Spontaneous respiratory plasticity following unilateral high cervical spinal cord injury in behaving rats

Abstract

Unilateral cervical C2 hemisection (C2Hx) is a classic model of spinal cord injury (SCI) for studying respiratory dysfunction and plasticity. However, most previous studies were performed under anesthesia, which significantly alters respiratory network. Therefore, the goal of this work was to assess spontaneous diaphragm recovery post-C2Hx in awake, freely behaving animals. Adult rats were chronically implanted with diaphragm EMG electrodes and recorded during 8 weeks post-C2Hx. Our results reveal that ipsilateral diaphragm activity partially recovers within days post-injury and reaches pre-injury amplitude in a few weeks. However, the full extent of spontaneous ipsilateral recovery is significantly attenuated by anesthesia (ketamine/xylazine, isoflurane, and urethane). This suggests that the observed recovery may be attributed in part to activation of NMDA receptors which are suppressed by anesthesia. Despite spontaneous recovery in awake animals, ipsilateral hemidiaphragm dysfunction still persists: i) Inspiratory bursts during basal (slow) breathing exhibit an altered pattern, ii) the amplitude of sighs - or augmented breaths - is significantly decreased, and iii) the injured hemidiaphragm exhibits spontaneous events of hyperexcitation. The results from this study offer an under-appreciated insight into spontaneous diaphragm activity and recovery following high cervical spinal cord injury in awake animals.

Keywords: Anesthesia; Phrenic; Plasticity; Respiration; Spinal cord injury.

Figure 1

A. An example of bilateral diaphragm EMG recording in a behaving rat. EMG_L – left EMG, EMG_R – right EMG, ∫ -integrated EMG activity, Freq – instant frequency of breathing in Hz; B. Histogram of instant breath to breath intervals during 5 minutes of recording. Red color represents breathing frequency of 5 or more breaths per second (>5 Hz), which occurs during sniffing behavior, blue color represents breathing frequency of 3 or less breaths per second (<3 Hz), which is observed during basal breathing, and green color represents intermediate range of breathing frequencies. C. Examples of raw traces and integrated diaphragm activity during basal and breathing that was observed during sniffing behavior.

Figure 2

A. An example of bilateral diaphragm EMG recording in an anesthetized rat before and after C2Hx surgery, demonstrating complete abolishment of diaphragm activity on the side of the injury. EMG_I – ipsilateral EMG, EMG_C – contralateral EMG, ∫-integrated EMG activity; B. Reconstraction of lesions after C2Hx spinal cord cross-section through the lesion epicenter demonstrating minimal white matter sparing (<15 %; see the thin band of tissue at the medial-dorsal location). C. Bar graph represents mean, standard deviation and percentage of speared tissue for each animal (n=7).

Figure 3

A. Examples of raw traces from bilateral diaphragm EMG recordings during basal breathing before (Naïve) and throughout experimental time points post-injury (1 day, 1, 2, 4, 6 and 8 weeks post-C2Hx). EMG_I – ipsilateral EMG, EMG_C – contralateral EMG; B. Averaged amplitudes of integrated diaphragm activity (EMGs) as a percent of pre-injury level in 7 animals recorded before (b) and 1 day (1d), 1 week (1w) through 8 weeks (8w) post-C2Hx for ipsi- and contra- sides

Figure 4

A. An example of a bilateral diaphragm EMG recording in a rat under isoflurane anesthesia (8 weeks post-C2Hx); B. An example of a bilateral diaphragm EMG recording 10 min post-isoflurane anesthesia (8 weeks post-C2Hx); C. An example of a bilateral diaphragm EMG recording in a rat under xylazine/ketamine anesthesia (8 weeks post-C2Hx); D. An example of a bilateral phrenic recording in a rat under urethane anesthesia (8 weeks post-C2Hx). Panels A–D represent recordings from the same rat. E. Summary histogram of averaged phrenic integrated amplitudes from the ipsilateral (PNI) and contralateral (PNC) sides. Ipsi- amplitude is normalized to the contrlateral side. EMG_I – ipsi EMG, EMG_C – contra EMG, PNI – ipsilateral phrenic neurogram, PNC – contralateral phrenic neurogram.

Figure 5

A.(1) An example of diaphragm activity (EMG) demonstrating different inspiratory burst shape on the ipsi and contralateral sides of the diaphragm during basal breathing 8 weeks post-C2Hx; (2) Overlapped averaged integrated diaphragm activity before injury (naïve) and 8 weeks post-C2Hx in the same animal. Note the plateau-like shape and increased background activity in the ipsilateral side of diaphragm. B. An example of diaphragm activity (EMG) during a sigh (*) at 8 weeks post-C2Hx (1) and quantification of diaphragm activity (2) presented as a ratio of activity during a sigh to the amplitude of the preceding breath, for ipsi and contralateral hemidiaphragms in n=7 animals. b = baseline, 1w = 1 week post C2Hx, 8w = 8weeks post C2Hx. Note the decrease in diaphragm amplitude during a sigh on the ipsilateral side of the diaphragm; C. An example of diaphragm activity (EMG) that occurs during a hyperexcitability event (**) on the ipsilateral (left) hemidiaphragm, representing spontaneous hyperexcitability seen on the injured side. In all panels, EMG_I – ipsi EMG, EMG_C – contra EMG, ∫-integrated EMG activity, Int Ampl – amplitudes of integrated activity.