RESUMEN
Respiratory deficits after C2 hemisection (C2Hx) have been well documented through single-sex investigations. Although ovarian sex hormones enable enhanced respiratory recovery observed in females 2 wk post-C2Hx, it remains unknown if sex impacts spontaneous respiratory recovery at chronic time points. We conducted a longitudinal study to provide a comprehensive sex-based characterization of respiratory neuromuscular recovery for 8 wk after C2Hx. We recorded ventilation and chronic diaphragm electromyography (EMG) output in awake, behaving animals, phrenic motor output in anesthetized animals, and performed diaphragm muscle histology in chronically injured male and female rodents. Our results show that females expressed a greater recovery of tidal volume and minute ventilation compared with males during subacute and chronic time points. Eupneic diaphragm EMG amplitude during wakefulness and phrenic motor amplitude are similar between sexes at all time points after injury. Our data also suggest that females have a greater reduction in ipsilateral diaphragm EMG amplitude during spontaneous deep breaths (e.g., sighs) compared with males. Finally, we show evidence for atrophy and remodeling of the fast, fatigable fibers ipsilateral to injury in females, but not in males. To our knowledge, the data presented here represent the first study to report sex-dependent differences in spontaneous respiratory recovery and diaphragm muscle morphology following chronic C2Hx. These data highlight the need to study both sexes to inform evidence-based therapeutic interventions in respiratory recovery after spinal cord injury (SCI).NEW & NOTEWORTHY In response to chronic C2 hemisection, female rodents display increased tidal volume during eupneic breathing compared with males. Females show a greater reduction in diaphragm electromyography (EMG) amplitude during spontaneous deep breaths (e.g., sighs) and atrophy and remodeling of fast, fatigable diaphragm fibers. Given that most rehabilitative interventions occur in the subacute to chronic stages of injury, these results highlight the importance of considering sex when developing and evaluating therapeutics after spinal cord injury.
Asunto(s)
Diafragma , Electromiografía , Recuperación de la Función , Traumatismos de la Médula Espinal , Animales , Femenino , Masculino , Diafragma/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Recuperación de la Función/fisiología , Electromiografía/métodos , Ratas , Ratas Sprague-Dawley , Caracteres Sexuales , Respiración , Nervio Frénico/fisiopatología , Nervio Frénico/fisiología , Médula Cervical/lesiones , Médula Cervical/fisiopatologíaRESUMEN
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.
Asunto(s)
Diafragma , Tórax , Ratas , Animales , Diafragma/fisiología , Respiración , Nervio Frénico/fisiología , Estimulación EléctricaRESUMEN
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.
Asunto(s)
Fenómenos Electrofisiológicos/fisiología , Hemodinámica/fisiología , Inhalación/fisiología , Plasticidad Neuronal/fisiología , Neuronas Aferentes/fisiología , Nervio Frénico/fisiología , Vías Aferentes/fisiología , Animales , Presión Arterial/fisiología , Análisis de los Gases de la Sangre , Femenino , Frecuencia Cardíaca/fisiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The spiny mouse (Acomys cahirinus) appears to be unique among mammals by showing little scarring or fibrosis after skin or muscle injury, but the Acomys response to spinal cord injury (SCI) is unknown. We tested the hypothesis that Acomys would have molecular and immunohistochemical evidence of reduced spinal inflammation and fibrosis following SCI as compared to C57BL/6 mice (Mus), which similar to all mammals studied to date exhibits spinal scarring following SCI. Initial experiments used two pathway-focused RT-PCR gene arrays ("wound healing" and "neurogenesis") to evaluate tissue samples from the C2-C6 spinal cord 3 days after a C3/C4 hemi-crush injury (C3Hc). Based on the gene array results, specific genes were selected for RT-qPCR evaluation using species-specific primers. The results supported our hypothesis by showing increased inflammation and fibrosis related gene expression (Serpine 1, Plau, and Timp1) in Mus as compared to Acomys (p < .05). RT-qPCR also showed enhanced stem cell and axonal guidance related gene expression (Bmp2, GDNF, and Shh) in Acomys compared to Mus (p < .05). Immunohistochemical evaluation of the spinal lesion at 4 weeks postinjury indicated less collagen IV immunostaining in Acomys (p < .05). Glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1(IBA1) immunostaining indicated morphological differences in the appearance of astrocytes and macrophages/microglia in Acomys. Collectively, the molecular and histologic results support the hypothesis that Acomys has reduced spinal inflammation and fibrosis following SCI. We suggest that Acomys may be a useful comparative model to study adaptive responses to SCI.
