Repeated brief isoflurane anesthesia during early postnatal development produces negligible changes on adult behavior in male mice

Brain development is a complex process regulated by genetic programs and activity-dependent neuronal connectivity. Anesthetics profoundly alter neuronal excitability, and anesthesia during early brain development has been consistently associated with neuroapoptosis, altered synaptogenesis, and persistent behavioral abnormalities in experimental animals. However, the depth, and even more the duration and developmental time point(s) of exposure to anesthesia determine the neuropathological and long-term behavioral consequences of anesthetics. Here, we have investigated adulthood phenotypic changes induced by repeated but brief (30 min) isoflurane anesthesia delivered during two distinct developmental periods in male mice. A set of animals were subjected to anesthesia treatments at postnatal days 7, 8 and 9 (P7-9) when the animals are susceptible to anesthesia-induced neuroapoptosis and reduced synaptogenesis. To control the potential influence of (handling) stress, a separate group of animals underwent repeated maternal separations of similar durations. Another set of animals were exposed to the same treatments at postnatal days 15, 16 and 17 (P15-17), a developmental time period when anesthetics have been shown to increase synaptogenesis. Starting from postnatal week 9 the mouse phenotype was evaluated using a battery of behavioral tests that assess general locomotor activity (home cage activity, open field), learning and memory (water maze) and depression- (saccharin preference, forced swim test), anxiety- (light-dark box, stress-induced hyperthermia) and schizophrenia- (nesting, prepulse inhibition) related endophenotypes. Apart from mild impairment in spatial navigation memory, exposure to anesthesia treatments during P7-9 did not bring obvious behavioral alterations in adult animals. Importantly, maternal separation during the same developmental period produced a very similar phenotype during the water maze. Mice exposed to anesthesia during P15-17 showed mild hyperactivity and risk-taking behavior in adulthood, but were otherwise normal. We conclude that significantly longer administration periods are needed in order for early-life repeated exposures to anesthetics to produce behavioral alterations in adult mice.Peer reviewe

Animals exposed to repeated brief isoflurane anesthesia or maternal separation at postnatal days 15–17 show varying circadian activity at adult age.

<p>Hourly average circadian activity during 7-day monitoring (<b>A, D</b>), average circadian activity during different light-cycles (<b>B, E</b>) and average circadian activity during the first dark period in comparison to remaining dark periods (<b>C, F</b>). Lights off (active period; grey) during 6:00 PM– 6:00 AM. Abbreviations: ISO, isoflurane; MS, maternal separation. ***<0.001 two-way ANOVA followed by Newman-Keuls <i>post hoc</i> test MS vs. Sham. ###<0.001, ##<0.01 two-way ANOVA followed by Newman-Keuls <i>post hoc</i> test ISO vs. MS. N = 6-8/group.</p

Early-life exposure to three consecutive and brief isoflurane anesthesias or maternal separations do not produce long-lasting depression-related behavior.

<p>Saccharin preference (<b>A, E</b>). Latency to immobility (<b>B, F</b>) and relative immobility during 2–6 min (<b>C, G</b>) in forced swimming test. Stress-induced hyperthermia (<b>D, H</b>). Abbreviations: ISO, isoflurane; MS, maternal separation; FST, forced swimming test; SIH, stress-induced hyperthermia. N = 6-8/group.</p

Experimental time-line.

<p>At postnatal days 7, 8 and 9 (P7-9) or 15, 16 and 17 (P15-17) male mice were subjected to 30 min maternal separation or 30 min isoflurane anesthesia, or were left unhandled (sham). Animals were weaned at P21 (week 3; W3). Beginning from age of 9-weeks animals were subjected to a behavioral phenotyping battery consisting of circadian activity, nest construction and saccharin preference (InfraMot monitoring system), light-dark box, open field, prepulse inhibition (PPI), water maze, stress-induced hyperthermia and forced swim test. N = 6-8/group.</p

Early-life exposure to three consecutive and brief isoflurane anesthesias or maternal separations do not produce long-lasting schizophrenia-related behavior.

<p>Nesting behavior (<b>A, C</b>). Prepulse Inhibition (prepulse 68dB or 80dB) (<b>B, D</b>). Abbreviations: ISO, isoflurane; MS, maternal separation; PPI, prepulse inhibition. N = 6-8/group.</p

Early-life exposure to three consecutive and brief isoflurane anesthesias or maternal separations at postnatal days 7–9 bring mild deficit in spatial navigation memory.

<p>Latency to escape (find the platform) (<b>A, E</b>) and time spent near the vicinity of pre-existing platform (quadrant, zone) (<b>B, F</b>) during the first learning trials and probe test, respectively. Latency to escape (find the platform) (<b>C, G</b>) and time spent near the vicinity of pre-existing platform (quadrant, zone) (<b>D, H</b>) during the reverse learning trials and probe test, respectively. Abbreviations: ISO, isoflurane; MS, maternal separation. *<0.05, **<0.01, two-way ANOVA followed by Newman-Keuls <i>post hoc</i> test. N = 6-8/group.</p

Early-life exposures to three consecutive and brief isoflurane anesthesias at postnatal days 15–17 produce mild long-lasting decrease in anxiety-related behavior.

<p>Distance travelled (and habituation) (<b>A, G</b>) and time in center (<b>B, H</b>) in a novel open environment (open field test). Latency to light (<b>C, I</b>), relative distance travelled (<b>D, J</b>), distance in light (<b>E, K</b>) and rearings in light (<b>F, L</b>) in the light-dark box test. Abbreviations: ISO, isoflurane; MS, maternal separation; OF, open field test; LD, light-dark box test. *<0.05, two-way ANOVA followed by Newman-Keuls <i>post hoc</i> test. N = 6-8/group.</p