There is concern that clinical use of anesthetic drugs may cause neurotoxicity in the developing brain and subsequent abnormal neurobehavior. We therefore evaluated neurotoxic effects of inhalation anesthetics in the neonatal rat brain, using in vivo histological and neurobehavioral outcomes. Wistar rats (
There is accumulating evidence that, during synaptogenesis, the brain is sensitive to toxicity from many environmental agents, potentially resulting in neurological injury manifesting as neuroapoptosis [
The importance of evaluating experimental and preclinical data on the extent and significance of anesthetic-induced neurotoxicity has recently propelled this issue to the forefront of research in anesthesiology prompting the US Food and Drug Administration (FDA) to establish an Expert Working Group [
We deliberately chose to use postnatal day 15 (PD15) male Wistar rats for all experiments in an effort to explore the link between piriform cortical neurotoxicity and neurobehavioral outcomes in an animal model exposed to clinically relevant concentrations of anesthetic agents. While PD15 immediately follows the most prominent brain growth spurt in rats, it is very difficult to perform behavioral studies in younger animals. All animals were administered either an experimental drug or placebo. They were then allowed to recover in the home cage for either 48 hours or 96 hours. Behavioral data were collected, and the animals were sacrificed. Their brains were extracted and stored for later analysis. The experimental procedures were approved by the Animal Ethics Review Committee of the Biomedical Facility, University College Dublin, where these animals were bred, and were carried out by an individual who held the appropriate license issued by the Irish Minister of Health and Children.
The animals were administered a single dose of anesthetic via a route appropriate to the agent employed (subcutaneous injection or inhalation), with vehicle-treated animals serving as controls. Urethane, isoflurane, and sevoflurane were tested for potential neurotoxicity, and the animals were exposed to these agents at drug concentrations and doses approximated to the clinical setting. The primary reason for using urethane as a positive control is that it is a prototypic agent that is well known to be neurotoxic to the piriform cortex [
Depth of anesthesia was determined by an a priori calculation of the inhaled volatile agent MAC, with measurement of end-tidal concentration to ensure that this MAC was being delivered. In the rat, minimum alveolar concentration (MAC) can be determined using the tail-clamp technique. There is an age-dependent relationship with respect to anesthetic requirements, with MAC being significantly higher in younger animals. The most up-to-date values for MAC in the neonatal rat are from Orliaguet and colleagues [
To determine behavioral manifestations of any possible acute developmental insults to the brain, behavioral parameters were collected for each animal prior to sacrifice, that is, on day 2 or day 4 after anesthetic exposure. The tests used in this experiment were based on available recommendations for the behavioral assessment of functional neurotoxicity in young rats [
The ultimate goal of this study was to quantify the total number of cells and the number of dying cells in a specific layer of the piriform cortex of animals who had received a study drug and to compare these counts with those of the control animals. Thus, the total number of cells in specific preassigned areas of sections of the piriform cortex was counted in 6 alternate 30
Each animal was sublethally anesthetized with sodium pentobarbitone (Euthatal, Merial Animal Health Ltd., Dublin; 200 mg/ml) at a dose of 50 mg/Kg. A deep surgical plane of anesthesia was identified when there were no response to deep stimulation, absent corneal and righting reflexes, and a shallow breathing pattern. The heart was then cannulated, and the brain was perfused and fixed with 4% buffered paraformaldehyde. After extraction of an animal brain from its skull, it was immediately placed in a specimen tube containing 30% sucrose, a cryoprotectant, and stored for 48 hours at 4°C. They were then “snap-frozen” in precooled
For differentiation, we used acid alcohol 1%. “Eosin Y aqueous” (Sigma Diagnostics, Missouri, USA) was then applied. Eosin Y is used as a fluorescent indicator, and its fluorescence can be seen with dark-ground illumination, without special filters. Stained sections were mounted with “Citifluor” (Agar Scientific, UK).
For cell counts, three anatomical regions of layer II of the piriform cortex on each side of the brain were selected, as this location has previously been described as being a particularly vulnerable area of the developing brain for anesthetic-induced neurotoxic injury [
The grid section that was used for counting cells was 60,000
Since six segments of piriform were counted in each brain section, the total volume of the piriform cortex analyzed per brain section was 10,800,000
The frequency of injured cells in layer II of the right and left piriform cortexes was then assessed. The total number of dying cells in each of the 6 segments as described above, in each of the six sections, from each of the brains was counted. These damaged granule cells were initially identified under regular light, with their pyknotic nuclei and eosinophilic cytoplasm, and then confirmed under UV light by their fluorescent yellow cytoplasm. The total number of dying cells per piriform cortex was then calculated for each brain and thus for each study group using the Cavalieri principle.
Hence, using measured cell counts, mean ± SEM values were calculated for each brain with the results expressed as the (a) total cell count per piriform cortex and (b) total eosinophilic cells per piriform cortex. These results were then used to generate the mean ± SEM values for each animal group. For statistical analysis, 6 brains were counted from each of the 10 study groups.
