Daily acute intermittent hypoxia elicits functional recovery of diaphragm and inspiratory intercostal muscle activity after acute cervical spinal injury

Abstract

A major cause of mortality after spinal cord injury is respiratory failure. In normal rats, acute intermittent hypoxia (AIH) induces respiratory motor plasticity, expressed as diaphragm (Dia) and second external intercostal (T2 EIC) long-term facilitation (LTF). Dia (not T2 EIC) LTF is enhanced by systemic adenosine 2A (A2A) receptor inhibition in normal rats. We investigated the respective contributions of Dia and T2 EIC to daily AIH-induced functional recovery of breathing capacity with/without A2A receptor antagonist (KW6002, i.p.) following C2 hemisection (C2HS). Rats received daily AIH (dAIH: 10, 5-min episodes, 10.5% O2; 5-min normoxic intervals; 7 successive days beginning 7days post-C2HS) or daily normoxia (dNx) with/without KW6002, followed by weekly (reminder) presentations for 8weeks. Ventilation and EMGs from bilateral diaphragm and T2 EIC muscles were measured with room air breathing (21% O2) and maximum chemoreceptor stimulation (

Mcs: 7% CO2, 10.5% O2). dAIH increased tidal volume (VT) in C2HS rats breathing room air (dAIH+vehicle: 0.47±0.02, dNx+vehicle: 0.40±0.01ml/100g; p<0.05) and MCS (dAIH+vehicle: 0.83±0.01, dNx+vehicle: 0.73±0.01ml/100g; p<0.001); KW6002 had no significant effect. dAIH enhanced contralateral (uninjured) diaphragm EMG activity, an effect attenuated by KW6002, during room air breathing and MCS (p<0.05). Although dAIH enhanced contralateral T2 EIC EMG activity during room air breathing, KW6002 had no effect. dAIH had no statistically significant effects on diaphragm or T2 EIC EMG activity ipsilateral to injury. Thus, two weeks post-C2HS: 1) dAIH enhances breathing capacity by effects on contralateral diaphragm and T2 EIC activity; and 2) dAIH-induced recovery is A2A dependent in diaphragm, but not T2 EIC. Daily AIH may be a useful in promoting functional recovery of breathing capacity after cervical spinal injury, but A2A receptor antagonists (e.g. caffeine) may undermine its effectiveness shortly after injury.

Keywords: Adenosine receptor; Breathing; Intermittent hypoxia; Motor neuron; Spinal cord injury; Spinal plasticity.

Fig. 1

Timeline describing the experimental design from day 1 (1d) until 52 days post-spinal cord injury (dps). C2HS: cervical hemisection in second segment, dAIH/dNx: daily acute intermittent hypoxia or normoxia for seven days depending on groups (see methods), R1-R5: weekly presentations or “reminders” of acute intermittent hypoxia or normoxia, M1-M6: weekly measurements including grooming test and simultaneous plethysmography and electromyography of bilateral diaphragm and second external intercostal muscles.

Fig. 2

Absolute values of tidal volume (Vt) per 100 gr rat during normoxia (Nx, A) and maximum chemoreceptor stimulation (MCS, B) in all groups 2 days before spinal injury and then, 1 day up to 52 days post-injury. Note: (1) all groups show reduced Vt 1 dps in A and B compared to shams; (2) AIH elicits an increase in Vt during Nx and MCS, which was not significantly affected by adenosine A2a antagonist. AIH: acute intermittent hypoxia, V: vehicle (DMSO), KW: KW6002, Nx: normoxia. Values are means ± SEM. # significantly different from controls (Nx + V and Nx + KW6002); p < 0.05.

Fig. 3

Absolute values of respiratory frequency during normoxia (Nx, A) and maximum chemoreceptor stimulation (MCS, B) in all groups studied 2 days before spinal cord injury, and one day up to 52 days post-injury. Respiratory frequency is significantly increased in all groups, compared to sham during normoxia and MCS. AIH: acute intermittent hypoxia, V: vehicle (DMSO), KW: KW6002.

Fig. 4

Changes in ipsilateral (injured) diaphragm (A) and second external intercostal muscle (T2 EIC, B) muscle amplitude during normoxia (Nx) expressed as percent change of pre-injury values one day up to 52 days post-injury. The spontaneous recovery is remarkable in ipsilateral T2 EIC in all groups (B) and not significant in ipsilateral diaphragm (A). Daily acute intermittent hypoxia (dAIH) alone or combined with A2a inhibition (KW6002) does not have an effect in neither diaphragm nor T2 EIC ipsilateral motor recovery. AIH: acute intermittent hypoxia, V: vehicle (DMSO), KW: KW6002.

Fig. 5

Representative integrated (A and B) and raw (C and D) EMG activity of right/left diaphragm (R/L_Dia) and second external intercostal muscle (R/L_T2 EIC) before (A, C) and one day after (B, D) spinal hemisection (C2HS) during maximum chemoreceptor stimulation (MCS). Note: (1) significantly reduced EMG activity in ipsilateral diaphragm and T2 external intercostal in (D), confirming the cervical hemisection; (2) absence of MCS response in injured (left) diaphragm and bilateral T2 EIC muscle in (B). (E) Representative longitudinal spinal cord slide (C1-C6) stained with Cresyl violet showing C2HS.

Fig. 6

Changes in contralateral (uninjured) diaphragm (A) and second external intercostal muscle (T2 EIC, B) muscle amplitude during normoxia (Nx) expressed as percent change of pre-injury values one day up to 52 days post-injury. Note: (1) all groups show significant increase in EMG amplitude compared to sham and pre-injury values during Nx in the diaphragm (A), demonstrating compensatory plasticity; (2) contralateral T2 EIC exhibits a transitory compensatory plasticity in all groups at 17 days post-injury; (3) daily acute intermittent hypoxia (dAIH) significantly increases EMG amplitude at 17 days post-injury in diaphragm and T2 EIC and this effect is impaired by A2a inhibition in diaphragm but not in T2 EIC muscle. AIH: acute intermittent hypoxia, V: vehicle (DMSO), KW: KW6002. Values are mean ± SEM.** significantly different from AIH + KW6002, * significantly different from time controls (TC: Nx + V and Nx + KW6002), # significantly different from sham; p < 0, 05.

Fig. 7

Changes in contralateral (uninjured) diaphragm during maximum chemoreceptor stimulation (MCS, A) and representative integrated EMG activity of right/left diaphragm (R/L_Dia) and second external intercostal muscle (R/L_T2 EIC) at 52 days post-injury during MCS (B). Note: (1) reduced but consistent MCS response in all groups in uninjured diaphragm (A); (2) daily acute intermittent hypoxia (dAIH) significantly increases EMG amplitude at 17 days post-injury and this effect is impaired by KW6002; (3) small MCS response in uninjured T2 EIC (B); (4) the MCS response in injured T2 EIC remains abolished at all this time-points (B).

Fig 8

Grooming Test scoring.