pathological alterations affecting these structures might underlie the respiratory impairment detected in DMSXL mice. Understanding mechanisms of respiratory deficiency should guide pharmaceutical and clinical research towards better therapy for the respiratory deficits associated with DM1.

Cardiorespiratory responses to exercise in CHF: a conspiracy of maladaptation

Chronic heart failure (CHF) patients have markedly impaired exercise performance in the face of reduced cardiac output, augmented sympathetically mediated systemic vasoconstriction and an excessive hyperventilation secondary to inefficient pulmonary gas exchange and excessive drive to breathe. Olson et al. in a recent article in The Journal of Physiology (Olson et al. 2010) have now shed light on this complex, maladaptive response in CHF by demonstrating that these patients experienced marked increases in stroke volume and cardiac output, and in limb vascular conductance and blood flow, during submaximal exercise when their work of breathing (WOB) was reduced via a mechanical ventilator (MV). There is much to be admired in their experimental design, including use of thermal dilution measures of limb blood flow and oesophageal pressures to quantify WOB. Furthermore, the use of MV to unload the respiratory muscles served to unmask the cardiovascular effects of the normally occurring excessive WOB in these difficult-to-study patients. The increased limb vascular conductance with respiratory muscle unloading is similar in direction (but smaller in magnitude) to that reported in healthy athletes; however, in the CHF patients this effect occurred at only 60% maximum exercise, whereas in health, near-maximum exercise was required to produce sufficient WOB to elicit any cardiovascular effects of respiratory muscle unloading (Dempsey et al. 2006). The augmented stroke volume (SV) and cardiac output observed with reduced inspiratory negative pleural pressure in CHF is opposite in direction to that reported in healthy humans and animals, apparently reflecting a critical dependency of SV on ventricular preload in health and on after-load in CHF. If the observed increase in limb blood flow with unloading does indeed imply a ‘steal’ of flow from the limbs by the diaphragm (which to date has been reported only in a rodent model of CHF), then this shift would probably require (a) activation of respiratory muscle metaboreflexes acting via type III–IV phrenic afferents (Dempsey et al. 2006) for feedback regulation of sympathetic vasoconstrictor outflow, plus (b) a marked difference in vasoconstrictor sensitivity of phrenic arterioles vs. those in limb musculature in response to noradrenaline (norepinephrine) (Aaker & Laughlin, 2002). However, we should not presume that the diaphragm is receiving all of the blood flow it requires during whole body exercise; to the contrary, the diaphragm fatigues during high intensity endurance exercise, but does not fatigue when the same high WOB is sustained voluntarily in a resting subject (Dempsey et al. 2006). These data imply that a strong competition for blood flow between respiratory and limb skeletal muscles does indeed prevail during exercise of heavy intensity. Studies are now required to provide more direct evidence of how alterations in the work of breathing alter this flow distribution between locomotor muscles and those of the chest and abdominal wall during exercise. We also need to determine the mechanisms responsible for activation of respiratory muscle metaboreflexes and, in turn, selective sympathetically mediated vasoconstriction. There are several potential sources of the augmented drive to breathe in CHF. Recent findings in animal models of CHF report upregulation at the level of the carotid chemoreceptor as a key source of both augmented ventilatory and sympathetic drives both at rest (Ding et al. 2008) and especially during exercise (Stickland et al. 2007). One of the links of WOB to limb blood flow and in turn, to exercise performance may be found in the reduced exercise-induced quadriceps fatigue obtained when WOB was reduced via MV during near-maximum exercise in health and during sub-maximum exercise in hypoxia or in patients with chronic obstructive pulmonary disease (COPD) (Amann et al. 2010). In turn, a clinical implication of Olson et al.'s findings would be the use of respiratory muscle unloading together with supplemental O2 in rehabilitation of exercising CHF patients, in order to reduce sympathetic vasoconstriction and effort perception, thereby promoting increased work rates and allowing a greater locomotor muscle training effect to be achieved. Finally it is common clinical practice to contrast an exercising patient's specific perceptions of dyspnoea vs. those of limb muscle discomfort to identify the source of exercise performance limitation (i.e. respiratory vs. limb muscle). However, given the dependence of limb blood flow and O2 transport, fatigue and effort perception on the work of breathing as shown by Olson et al. and in related studies (Dempsey et al. 2006; Amann et al. 2010), this practice is likely to be highly misleading.