Data were entered in a database using GraphPad Prism v.4.0 (Graph Pad Inc., San Diego, CA) and examined for distribution using the Kolmogorov–Smirnov test. Normally distributed parametric data were compared using analysis of variance with post hoc Dunnett’s test for a defined control group. The Wilcoxon rank test was used for comparison of all the groups when data were not normally distributed. Data were expressed as mean ± SEM, and
Data were collected on a total of 69 animals, out of a possible 79 experimental animals (this was due to 6 animals not surviving anesthesia and 4 animals being omitted from the behavioral stage in error). Of the six animals that died under anesthesia, 2 were from 18 animals under isoflurane anesthesia and 4 from 21 under urethane anesthesia. No animals died under sevoflurane anesthesia. The animals were weighed at the start of the study on day 1 and on day 5 prior to sacrifice (i.e., the 96-hour group). As expected, the control animals gained weight over the experiment duration, as did the sevoflurane-exposed and isoflurane-exposed animals. The urethane animals however failed to thrive and had no significant weight gain over the 5-day experiment duration (28.81 ± 5.47 g versus 27.5 ± 8.87 g,
Animal weights on day 1 and day 5 after anesthetic exposure. The indicated treatments were administered in a single dose to PD15 rats for 4 hours (for inhalational agents) or as a single IV bolus (urethane), as indicated in Methods. Data represent mean ± SEM, with the number of animals indicated on the graph. Control animals were treated with air or saline, depending on the delivery route. Symbols indicate day 5 values that differ significantly from the day 1 values for that group (
To determine behavioral manifestations of any possible acute developmental insults to the brain, behavioral parameters were collected for each animal prior to sacrifice, as described in Methods. An individual behavioral data sheet was filled out for each animal, and the animal’s behavior was assessed immediately prior to terminal anesthesia, that is, day 2 or day 4 after anesthetic exposure. This two-stage behavioral assessment comprised 5-minute observation in a Perspex activity box and 30-minute observation of maternal-pup interaction in the home cage. Parameters assessed were as follows: Locomotion/activity Grooming Rearing Pivoting Vocalization Retrieval efficiency Maternal grooming Suckling score Maternal resting time
Overall, the behaviors observed in control and treated groups fell within normal limits. However, some parameters displayed a statistically significant change in drug-treated animals, when compared to control animals (Table Locomotor activity at 48 and 96 hours Grooming at 48 hours Rearing at 48 and 96 hours Suckling at 48 and 96 hours
Behavioral parameters of neonatal rats exposed to the anesthetic agents sevoflurane and isoflurane at 1 MAC for 4 h on postnatal day 15 and sacrificed 48 or 96 h later.
Behavioral parameter | Time (h) | Control | Sevoflurane | Isoflurane | Urethane |
|
---|---|---|---|---|---|---|
Locomotion/activity score | 48 | 9.9 ± 1.66 ( |
11.7 ± 2.70 ( |
9.38 ± 3.21 ( |
5.50 ± 1.32 ( |
0.03 |
Locomotion/activity score | 96 | 11.2 ± 2.41 ( |
15.8 ± 1.56† ( |
15.4 ± 2.03† ( |
6.78 ± 2.17 |
0.02 |
Rearing score | 48 | 2.60 ± 0.68 | 6.78 ± 1.04† | 3.38 ± 0.78 | 1.25 ± 0.25 |
0.03 |
Rearing score | 96 | 3.67 ± 0.68 | 7.56 ± 1.21† | 4.25 ± 0.64 | 4.11 ± 1.31 | 0.01† |
Suckling score | 48 | 3.20 ± 0.53 | 1.67 ± 0.33 |
2.63 ± 0.37 | 3.00 ± 1.08 | 0.04 |
Suckling score | 96 | 2.42 ± 0.36 | 0.89 ± 0.26 |
1.13 ± 0.23 | 4.56 ± 0.71 | 0.04 |
Grooming score | 48 | 2.80 ± 0.53 | 2.22 ± 0.43 | 2.5 ± 0.57 | 1.50 ± 1.19 |
0.04 |
Animals were assessed behaviorally immediately prior to sacrifice as described in Methods. The
Specifically, in sevoflurane-exposed animals, locomotion was not different at 48 hours when compared to controls; however, at 96 hours, there was a statistically significant increase in animal activity (Table
In urethane-exposed animals, activity significantly decreased at both 48 and 96 h, as was rearing at 48 h when compared to control animals. However, rearing was comparable to controls at 96 h. Suckling scores were similar to controls at both timepoints; however, suckling time significantly increased in the urethane-exposed animals (
Total cell counts were calculated for layer II of the piriform cortex of each of the drug-treated animals, and a mean ± SEM value was calculated for each study group. When these were compared to those of the control brains, a statistically significant reduction was seen in all three drug groups (
Neuronal integrity in the piriform cortex following anesthetic exposure. Data represent the mean ± SEM of total cell numbers in layer II of the piriform cortex in animals sacrificed 48 h and 96 h after anesthetic exposure on PD15. Values were calculated using unbiased stereological techniques, and those differing significantly from vehicle-exposed controls are indicated by an asterisk (
Mean values were generated for each of the study groups for the number of dying cells per piriform cortex, and drug groups were compared to controls. In comparison with controls, there was a statistically significant increase in the number of apoptotic cells in the isoflurane- and urethane-exposed brains at both timepoints, that is, at 48 and 96 h (