Asunto(s)
Murinae , Traumatismos de la Médula Espinal/patología , Animales , Vértebras Cervicales , Fibrosis/patología , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Especificidad de la Especie , TranscriptomaRESUMEN
Hypoxia elicits complex cell signaling mechanisms in the respiratory control system that can produce long-lasting changes in respiratory motor output. In this article, we review experimental approaches used to elucidate signaling pathways associated with hypoxia, and summarize current hypotheses regarding the intracellular signaling pathways evoked by intermittent exposure to hypoxia. We review data showing that pharmacological treatments can enhance neuroplastic responses to hypoxia. Original data are included to show that pharmacological modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) function can reveal a respiratory neuroplastic response to a single, brief hypoxic exposure in anesthetized mice. Coupling pharmacologic treatments with therapeutic hypoxia paradigms may have rehabilitative value following neurologic injury or during neuromuscular disease. Depending on prevailing conditions, pharmacologic treatments can enable hypoxia-induced expression of neuroplasticity and increased respiratory motor output, or potentially could synergistically interact with hypoxia to more robustly increase motor output.
Asunto(s)
Hipoxia/complicaciones , Plasticidad Neuronal/fisiología , Trastornos Respiratorios/tratamiento farmacológico , Trastornos Respiratorios/etiología , Animales , Humanos , Plasticidad Neuronal/efectos de los fármacos , Receptores AMPA/efectos de los fármacos , Receptores AMPA/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
Neuronas Aferentes/fisiología , Nervio Frénico/fisiología , Animales , Diafragma/inervación , Diafragma/fisiología , Humanos , Plasticidad Neuronal , Nocicepción , Nervio Frénico/citología , RespiraciónRESUMEN
Brief, intermittent oxygen reductions [acute intermittent hypoxia (AIH)] evokes spinal plasticity. Models of AIH-induced neuroplasticity have focused on motoneurons; however, most midcervical interneurons (C-INs) also respond to hypoxia. We hypothesized that AIH would alter the functional connectivity between C-INs and induce persistent changes in discharge. Bilateral phrenic nerve activity was recorded in anesthetized and ventilated adult male rats and a multielectrode array was used to record C4/5 spinal discharge before [baseline (BL)], during, and 15 min after three 5 min hypoxic episodes (11% O2, H1-H3). Most C-INs (94%) responded to hypoxia by either increasing or decreasing firing rate. Functional connectivity was examined by cross-correlating C-IN discharge. Correlograms with a peak or trough were taken as evidence for excitatory or inhibitory connectivity between C-IN pairs. A subset of C-IN pairs had increased excitatory cross-correlations during hypoxic episodes (34%) compared with BL (19%; p < 0.0001). Another subset had a similar response following each episode (40%) compared with BL (19%; p < 0.0001). In the latter group, connectivity remained elevated 15 min post-AIH (30%; p = 0.0002). Inhibitory C-IN connectivity increased during H1-H3 (4.5%; p = 0.0160), but was reduced 15 min post-AIH (0.5%; p = 0.0439). Spike-triggered averaging indicated that a subset of C-INs is synaptically coupled to phrenic motoneurons and excitatory inputs to these "pre-phrenic" cells increased during AIH. We conclude that AIH alters connectivity of the midcervical spinal network. To our knowledge, this is the first demonstration that AIH induces plasticity within the propriospinal network.SIGNIFICANCE STATEMENT Acute intermittent hypoxia (AIH) can trigger spinal plasticity associated with sustained increases in respiratory, somatic, and/or autonomic motor output. The impact of AIH on cervical spinal interneuron (C-IN) discharge and connectivity is unknown. Our results demonstrate that AIH recruits excitatory C-INs into the spinal respiratory (phrenic) network. AIH also enhances excitatory and reduces inhibitory connections among the C-IN network. We conclude that C-INs are part of the respiratory, somatic, and/or autonomic response to AIH, and that propriospinal plasticity may contribute to sustained increases in motor output after AIH.
