Early exposure to general anesthesia impairs social and emotional development in rats

Abstract

Several animal and emerging human studies suggest an association between an early exposure to general anesthesia (GA) and long-lasting problems with complex social and emotional behaviors such as inattentiveness, impulsivity, anxiogenic tendencies, as well as difficulties engaging in proper social intercourse, with significant increase in attention deficit and hyperactivity-type behaviors. To further investigate these behaviors, and to examine the potential of presently available rodent behavioral models to guide future assessments of long-term socio-emotional impairments in humans, we examined the long-term effects of GA on anxiety/fear and social behaviors. We exposed male and female Sprague-Dawley infant rats at the peak of their synaptogenesis to either GA containing midazolam (9 mg/kg, i.p.), 70% nitrous oxide (N₂O) and 0.75% isoflurane (Iso) administered in 29-30% oxygen (experimental), or air (with 30% oxygen) plus the vehicle, 0.1% dimethyl sulfoxide (Sham) for 6 h. Behavioral experiments were conducted at adolescence (the open-field test) and young adulthood (the open-field test, the elevated plus-maze and the social novelty test). We report that an early exposure to GA during critical stages of brain development results in long-lasting increase in risk-taking tendencies and significant changes in the anxiety-related behaviors when tested in young adult rats. In addition, we noted novelty-seeking tendencies/less guarded behavior with changes in social discrimination. We conclude that early exposure to anesthesia may have lasting influences on emotional and social development. Importantly, our results show that currently used rodent behavioral models could be a good correlate to assess long-term socio-emotional GA-induced impairments observed in humans.

Keywords: Developmental neurotoxicity; Rodents; Sex differences; Social interactions; Thigmotaxic behavior.

Figure 1.

The summary of experimental protocol and timeline of behavioral experiments in rat pups (P7), adolescent (P27-P37) and young adult (P61-P87) rats.

Figure 2.. Open-field behavior of rats exposed to general anesthesia at P7.

A-B. Total distance traveled in the open-field arena during 10 min, measured in adolescent (A) and young adult rats (B). Female rats traveled longer distances than male littermates at both time points, as assessed by two-way ANOVA (adolescent – factor sex: F₁,₇₉=20.58, p<0.001; young adult – factor sex: F₁,₇₉=39.58, p<0.001; interaction and factor treatment not significant for both age groups). The number of animals in each group is indicated at the bottom of each bar. C-D. The distance traveled in the border zone (36% of the entire arena), measured in adolescent (C) and young adult rats (D). Female rats traveled longer distances in the border zone than male littermates at both time points (adolescent – factor sex: F₁,₇₉=8.49, p=0.005; young adult – factor sex: F₁,₇₉=6.03, p=0.016; interaction and factor treatment not significant for both age groups). E-F. The number of entries to the inner zone, measured in adolescent (E) and young adult rats (F). Both male and female young adult rats exposed to GA at P7 had more entries to the inner zone of the open-field arena (64% of the entire arena), as compared to the Sham group (factor treatment: F₁,₇₉=6.58, p=0.012; interaction not significant). Female rats had overall fewer entries to the inner zone (factor sex: F₁,₇₉=9.88, p=0.002). *p<0.05, **p<0.01 and ***p<0.001, two-way ANOVA. Each bar represents the mean + SEM.

Figure 3.. Anxiety-related behavior of Sham and GA-exposed rats in the elevated plus-maze.

A. Total distance traveled in the elevated plus-maze during 5 min, measured in young adult rats. Female rats, irrespective of treatment, traveled longer distances than male littermates at both time points, as assessed by two-way ANOVA (factor sex: F₁,₇₉=73.50, p<0.001; interaction and factor treatment not significant). The number of animals in each group is indicated at the bottom of each bar. B. The percent of time young adult rats spent in open arms of the elevated plus-maze. Female rats, regardless of treatment, showed lower anxiety than male littermates (factor sex: F₁,₇₉=81.44, p<0.001; interaction and factor treatment not significant). C. The distance traveled in the distal zone of the open arms measured in young adult rats. Female rats, regardless of treatment, had longer distances in the distal parts of the open arms than male littermates (factor sex: F₁,₇₉=98.35, p<0.001; interaction not significant). Both male and female young adult rats exposed to GA at P7 traveled shorter distances in the distal zone, relative to Sham controls (factor treatment: F₁,₇₉=4.46, p=0.038). D. The number of entries to the distal zone of the open arms, measured in young adult rats. Female rats, regardless of treatment, explored the distal parts of the open arms more freely than male littermates (factor sex: F₁,₇₉=108.00, p<0.001; interaction not significant). Both male and female young adult rats exposed to GA at P7 had fewer entries to the distal zone than Sham controls (factor treatment: F₁,₇₉=4.92, p=0.030). *p<0.05 and ***p<0.001, two-way ANOVA. Each bar represents the mean + SEM.