Phrenicotomy alters phrenic long-term facilitation following intermittent hypoxia in anesthetized rats

Intermittent hypoxia (IH) can induce a persistent increase in neural drive to the respiratory muscles known as long-term facilitation (LTF). LTF of phrenic inspiratory activity is often studied in anesthetized animals after phrenicotomy (PhrX), with subsequent recordings being made from the proximal stump of the phrenic nerve. However, severing afferent and efferent axons in the phrenic nerve has the potential to alter the excitability of phrenic motoneurons, which has been hypothesized to be an important determinant of phrenic LTF. Here we test the hypothesis that acute PhrX influences immediate and long-term phrenic motor responses to hypoxia. Phrenic neurograms were recorded in anesthetized, ventilated, and vagotomized adult male rats with intact phrenic nerves or bilateral PhrX. Data were obtained before (i.e., baseline), during, and after three 5-min bouts of isocapnic hypoxia. Inspiratory burst amplitude during hypoxia (%baseline) was greater in PhrX than in phrenic nerve-intact rats (P < 0.001). Similarly, burst amplitude 55 min after IH was greater in PhrX than in phrenic nerve-intact rats (175 + or - 9 vs. 126 + or - 8% baseline, P < 0.001). In separate experiments, phrenic bursting was recorded before and after PhrX in the same animal. Afferent bursting that was clearly observable in phase with lung deflation was immediately abolished by PhrX. The PhrX procedure also induced a form of facilitation as inspiratory burst amplitude was increased at 30 min post-PhrX (P = 0.01 vs. pre-PhrX). We conclude that, after PhrX, axotomy of phrenic motoneurons and, possibly, removal of phrenic afferents result in increased phrenic motoneuron excitability and enhanced LTF following IH.

Phrenic nerve afferent activation of neurons in the cat SI cerebral cortex.

Stimulation of respiratory afferents elicits neural activity in the somatosensory region of the cerebral cortex in humans and animals. Respiratory afferents have been stimulated with mechanical loads applied to breathing and electrical stimulation of respiratory nerves and muscles. It was hypothesized that stimulation of the phrenic nerve myelinated afferents will activate neurons in the 3a and 3b region of the somatosensory cortex. This was investigated in cats with electrical stimulation of the intrathoracic phrenic nerve and C(5) root of the phrenic nerve. The somatosensory cortical response to phrenic afferent stimulation was recorded from the cortical surface, contralateral to the phrenic nerve, ispilateral to the phrenic nerve and with microelectrodes inserted into the cortical site of the surface dipole. Short-latency, primary cortical evoked potentials (1 degrees CEP) were recorded with stimulation of myelinated afferents of the intrathoracic phrenic nerve in the contralateral post-cruciate gyrus of all animals (n = 42). The mean onset and peak latencies were 8.5 +/- 5.7 ms and 21.8 +/- 9.8 ms, respectively. The rostro-caudal surface location of the 1 degrees CEP was found between the rostral edge of the post-cruciate dimple (PCD) and the rostral edge of the ansate sulcus, medio-lateral location was between 2 mm lateral to the sagittal sulcus and the lateral end of the cruciate sulcus. Histological examination revealed that the 1 degrees CEP sites were recorded over areas 3a and 3b of the SI somatosensory cortex. Intracortical activation of 16 neurons with two patterns of neural activity was recorded: (1) short-latency, short-duration activation of neurons and (2) long-latency, long-duration activation of neurons. Short-latency neurons had a mean onset latency of 10.4 +/- 3.1 ms and mean burst duration of 10.1 +/- 3.2 ms. The short-latency units were recorded at an average depth of 1.7 +/- 0.5 mm below the cortical surface. The long-latency neurons had a mean onset latency of 36.0 +/- 4.2 ms and mean burst duration of 32.2 +/- 8.4 ms. The long-latency units were recorded at an average depth of 2.4 +/- 0.2 mm below the cortical surface. The results of the study demonstrated that phrenic nerve afferents have a short-latency central projection to the SI somatosensory cortex. The phrenic afferents activated neurons in lamina III and IV of areas 3a and 3b. The cortical representation of phrenic nerve afferents is medial to the forelimb, lateral to the hindlimb, similar to thoracic loci, hence the phrenic afferent SI site in the cat homunculus is consistent with body position (thoracic region) rather than spinal segment (C(5)-C(7)). The phrenic afferent activation of the somatosensory cortex is bilateral, with the ipsilateral cortical activation occurring subsequent to the contralateral. These results support the hypothesis that phrenic afferents provide somatosensory information to the cerebral cortex which can be used for diaphragmatic proprioception and somatosensation.