Asunto(s)
Potenciales de Acción/fisiología , Hipoxia de la Célula/fisiología , Médula Cervical/fisiología , Interneuronas/fisiología , Plasticidad Neuronal/fisiología , Oxígeno/metabolismo , Animales , Masculino , Ratas , Ratas Sprague-Dawley , Sinapsis/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
C2 spinal hemilesion (C2Hx) paralyzes the ipsilateral diaphragm, but recovery is possible through activation of "crossed spinal" synaptic inputs to ipsilateral phrenic motoneurons. We tested the hypothesis that high-frequency epidural stimulation (HF-ES) would potentiate ipsilateral phrenic output after subacute and chronic C2Hx. HF-ES (300 Hz) was applied to the ventrolateral C4 or T2 spinal cord ipsilateral to C2Hx in anesthetized and mechanically ventilated adult rats. Stimulus duration was 60 s, and currents ranged from 100 to 1,000 µA. Bilateral phrenic nerve activity and ipsilateral hypoglossal (XII) nerve activity were recorded before and after HF-ES. Higher T2 stimulus currents potentiated ipsilateral phasic inspiratory activity at both 2 and 12 wk post-C2Hx, whereas higher stimulus currents delivered at C4 potentiated ipsilateral phasic phrenic activity only at 12 wk (P = 0.028). Meanwhile, tonic output in the ipsilateral phrenic nerve reached 500% of baseline values at the high currents with no difference between 2 and 12 wk. HF-ES did not trigger inspiratory burst-frequency changes. Similar responses occurred following T2 HF-ES. Increases in contralateral phrenic and XII nerve output were induced by C4 and T2 HF-ES at higher currents, but the relative magnitude of these changes was small compared with the ipsilateral phrenic response. We conclude that following incomplete cervical spinal cord injury, HF-ES of the ventrolateral midcervical or thoracic spinal cord can potentiate efferent phrenic motor output with little impact on inspiratory burst frequency. However, the substantial increases in tonic output indicate that the uninterrupted 60-s stimulation paradigm used is unlikely to be useful for respiratory muscle activation after spinal injury.NEW & NOTEWORTHY Previous studies reported that high-frequency epidural stimulation (HF-ES) activates the diaphragm following acute spinal transection. This study examined HF-ES and phrenic motor output following subacute and chronic incomplete cervical spinal cord injury. Short-term potentiation of phrenic bursting following HF-ES illustrates the potential for spinal stimulation to induce respiratory neuroplasticity. Increased tonic phrenic output indicates that alternatives to the continuous stimulation paradigm used in this study will be required for respiratory muscle activation after spinal cord injury.
Asunto(s)
Diafragma/inervación , Plasticidad Neuronal , Nervio Frénico/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Estimulación de la Médula Espinal/métodos , Animales , Diafragma/fisiología , Femenino , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/terapiaRESUMEN
While rodent gait analysis can quantify the behavioral consequences of disease, significant methodological differences exist between analysis platforms and little validation has been performed to understand or mitigate these sources of variance. By providing the algorithms used to quantify gait, open-source gait analysis software can be validated and used to explore methodological differences. Our group is introducing, for the first time, a fully-automated, open-source method for the characterization of rodent spatiotemporal gait patterns, termed Automated Gait Analysis Through Hues and Areas (AGATHA). This study describes how AGATHA identifies gait events, validates AGATHA relative to manual digitization methods, and utilizes AGATHA to detect gait compensations in orthopaedic and spinal cord injury models. To validate AGATHA against manual digitization, results from videos of rodent gait, recorded at 1000 frames per second (fps), were compared. To assess one common source of variance (the effects of video frame rate), these 1000 fps videos were re-sampled to mimic several lower fps and compared again. While spatial variables were indistinguishable between AGATHA and manual digitization, low video frame rates resulted in temporal errors for both methods. At frame rates over 125 fps, AGATHA achieved a comparable accuracy and precision to manual digitization for all gait variables. Moreover, AGATHA detected unique gait changes in each injury model. These data demonstrate AGATHA is an accurate and precise platform for the analysis of rodent spatiotemporal gait patterns.
Asunto(s)
Marcha , Miembro Posterior/fisiopatología , Procesamiento de Imagen Asistido por Computador/métodos , Traumatismos de la Médula Espinal/fisiopatología , Grabación en Video , Animales , RatasRESUMEN
Power spectral analyses of electrical signals from respiratory nerves reveal prominent oscillations above the primary rate of breathing. Acute exposure to intermittent hypoxia can induce a form of neuroplasticity known as long-term facilitation (LTF), in which inspiratory burst amplitude is persistently elevated. Most evidence indicates that the mechanisms of LTF are postsynaptic and also that high-frequency oscillations within the power spectrum show coherence across different respiratory nerves. Since the most logical interpretation of this coherence is that a shared presynaptic mechanism is responsible, we hypothesized that high-frequency spectral content would be unchanged during LTF. Recordings of inspiratory hypoglossal (XII) activity were made from anesthetized, vagotomized, and ventilated 129/SVE mice. When arterial O2 saturation (SaO2) was maintained >96%, the XII power spectrum and burst amplitude were unchanged for 90 min. Three, 1-min hypoxic episodes (SaO2 = 50 ± 10%), however, caused a persistent (>60 min) and robust (>400% baseline) increase in burst amplitude. Spectral analyses revealed a rightward shift of the signal content during LTF, with sustained increases in content above â¼125 Hz following intermittent hypoxia and reductions in power at lower frequencies. Changes in the spectral content during LTF were qualitatively similar to what occurred during the acute hypoxic response. We conclude that high-frequency content increases during XII LTF in this experimental preparation; this may indicate that intermittent hypoxia-induced plasticity in the premotor network contributes to expression of XII LTF.