Adenosine 2A receptor inhibition enhances intermittent hypoxia-induced diaphragm but not intercostal long-term facilitation

Abstract

Acute intermittent hypoxia (AIH) elicits diaphragm (Dia) and second external intercostal (T2 EIC) long-term facilitation (LTF) in normal unanesthetized rats. Although AIH-induced phrenic LTF is serotonin dependent, adenosine constrained in anesthetized rats, this has not been tested in unanesthetized animals. Cervical (C2) spinal hemisection (C2HS) abolishes phrenic LTF because of loss of serotonergic inputs 2 weeks post-injury, but LTF returns 8 weeks post-injury. We tested three hypotheses in unanesthetized rats: (1) systemic adenosine 2aA (A2A) receptor inhibition with intraperitoneal (IP) KW6002 enhances Dia and T2 EIC LTF in normal rats; (2) Dia and T2 EIC LTF are expressed after chronic (8 weeks), but not acute (1 week) C2HS; and (3) KW6002 enhances Dia and T2 EIC LTF after chronic (not acute) C2HS. Electromyography radiotelemetry was used to record Dia and T2 EIC activity during normoxia (21% O2), before and after AIH (10, 5-min 10.5% O2, 5-min intervals). In normal rats, KW6002 enhanced DiaLTF versus AIH alone (33.1±4.6% vs. 22.1±6.4% baseline, respectively; p<0.001), but had no effect on T2 EIC LTF (p>0.05). Although Dia and T2 EIC LTF were not observed 2 weeks post-C2HS, LTF was observed in contralateral (uninjured) Dia and T2 EIC 8 weeks post-C2HS (18.7±2.7% and 34.9±4.9% baseline, respectively; p<0.05), with variable ipsilateral expression. KW6002 had no significant effects on contralateral Dia (p=0.447) or T2 EIC LTF (p=0.796). We conclude that moderate AIH induces Dia and T2 EIC LTF after chronic, but not acute cervical spinal injuries. A single A2A receptor antagonist dose enhances AIH-induced Dia LTF in normal rats, but this effect is not significant in chronic (8 weeks) C2HS unanesthetized rats.

Keywords: cervical; diaphragm; intercostal muscle; intermittent hypoxia; long-term facilitation; respiratory plasticity; spinal cord injury.

FIG. 1.

Summary of changes in diaphragm (Dia) (A) and second external intercostal (T2 EIC) muscle (B) integrated burst amplitude, and respiratory frequency (C) in unanesthetized, normal rats; variables are expressed as a percent change from baseline averaged between 0 and 60 min after acute intermittent hypoxia (AIH) or normoxia (Nx). Representative integrated electromyography (EMG) activity of AIH plus KW6002-treated group in Dia (D, upper trace) and T2 EIC muscle (D, lower trace). A significant increase in EMG integrated burst amplitude was observed in Dia (A) and T2 EIC muscle (B) 0 to 60 min post-AIH plus vehicle (V, dimethyl sulfoxide), but not in control rats (NX+KW6002 and NX+V), indicative of robust Dia and T2 EIC long-term facilitation (DiaLTF and T2 EIC LTF, respectively). A significantly greater DiaLTF was observed in rats treated with AIH plus KW6002 versus AIH plus vehicle (i.e., enhanced DiaLTF) (A and D). Similar enhancement was not observed in T2 EIC muscle (B and D). Respiratory frequency was significantly increased in AIH plus KW6002 and AIH plus vehicle-treated rats 0–60 min post-AIH compared with control rats. Values are means±1 standard error of the mean. *Significantly different from time control rats; †significantly different from baseline; # significantly different from AIH plus vehicle-treated group; p<0.001.

FIG. 2.

Changes in minute activity (amplitude×frequency) in diaphragm (Dia) (A) and second external intercostal (T2 EIC) muscles (B) in unanesthetized, normal rats; variables are expressed as a percent change from baseline averaged between 0 and 60 min after acute intermittent hypoxia (AIH) or normoxia (Nx). Note significant increases in minute activity in both Dia and T2 EIC muscle after AIH plus vehicle (vehicle, dimethyl sulfoxide). A2A antagonist (KW6002) significantly enhances Dia (A) but not T2 EIC (B) minute activity long-term facilitation. Values are means±1 standard error of the mean. *Significantly different from controls (NX+V, NX+KW6002); ^†significantly different from baseline; ^#significantly different from AIH plus vehicle; p<0.001.

FIG. 3.

