Anxiogenic CO2 stimulus elicits exacerbated hot flash-like responses in a rat menopause model and hot flashes in postmenopausal women

Abstract

Objective: As longitudinal studies determined that anxiety is a strong risk factor for hot flashes, we hypothesized that an anxiogenic stimulus that signals air hunger (hypercapnic, normoxic gas) would trigger an exacerbated hot flash-associated increase in tail skin temperature (TST) in a rat ovariectomy (OVEX) model of surgical menopause and hot flashes in symptomatic postmenopausal women. We also assessed TST responses in OVEX serotonin transporter (SERT) rats that models a common polymorphism that is associated with increased climacteric symptoms in postmenopausal women and increases in anxiety traits.

Methods: OVEX and sham-OVEX rats (initial experiment) and wildtype and SERT OVEX rats (subsequent experiment) were exposed to a 5-minute infusion of 20% carbon dioxide (CO2) normoxic gas while measuring TST. Postmenopausal women were given brief 20% and 35% CO2 challenges, and hot flashes were self-reported and objectively verified.

Results: Compared to controls, OVEX rats had exacerbated increases in TST, and SERT OVEX rats had prolonged TST increases following CO2. Most women reported mild/moderate hot flashes after CO2 challenges, and the hot flash severity to CO2 was positively correlated with daily hot flash frequency.

Conclusions: The studies demonstrate that this anxiogenic stimulus is capable of inducing cutaneous vasomotor responses in OVEX rats, and eliciting hot flashes in postmenopausal women. In rats, the severity of the response was mediated by loss of ovarian function and increased anxiety traits (SERT), and, in women, by daily hot flash frequency. These findings may provide insights into anxiety-related triggers and genetic risk factors for hot flashes in thermoneutral environments.

Figure 1. Rats modeling surgical menopause exhibit exacerbated hot flash-associated tail skin temperature responses

Effects of ovariectomy or sham surgery on baseline tail skin temperature in response to hypercapnic or atmospheric air infusion. a) Representative thermal images with scale (to the right) of a sham-OVEX (top) or OVEX rat (bottom) after 5 min exposure to atmospheric air (left) or 20% CO₂ (right). b) Line graph with error bars (SEM) represents mean tail skin temperature prior to and following atmospheric air or hypercapnic gas challenge in OVEX or sham-OVEX groups assessed with a tail thermistor at the base of the tail (n=15/group for CO₂ challenge and n=6/group for atmospheric air challenge). *denotes significance of surgical treatment at specific time points in (b) with Fisher’s Least Significant Difference test protected by an ANOVA, and + denotes significant differences over time from t-1 as measured by Dunnett’s test.

Figure 2. Small clinical study of hot flash provocation with CO₂ challenges in highly symptomatic women

a) Timeline for the study procedures; a room air acclimation (through a mask) was given for 10 min prior to a rest period before a 40s, 20% CO₂ (normoxic) challenge. Participants rested for 15-20 min prior to the 35% CO₂ administration. b) Table illustrates participants assigned number, age, total hot flashes during the day and night for a 7 day period prior to the study, average hot flashes per day, baseline STAI and NRS anxiety, NRS anxiety post control air, NRS hot flash and anxiety (respectively) severity post 20% and 35% CO₂ inhalation, followed by the NRS Anxiety and STAI anxiety at rest. Participants are ranked in order of increasing daily hot flashes. 3 of the 4 participants, and 2 of the 3 participants with confirmed inhalations of 20% CO₂ and 35%CO₂ reported hot flashes within 5 min, respectively; hot flashes were rated from mild to moderate. Numbers below NRS hot flashes or anxiety (columns 9 and 10, respectively) post CO₂ respectively represent 20% CO₂ and 35% CO₂ with the parenthetical number representing the mean of the confirmed inhalations (“-” indicates no confirmed inhalation for respective challenge). Correlation analyses revealed that the frequency of daily hot flashes (total per 24h day) was positively correlated with the c) mean severity of the hot flash post confirmed CO₂ inhalations and d) mean severity of the anxiety response post confirmed CO₂ inhalations. Correlation analysis also demonstrated that the e) mean hot flash severity following verified CO₂ inhalation was positively correlated with the mean anxiety rating following verified CO₂ inhalation.

Figure 3. Rats with a heterozygous null mutation of the serotonin transporter (SERT^+/−) have prolonged hot flash-associated tail responses to hypercapnic gas infusion

Effects of ovariectomy in SERT^+/− and WT rats tail skin temperature in response to hypercapnic gas infusion. a) Representative thermal image with scale (to the right) of a WT-ovex (top) or SERT^+/− rat (bottom) after 5 min exposure to 20% CO₂. b) Line graph with error bars (SEM) represents mean tail skin temperature prior to and following hypercapnic gas challenge in ovex SERT^+/− or WT rats assessed with a tail thermistor at the base of the tail (n=8,7). *denotes significant effect of estrogen treatment with a two-way ANOVA p=0.001 in (a). *denotes significance of genotype at specific time points in (c) with Fisher’s Least Significant Difference test protected by an ANOVA, and + denotes significant differences over time from t-1 as measured by Dunnett’s test.