Neurodegeneration in the piriform cortex following anesthetic exposure. Data represent the mean ± SEM of total dying cells in layer II of the piriform cortex in animals sacrificed 48 h and 96 h after anesthetic exposure on PD15 (numbers of animals as per Figure
We have demonstrated in this in vivo rat model of the neonatal developing brain that a single 4-hour exposure to isoflurane produces piriform cortical toxicity similar to that reported previously with the antiquated anesthetic, urethane. Sevoflurane, in contrast, was shown to induce significantly less neurotoxicity, which recovered to baseline at 96 hours. Thus, although our data showed that all three anesthetic drugs cause some degree of neurological injury, sevoflurane appears to be least neurotoxic. This is consistent with our previous in vitro observation that isoflurane and enflurane produce pronounced antiproliferative effects in comparison with sevoflurane [
The piriform cortex receives input from the olfactory bulb and sends efferents to the hippocampus via the entorhinal cortex. It thereby connects the two canonical neurogenic regions of the adult rodent brain. For over two decades, this cortical region has been known to contain a population of neurons immunoreactive for markers of neuroplasticity including the polysialylated neural cell adhesion molecule, which is usually highly expressed in newly generated neurons [
The reduced neurotoxicity associated with the newer agent, sevoflurane, may reflect its lower solubility in blood [
In contrast, the isoflurane- and urethane-treated animals did have evidence (albeit limited) of neuroapoptosis. However, it is obvious that the small number of dying cells we counted four days after anesthetic exposure could not, in itself, account for the large amount of cell loss we documented. Interestingly, Eidt et al. demonstrated that cell death in the rat piriform cortex peaks during 12–24 h following a neurological insult (status epilepticus) and that, after this period, very few dying cells are apparent at any given timepoint, despite a significant loss of neuronal integrity [
It is generally accepted that all general anesthetics in current clinical practice have either
In chicken B lymphocytes, isoflurane triggers apoptosis by activating the endoplasmic reticulum (ER) membrane inositol 1,4,5-triphosphate (IP3) receptors, which results in excessive calcium release from the ER [
Our behavioral data demonstrated decreased activity in the urethane animals at 48 hours and 96 hours. These effects, along with the observed decrease in rearing and grooming, are typical manifestations of drugs that induce gross toxicity and most likely correlate with the failure of these animals to thrive. In contrast, increased activity was demonstrated in the sevoflurane and isoflurane animals at 96 hours. The sevoflurane animals also showed increased rearing and decreased suckling scores at 48 and 96 hours. The clinical significance of this overall pattern is difficult to interpret. However, it most likely reflects the distinct recovery characteristics of the different volatile agents. Since sevoflurane is more rapidly cleared than other anesthetics, patients recover faster from sevoflurane anesthesia, and this may account for the increased agitation and rearing observed since, in humans, sevoflurane is associated with distinct emergence agitation, particularly in children [
We studied the effect of these volatile anesthetics on neuroapoptosis on postnatal day 15, somewhat later than many previous reports. One previous report did examine synaptic alterations induced by a 2-hour anesthetic exposure to isoflurane, sevoflurane, and desflurane on postnatal day 16 [
Numerous studies have shown that many anesthetics, and other drugs currently in clinical use, can produce widespread apoptosis in the developing rodent brain [
Anand and Soriano question whether these animal findings are attributable to the effects of anesthetics or whether other clinical factors (surgery, starvation, hypoxia, etc.) may be influential and whether any of these findings in rat and mice pups can be extrapolated to humans [
It is clear that a multidisciplinary effort, such as that epitomized by the SmartTots collaboration between the International Anaesthesia Research Society and the US FDA, is required to determine exactly how safe anesthetics are in human infants and children [
With respect to the dosing regimen used in this study, we attempted to approximate the clinical setting throughout and importantly only subjected the animals to a single dose of drug. We exposed the animals to only 1.0 MAC volatile agent; however, it could be argued that the 4-hour exposure time, despite being similar or markedly reduced in comparison with other relevant studies, may have been excessive, as the PD15 rat brain has a greatly accelerated rate of cell turnover when compared to the human infant [
In this rat model of the neonatal developing brain, we have shown that volatile anesthetics are neurotoxic to the piriform cortex using behavioral and histochemical techniques, but that this effect is significantly less marked and may recover completely with sevoflurane. Further experimental and clinical studies are warranted to fully understand the mechanism of this effect in order to minimize it and to identify those drugs that have the least potential for clinical neurotoxicity.
The authors declare that they have no conflicts of interest.
The authors acknowledge financial support from the UCD School of Medicine.