Representative motor electromyography (EMG) activity of right/left diaphragm (R/L Dia) and second thoracic external intercostal muscle (R/L T2 EIC) and changes in contralateral (uninjured) Dia (C) and T2 EIC (D) EMG amplitude (expressed as percent change of pre-injury values) 7 days post-injury. At this time point, raw recordings show a remarkable spontaneous recovery in left (injured) T2 EIC muscle but only modest in the Dia (A). In contralateral Dia (C), all groups show significant increases in EMG burst amplitude (above 100%) during pre-treatment (normoxia [NX], grey bars) compared with pre-injury and Sham values, demonstrating a degree of spontaneous compensatory motor activity, which is not observed in contralateral T2 EIC muscle (D). Unlike normal rats (Fig. 1) and chronic C2 hemisected rats (Fig. 5), AIH alone or combined with KW6002 does not elicit long-term facilitation (LTF) in Dia and T2 EIC muscle (0 to 60 min post-AIH/NX, black bars; B, C, D). AIH, acute intermittent hypoxia; KW, KW6002; V, vehicle (dimethyl sulfoxide). Values are means±1 standard error of the mean. *Significantly different from pre-injury values; #significantly different from Sham; p<0.05.

FIG. 4.

Changes in ipsilateral (injured) diaphragm (Dia, A) and second external intercostal (T2 EIC, B) electromyography (EMG) amplitude expressed as percent change of pre-injury values for pre-treatment (during baseline, grey bars) and post-treatment (0 to 60 min post-acute intermittent hypoxia/normoxia (AIH/NX, black bars) at 7 days post-injury. Note: (1) In A, all experimental groups exhibit a small degree of spontaneous recovery in ipsilateral Dia EMG peak amplitude; (2) in B, all groups show remarkable spontaneous recovery of ipsilateral T2 EIC EMG peak amplitude, although these values remain significantly below Sham rats; and (3) no AIH-induced long-term facilitation was observed 7 days post-injury in either left Dia or T2 EIC muscle. KW, KW6002; V, vehicle (dimethyl sulfoxide). Values are means±1 standard error of the mean. *Significantly different from pre-injury values; #significantly different from Sham; p<0.05.

FIG. 5.

Representative raw (A) and integrated (B) traces of right/left diaphragm (R/L Dia) and second thoracic external intercostal muscle (R/L T2 EIC) and changes in contralateral (uninjured) Dia (C) and T2 EIC (D) electromyography (EMG) amplitude (expressed as percent change of pre-injury values) at 8 weeks post-injury. In contralateral Dia (C), all groups show significant increases (above 100%) in EMG amplitude during pre-treatment (normoxia [NX], grey bars) compared with pre-injury and Sham values demonstrating spontaneous compensatory motor activity of Dia on the intact side. Similar increases were not observed in T2 EIC muscle activity (D). Raw trace (A) illustrates complete ipsilateral T2 EIC spontaneous recovery, but only modest spontaneous recovery of ipsilateral Dia activity. Unlike acute C2 hemisected rats (Fig. 3), at this time point, acute intermittent hypoxia (AIH)-induced long-term facilitation (LTF) is restored in contralateral Dia (i.e., DiaLTF; B, C) and T2 EIC muscle (i.e., T2 EIC LTF; B, D), as in normal unanesthetized rats (Fig. 1). Although A2A receptor inhibition with KW6002 tends to increase DiaLTF, this effect was not statistically significant. A2A inhibition had no apparent effect on T2 EIC LTF. KW, KW6002; V, vehicle (dimethyl sulfoxide). Values are means±1 standard error of the mean. *Significantly different from pre-injury values; #significantly different from Sham; ≠significantly different from pre-treatment values; p<0.05.

FIG. 6.

Changes in ipsilateral (injured) diaphragm (Dia, A) and second external intercostal (T2 EIC, B) electromyography (EMG) amplitude expressed as percent change of pre-injury values for pre-treatment (baseline, grey bars) and post-treatment (after acute intermittent hypoxia/normoxia [AIH/NX], black bars) at 8 weeks post-injury. Note: (1) in (A), all experimental groups show modest spontaneous recovery of ipsilateral Dia activity versus pre-injury values and sham; (2) in (B), complete spontaneous recovery of ipsilateral T2 EIC activity is observed at this time post-injury; (3) AIH-induced Dia long-term facilitation was not observed in either ipsilateral Dia or T2 EIC activity (see text). KW, KW6002; V, vehicle (dimethyl sulfoxide). Values are means±1 standard error of the mean. *Significantly different from pre-injury values; #significantly different from Sham; p<0.001.