Figure 4.. General activity in the three stages of the social novelty test.

A. Total distance traveled during 5 min of habituation, measured in Sham and GA-treated young adult rats. Female rats, irrespective of treatment, traveled longer distances than male littermates, as assessed by two-way ANOVA (factor sex: F₁,₇₃=109.70, p<0.001; interaction and factor treatment not significant). The number of animals in each group is indicated at the bottom of each bar. B. Total distance traveled during 10 min of the sociability phase, measured in Sham and GA-treated young adult rats. Only female rats in the GA group traveled longer distances than Sham controls (interaction: F₁,₇₃=6.37, p=0.014; Sidak’s multiple comparisons test: p=0.009). C. Total distance traveled during 10 min of the social novelty phase, measured in Sham and GA-treated young adult rats. Female rats, irrespective of treatment, traveled longer distances than male littermates (factor sex: F_1,73=15.07, p<0.001; interaction and factor treatment not significant). **p<0.01 and ***p<0.001, two-way ANOVA or Sidak’s test. Each bar represents the mean + SEM.

Figure 5.. Rat behavior during the sociability stage of the social novelty test.

A. Time spent exploring (upper panel) and number of explorations (lower panel) of an unfamiliar conspecific (Stranger 1), measured in Sham and GA-treated young adult rats. The number of animals in each group is indicated at the bottom of each bar. Female rats, irrespective of treatment, had fewer exploration episodes than male littermates, as assessed by two-way ANOVA (factor sex: F₁,₇₃=9.16, p=0.003; interaction and factor treatment not significant). B. Time spent exploring (upper panel) and number of explorations of an empty wire cage (lower panel), measured in Sham and GA-treated young adult rats. Female rats, irrespective of treatment, spent more time exploring an empty cage than male littermates (factor sex: F₁,₇₃=4.61, p=0.035; interaction and factor treatment not significant). C. Time spent exploring (upper panel) and number of entries (lower panel) to the chamber containing an unfamiliar conspecific (Chamber 1), measured in Sham and GA-treated young adult rats. Female rats in the GA group had more entries to Chamber 1 than Sham controls (interaction: F₁,₇₃=4.09, p=0.047; Sidak’s multiple comparisons test: p=0.033). D. Time spent exploring (upper panel) and number of entries (lower panel) to the chamber containing an empty wire cage (Chamber 2), measured in Sham and GA-treated young adult rats. Female rats, irrespective of treatment, spent more time exploring Chamber 2 than male littermates (factor sex: F₁,₇₃=4.77, p=0.032; interaction and factor treatment not significant). Female rats in the GA group had more entries to Chamber 2 than Sham controls (interaction: F₁,₇₃=6.67, p=0.012; Sidak’s multiple comparisons test: p=0.010). *p<0.05 and **p<0.01, two-way ANOVA or Sidak’s test. Each bar represents the mean + SEM.

Figure 6.. Social novelty discrimination of rats exposed to general anesthesia at P7.

A. Time spent exploring (upper panel) and number of explorations (lower panel) of a familiar conspecific (Stranger 1), measured in Sham and GA-treated young adult rats. The number of animals in each group is indicated at the bottom of each bar. B. Time spent exploring (upper panel) and number of explorations (lower panel) of a novel conspecific (Stranger 2). Both male and female rats in the GA group spent more time (factor treatment: F₁,₇₃=6.43, p=0.013) and had more exploration episodes than Sham controls (factor treatment: F₁,₇₃=4.47, p=0.038; interaction and factor sex not significant for both parameters measured). C. Time spent exploring (upper panel) and number of entries (lower panel) to the chamber containing a familiar conspecific (Chamber 1). Female rats, irrespective of treatment, had more entries to Chamber 1 than male littermates (factor sex: F₁,₇₃=11.43, p=0.001; interaction and factor treatment not significant). D. Time spent exploring (upper panel) and number of entries (lower panel) to the chamber containing a novel conspecific (Chamber 2), measured in Sham and GA-treated young adult rats. Both male and female rats in the GA group spent more time exploring Chamber 2 than Sham controls (factor treatment: F₁,₇₃=4.76, p=0.032; interaction and factor sex not significant for both parameters measured). Female rats in the GA group had more entries to Chamber 2 than Sham controls (interaction: F₁,₇₃=4.75, p=0.033; Sidak’s multiple comparisons test: p=0.021). *p<0.05 and **p<0.01, two-way ANOVA or Sidak’s test. Each bar represents the mean + SEM.