Long-term reorganization of respiratory pathways after partial cervical spinal cord injury

High cervical spinal cord injury (SCI) interrupts bulbospinal respiratory pathways innervating phrenic motoneurons, and induces an inactivation of phrenic nerves (PN) and diaphragm. We have previously shown that the ipsilateral (ipsi) PN was inactivated following a lateral C2 SCI, but was spontaneously partially reactivated 7 days post-SCI. This phrenic reactivation depended on contralateral (contra) descending pathways, located laterally, that cross the spinal midline. We analysed here whether long-term post-lesional changes may occur in the respiratory network. We showed that ipsi PN reactivation was greater at 3 months compared with 7 days post-SCI, and that it was enhanced after acute contra phrenicotomy (Phx), which also induced a substantial reactivation of the ipsi diaphragm (not detected at 7 days post-SCI). At 3 months post-SCI (compared with 7 days post-SCI), ipsi PN activity was only moderately affected by ipsi Phx or by gallamine treatment, a nicotinic neuromuscular blocking agent, indicating that it was less dependent on ipsi sensory phrenic afferents. After an additional acute contra SCI (C1) performed laterally, ipsi PN activity was abolished in rats 7 days post-SCI, but persisted in rats 3 months post-SCI. This activity thus depended on new functional descending pathways located medially rather than laterally. These may not involve newly recruited neurons as retrograde labelling showed that ipsi phrenic motoneurons were innervated by a similar number of medullary respiratory neurons after a short and long post-lesional time. These results show that after a long post-lesional time, phrenic reactivation is reinforced by an anatomo-functional reorganization of spinal respiratory pathways.

Insights into the role of the respiratory muscle metaboreflex

Factors affecting exercise tolerance have been of interest to respiratory physiologists for several decades. Among these limiting factors is diaphragmatic fatigue, which occurs during sustained exercise of high intensities (> 80% maximal oxygen consumption ()) (Babcock et al. 1995). Exercise-induced diaphragmatic fatigue is, in part, due to the high levels of respiratory muscle work that must be sustained throughout high intensity exercise. The consequences of a fatiguing diaphragm and other respiratory muscles have been addressed in a recent review by Dempsey et al. (2006). Briefly, recent reports have suggested that accumulation of metabolites in the inspiratory and expiratory muscles when respiratory muscles are fatigued activates unmylenated type IV phrenic afferents (Hill, 2000), which in turn increases sympathetic vasoconstrictor activity via a supra-spinal reflex. Why is this reflex effect important during exercise? During strenuous exercise, when the work of breathing is reduced by ∼50% via proportional assist ventilation in healthy subjects, cardiac output is reduced and vascular conductance and blood flow to the exercising legs are increased and whole body exercise performance increases (Harms et al. 1997, 2000). Presently, it is not known what specific mechanisms activate the respiratory muscle metaboreflex sufficiently to increase sympathetic vasoconstrictor activity and affect cardiovascular indices. In the current issue of The Journal of Physiology, Witt et al. (2007) provide significant insight into the role of the respiratory muscle metaboreflex on cardiovascular consequences during heavy exercise. These researchers utilized five weeks of inspiratory muscle training (IMT) in an attempt to increase respiratory muscle strength and presumably reduce or delay respiratory muscle fatigue. In this study, IMT reduced the increase in heart rate and mean arterial pressure that typically occurs with the increased work of breathing during heavy exercise in healthy subjects. What was the mechanism for this reduced cardiovascular responsiveness to high levels of inspiratory work? The authors speculated that the decreased cardiovascular response was due to: (a) reduced activity of chemically sensitive afferent fibres innervating the inspiratory muscles or to a desensitization, or (b) a decline in responsiveness of the type III and IV afferents to chemical stimulants resulting from a conditioned response to repeated exposure to the accumulated metabolites associated with IMT, although either mechanism has yet to be established. The significance of these findings by Witt et al. (2007) addresses broader questions of the importance of respiratory muscle work to exercise performance. Recent studies have demonstrated that the respiratory muscle metaboreflex is a key player in increased sympathetic tone and blood flow redistribution during heavy exercise (Sheel et al. 2001; Rodman et al. 2003). Importantly, there appears to be a threshold for activation of this reflex that does not occur during exercise of low to moderate intensities or non-fatiguing inspiratory efforts. The authors suggest that IMT may have raised the threshold required to elicit significant increases in mean arterial pressure. Future work is necessary to determine specifically how increases in inspiratory muscle strength led to these improvements. Additionally, inspiratory muscle training has received considerable interest and enthusiasm in recent years as a method to improve whole body exercise performance in healthy subjects. However, to date, the literature appears inconclusive. This may be due in part to differences in training strategies, subject populations or inappropriate control groups. In those studies that have shown significant gains in performance, mechanisms responsible are still unresolved. Results from the present study of Witt et al. (2007) provide novel insights into a potential role of the metaboreflex for the possible benefit of IMT. Reduced cardiovascular responsiveness to high levels of inspiratory muscle work with IMT in this study points to a plausible explanation for the improvements in exercise performance with IMT. Alternatively, IMT has often been shown to be an effective tool for patients with chronic obstructive pulmonary disease (COPD) and congestive heart failure (CHF). Based on Witt et al. (2007), future studies investigating the role of IMT on the inspiratory muscle metaboreflex could help determine the mechanistic basis for the improvements in this population. In summary, Witt et al. (2007) have provided a significant step forward in our understanding of the inspiratory muscle metaboreflex by their careful, quantitative approach in determining the mechanisms responsible for the cardiovascular responses that occur with respiratory muscle work during strenuous exercise. The results from this study substantiate the importance of this reflex to competing vascular beds in healthy subjects during high intensity exercise. Hopefully, these findings will provide an impetus for further, insightful investigations into the cardiopulmonary consequences of respiratory muscle work during exercise.

