The long-term impact of ovariectomy on ventilation and expression of phrenic long-term facilitation in female rats

Abstract

New findings: What is the central question of this study? Would ovariectomy cause prolonged changes in ventilation and sustained loss of acute, intermittent hypoxia-induced neuroplasticity or would these outcomes be restored with time? What is the main finding and its importance? Our main findings demonstrate that ovariectomy elicits minimal alteration in overall breathing function but impairs acute, intermittent hypoxia-induced plasticity for ≤ 12 weeks.

Abstract: Sex hormones are necessary to enable respiratory neuroplasticity, including phrenic long-term facilitation (pLTF), a form of respiratory motor plasticity elicited by acute, intermittent hypoxia (AIH). Female rats exhibit a progressive increase in phrenic nerve amplitude after AIH characteristic of pLTF only during pro-oestrus, the stage of the oestrous cycle notable for elevated circulating oestradiol levels. Removal of the ovaries [ovariectomy (OVX)], the primary source of circulating oestradiol, also eliminates AIH-induced pLTF after 1 week. Ovariectomy is used routinely as a model to examine the impact of sex hormones on CNS structure and function, but the long-term impact of OVX is rarely examined. Extra-ovarian sites of oestradiol synthesis, including multiple CNS sites, have been identified and might possess the capacity to restore oestradiol levels, in part, over time, impacting respiratory function and the expression of respiratory neuroplasticity. We examined both ventilation in awake, freely behaving female rats, using barometric plethysmography, and the expression of AIH-induced pLTF in anaesthetized, ventilated female rats 2 and 12 weeks after OVX and compared them with age-matched ovarian-intact female rats. Our findings indicate that chronic OVX had little impact on baseline breathing or in the response to respiratory challenge (10% O₂ , 5% CO₂ , balance N₂ ) during plethysmography. However, OVX rats at both 2 and 12 weeks demonstrated a persistent loss of AIH-induced pLTF relative to control animals (P < 0.01), suggesting that other sources of oestradiol synthesis were insufficient to restore pLTF. These data are consistent with our previous work indicating that oestradiol plays a key role in expression of AIH-induced respiratory neuroplasticity.

Keywords: long-term facilitation; oestrogen; oevarectomy; respiratory neuroplasticity.

FIGURE 1

Study design. Experiments were designed to ensure that rats were similar in age during plethysmographic and phrenic neurophysiological testing. Ovariectomy (OVX) was completed either 2 weeks (2wks) or 12 weeks (12wks) before plethysmographic testing. Sham OVX surgeries were completed 12 weeks before testing. Sham rats were staged daily leading up to plethysmography and phrenic nerve recordings to ensure that testing was completed during pro‐oestrus, the stage of highest circulating oestradiol, in accordance with our previous study (Dougherty et al., 2017a)

FIGURE 2

Ventilation measured with barometric plethysmography is similar after ovariectomy (OVX). Ventilatory measures were recorded during baseline conditions (BL; 20.9% O₂, balance N₂) and hypercapnic hypoxia (Max; 10% O₂, 5% CO₂, balance N₂) for each treatment: Sham controls (n = 5) and female rats 2 weeks (2wk) post‐OVX (n = 5) and 12 weeks (12wk) post‐OVX (n = 5). Brething frequency (a), tidal volume (V _T; b) and minute ventilation (${\dot{V}}_{\mathrm{E}}$; c) were similar during BL (P > 0.05). All groups showed statistical increases in frequency and ${\dot{V}}_{\mathrm{E}}$ in response to Max challenge (i.e., condition effect; P < 0.001), but only the 12wk OVX group showed an increase in V _T with Max relative to BL (P < 0.001). Data represent two‐way ANOVA with repeated measures and Bonferroni post hoc tests. ⁺⁺ P ≤ 0.001 compared with BL

FIGURE 3

Phrenic long‐term facilitation (pLTF) is lost for 12 weeks (12wk) after ovariectomy (OVX). Phrenic nerve recordings were assessed in anaesthetized, mechanically ventilated rats (n = 5 per group). (a) Raw phrenic amplitudes in baseline (BL) recording conditions were similar between experimental groups (one‐way ANOVA; P = 0.09). (b) To discern expression of pLTF, phrenic amplitudes 60 min post‐acute intermittent hypoxia (AIH) were expressed relative to BL and compared using two‐way ANOVA. Significant effects of time (P < 0.001) and treatment (P = 0.018) and a significant time × treatment interaction (P = 0.018) were identified. Sham control rats showed a persistent increase in phrenic amplitude above BL (ΔmV, 2.41 mV; 89 ± 42%; P < 0.001) indicative of pLTF, whereas OVX groups showed no evidence of pLTF (2wk: ΔmV, 2.96 mV, 29 ± 13%; 12wk: ΔmV, 1.693 mV, 22 ± 33%; both P > 0.05). Phrenic amplitude in Sham rats 60 min post‐AIH was also elevated significantly above that of both OVX groups (P < 0.01). ⁺⁺⁺ P < 0.001 compared with BL; ^## P < 0.01 compared with both OVX groups

FIGURE 4

Phrenic hypoxic responses are modestly impacted by ovariectomy (OVX). (a) We compared the phrenic neural response to hypoxia between experimental groups (n = 5 per group). Raw phrenic nerve amplitudes during hypoxia were similar between experimental groups (one‐way ANOVA; P = 0.29). (b) When compared as a relative increase above baseline (BL) amplitudes, 2 week (2wk) OVX rats exhibited a significantly reduced hypoxic response relative to Sham (P = 0.03), but this difference was lost by 12 weeks (12wk) post‐OVX (one‐way ANOVA; P = 0.084). ^* P < 0.05 compared with Sham

FIGURE 5

Serum oestradiol concentrations are chronically reduced after ovariectomy (OVX) and are correlated with phrenic long‐term facilitation (pLTF). (a) One‐way ANOVA indicated a significant difference in serum oestradiol concentrations between experimental groups (P < 0.001). Ovariectomy caused a persistent reduction in serum oestradiol concentrations at both 2 weeks (2wk; P < 0.001) and 12 weeks (12wk; P < 0.01) compared with Sham rats. (b) Linear regression analysis demonstrated a significant relationship between serum oestradiol and the magnitude of pLTF expression across groups [P = 0.039, r ² = 0.36, F = 5.618; degrees of freedom numerator, degrees of freedom demoninator (1,10); shown with 95% confidence intervals]. ^** P ≤ 0.01, ^*** P ≤ 0.001 compared with Sham