Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits

Abstract

Recently it was demonstrated that exposure of the developing brain during the period of synaptogenesis to drugs that block NMDA glutamate receptors or drugs that potentiate GABA(A) receptors can trigger widespread apoptotic neurodegeneration. All currently used general anesthetic agents have either NMDA receptor-blocking or GABA(A) receptor-enhancing properties. To induce or maintain a surgical plane of anesthesia, it is common practice in pediatric or obstetrical medicine to use agents from these two classes in combination. Therefore, the question arises whether this practice entails significant risk of inducing apoptotic neurodegeneration in the developing human brain. To begin to address this problem, we have administered to 7-d-old infant rats a combination of drugs commonly used in pediatric anesthesia (midazolam, nitrous oxide, and isoflurane) in doses sufficient to maintain a surgical plane of anesthesia for 6 hr, and have observed that this causes widespread apoptotic neurodegeneration in the developing brain, deficits in hippocampal synaptic function, and persistent memory/learning impairments.

Fig. 1.

Triple anesthetic cocktail induces apoptotic neurodegeneration. a–l are light micrographic scenes from various brain regions of either a control rat (a,f, h, j) or a rat exposed to the triple anesthetic cocktail (b–e,g, i, k,l). Some sections are stained by the DeOlmos silver method (a, b, d,f, g, k), and the remainder are immunocytochemically stained to reveal caspase-3 activation (c, e, h–j,l). The regions illustrated are the posterior cingulate/retrosplenial cortex (a–c), subiculum (d, e), anterior thalamus (f, g), rostral CA1 hippocampus (h, i), and parietal cortex (j–l). The individual nuclei shown in the anterior thalamus (f, g) are laterodorsal (LD), anterodorsal (AD), anteroventral (AV), anteromedial (AM), and nucleus reuniens (NR).m and n are electron micrographic scenes depicting the ultrastructural appearance of neurons undergoing apoptosis. The cell in m displays a very early stage of apoptosis in which dense spherical chromatin balls are forming in the nucleus at a time when the nuclear membrane remains intact and very few changes are evident in the cytoplasm. The cell in nexhibits a much later stage of apoptosis in which the entire cell is condensed, the nuclear membrane is absent, and there is intermixing of nuclear and cytoplasmic constituents. These are hallmark characteristics of neuronal apoptosis as it occurs in the in vivo mammalian brain.

Fig. 2.

Effects of anesthetic exposure on LTP in the CA1 region of rat hippocampal slices. a, The graph depicts the time course of change in field EPSP slope (± SEM) in hippocampal slices from control rats treated with DMSO (●) and rats exposed to the triple anesthetic combination (triple c., ○). A single 100 Hz × 1 sec tetanus was delivered at time 0 (arrow). b, Traces to theright of the graph show examples of EPSPs before (solid traces) and 60 min after (dashed traces) the tetanus in slices from animals treated with the various anesthetics. Hippocampal slices were prepared at P29–P33 from rats treated at P7.

Fig. 3.

Effects of neonatal triple anesthetic cocktail treatment on spatial learning. a, Rats were tested at P32 for their ability to learn the location of a submerged (not visible) platform. An ANOVA of the escape path length data yielded a significant main effect of treatment (p = 0.032) and a significant treatment by blocks of trials interaction (p = 0.024), indicating that the performance of the rats that received the anesthetic cocktail was significantly inferior to that of control rats during place training. Subsequent pairwise comparisons indicated that differences were greatest during blocks 4, 5, and 6 (p = 0.003, 0.012, and 0.019, respectively). However, the rats receiving the anesthetic cocktail improved their performance to control-like levels during the last four blocks of trials. b, Rats were retested as adults (P131) for their ability to learn a different location of the submerged platform. The graph on the left represents the path length data from the first five place trials when all rats were tested. An ANOVA of these data yielded a significant main effect of treatment (p = 0.013), indicating that the control rats, in general, exhibited significantly shorter path lengths in swimming to the platform compared with anesthetic cocktail rats. Subsequent pairwise comparisons showed that differences were greatest during block 4 (p = 0.001). The graph on theright shows the data from study 2 rats that received 5 additional training days as adults. During these additional trials, the control group improved their performance and appeared to reach asymptotic levels, whereas the anesthetic cocktail rats showed no improvement. An ANOVA of these data yielded a significant main effect of treatment (p = 0.045) as well as a significant treatment by blocks of trials interaction (p = 0.001). Additional pairwise comparisons showed that group differences were greatest during blocks 7, 8, and 10 (p = 0.032, 0.013, and 0.017, respectively).c, Probe trial performance of anesthetic cocktail and control rats during adult testing. Search behavior of the rats was quantified when the submerged platform was removed from the pool after the last place trials in blocks 5 and 10. The histogram on theleft presents data for rats of both studies 1 and 2 combined after five blocks of place trials were completed. The histogram on the right presents data for rats of study 2 alone, after 10 blocks of place trials were completed. Thedotted line represents the amount of time that animals would be expected to spend in the target quadrant based on chance alone. Both histograms show that the control rats spent significantly more time in the target quadrant than the anesthesia-exposed rats, regardless of whether the probe tests were performed on both study groups after five blocks or only on the study 2 rats after 10 trials.d, e, Data from the radial arm maze test performed on P53 to evaluate spatial working memory capabilities are shown.d, A histogram showing that the anesthetic cocktail rats required significantly more days to reach a criterion demonstrating learning (8 correct responses out of the first 9 responses for 4 consecutive days) compared with controls. e, Plotting the days to criterion data as the cumulative percentage of rats reaching criterion in each group as a function of blocks of training days shows that the acquisition rate of the anesthetic cocktail rats began to slow around the fourth block of trials and remained slower throughout the rest of the experiment. Numbers inparentheses in each graph indicate sample sizes. *p < 0.05; Bonferroni corrected level:^†p < 0.005 in a;^†p < 0.01 in b.