Restorative respiratory pathways after partial cervical spinal cord injury: role of ipsilateral phrenic afferents

After disruption of the descending respiratory pathways induced by unilateral cervical spinal cord injury (SCI) in rats, the inactivated ipsilateral (ipsi) phrenic nerve (PN) discharge may partially recover following some specific experimental procedures [such as contralateral (contra) phrenicotomy (Phx)]. This phrenic reactivation involves normally silent contra pathways decussating at the level of the phrenic nucleus, but the mechanisms of this crossed phrenic activation are still poorly understood. The present study investigates the contribution of sensory phrenic afferents to this process by comparing the acute effects of ipsi and contra Phx. We show that the phrenic discharge (recorded on intact PNs) was almost completely suppressed 0 h and 3 h after a lateral cervical SCI, but was already spontaneously reactivated after 1 week. This ipsi phrenic activity was enhanced immediately after contra Phx and was completely suppressed by an acute contra cervical section, indicating that it is triggered by crossed phrenic pathways located laterally in the contra spinal cord. Ipsi phrenic activity was also abolished immediately after ipsi Phx that interrupts phrenic sensory afferents, an effect prevented by prior acute ablation of the cervical dorsal root ganglia, indicating that crossed phrenic activation depends on excitatory phrenic sensory afferents but also putatively on inhibitory non-phrenic afferents. On the basis of these data, we propose a new model for crossed phrenic activation after partial cervical injury, with an essential role played by ipsi-activating phrenic sensory afferents.

Activation of thalamic ventroposteriolateral neurons by phrenic nerve afferents in cats and rats.

It has been demonstrated that phrenic nerve afferents project to somatosensory cortex, yet the sensory pathways are still poorly understood. This study investigated the neural responses in the thalamic ventroposteriolateral (VPL) nucleus after phrenic afferent stimulation in cats and rats. Activation of VPL neurons was observed after electrical stimulation of the contralateral phrenic nerve. Direct mechanical stimulation of the diaphragm also elicited increased activity in the same VPL neurons that were activated by electrical stimulation of the phrenic nerve. Some VPL neurons responded to both phrenic afferent stimulation and shoulder probing. In rats, VPL neurons activated by inspiratory occlusion also responded to stimulation on phrenic afferents. These results demonstrate that phrenic afferents can reach the VPL thalamus under physiological conditions and support the hypothesis that the thalamic VPL nucleus functions as a relay for the conduction of proprioceptive information from the diaphragm to the contralateral somatosensory cortex.