As another variable to quantify possible detrimental effects of isoflurane on human neuron-like cells, we used an anticaspase 3 antibody in a Western blotting study. Per company data, this antibody can bind to the procaspase 3 and its fragments at 20 kDa and 17 kDa. These fragments are subunits of activated caspase 3.13 We detected a protein band at 32 kDa with this antibody in the differentiated SH-SY5Y cells. This molecular weight corresponds to that of procaspase 3. Isoflurane exposure did not significantly change the expression of this procaspase 3 protein (Fig. 3). We could not detect any protein bands corresponding to the 20- and 17-kDa subunits under control and isoflurane exposure conditions. Isoflurane exposure also did not change the expression of synaptophysin and drebrin in the differentiated SH-SY5Y cells (Fig. 3).
Volatile anesthetics have been frequently used in clinical practice for many decades. However, there is a growing concern in recent years that these drugs can have detrimental effects on the brain. The learning and memory functions of middle-aged rats and old rats were impaired after a 2-hour exposure to clinically relevant concentrations of general anesthetics.4 It has also been shown that isoflurane increases activated caspase 3 expression and Aβ production in mouse brain.5 Volatile anesthetics at clinically relevant concentrations can also induce Aβ oligomerization in vitro,14 a process that is considered to be important for the development of Alzheimer disease. These studies suggest that volatile anesthetics can cause cell injury and functional impairment of brain cells. Consistent with this suggestion, it has been proposed that anesthesia may contribute to the acute phase of POCD,1 a well established clinical syndrome. However, in clinical studies, it has been very difficult to separate anesthesia from other factors, such as surgery and the underlying diseases for which surgery is required. Also, patients who received general anesthesia or regional anesthesia were not different in cognitive functions assessed at 3 months or 6 months after surgery.15–17 Thus, it is not known whether general anesthesia/anesthetics can cause brain cell injury in humans. We showed here that 2% to 4% isoflurane did not cause significant injury to human neuron-like cells and did not increase the expression of the activated caspase 3 in these cells. Desflurane and sevoflurane at high concentrations also did not cause significant cell injury as assessed by LDH release. These results suggest that volatile anesthetics at clinically relevant concentrations may not cause a direct damaging effect on these cells. Consistent with our results, incubation of the differentiated SH-SY5Y cells with 2.5% isoflurane for 16 hours did not cause injury to these cells as assessed by the trypan blue exclusion, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining analysis, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.18 However, these cells could have been injured by isoflurane in our study because isoflurane at a very high concentration (5%) increased LDH release from them.
We used human neuron-like cultures because it is almost impossible to determine anesthetic-induced human neuronal injury in vivo or in primary human neuronal cultures. We used 2 cell lines in this study to make it less likely that the observed noninjury effect is specific to 1 cell line.
Dendritic spines and synapses are unique structures of neurons and the fundamental structures for interneuronal communication and learning and memory functions.19,20 Anesthetic effects on learning and memory functions could be attributable to their effects on dendritic spines and synapses. Our results showed that isoflurane did not affect the expression of synaptophysin and drebrin, proteins used to quantify synapses and dendritic spines, respectively, in studies.7,8 These results suggest that isoflurane does not have a significant effect on the quantity of dendritic spines and synapses in these human cells. A previous study showed that exposure of primary rat neuronal culture to isoflurane decreased drebrin expression and that exposure of 7-day-old mice to isoflurane decreased synaptic density.8 Interestingly, a recent study showed that volatile anesthetics increased dendritic spine density in rats when they were exposed to isoflurane at 16 days old.21 The reason for these different findings is that 7-day-old rodents are in peak time of synaptogenesis and 16-day-old rodents are not. This difference may change their vulnerability/responsiveness to volatile anesthetics.22 However, it is not clear whether age-dependent changes of synaptic vulnerability to volatile anesthetics occur in humans.
Our results do not suggest a significant detrimental effect of volatile anesthetics on human neuron-like cells. Consistent with this suggestion, the role of general anesthesia/anesthetics per se in POCD has not yet been established. Although 2 early studies suggest that general anesthesia may contribute to the development or speed up the development of Alzheimer disease,23,24 the majority of studies have not shown a relationship between general anesthesia/surgery and Alzheimer disease.25–28 Our recent study suggests that spine surgery under general anesthesia may not increase the risk for Alzheimer disease.29 In addition, a recent study did not show that noncardiac surgery was a risk for long-term cognitive decline after surgery.30 Finally, anesthetic toxicity on developing brain has been a research focus because of the concern that brain in the peak synaptogenesis phase may be vulnerable to anesthetics. A recent study showed that children exposed to 1 surgery under general anesthesia before 4 years old did not have learning deficits but children subjected to >1 surgery under anesthesia had higher chances of learning deficits later in their lives.31 However, another recent study using genetically identical twins showed that anesthesia and surgery exposure before 3 years old did not cause learning and memory deficits. Rather, the need for anesthesia and surgery early in life is a marker of vulnerability for developing learning problems later.32 Thus, additional studies are needed to define the potential detrimental anesthetic effects, such as their roles in POCD, and the possible molecular mechanisms/structure changes for these effects in humans.
Our studies have limitations. We used human neuron-like cell cultures. These cells are obviously different from in situ human neurons. It is also impossible to provide cell cultures with an environment identical to what the in situ human neurons have, such as local pH, oxygen and electrolyte concentrations, and the support and interaction from other cells. Thus, our cell cultures may have a lower vulnerability to volatile anesthetics than in situ human neurons. However, mouse neuronal cultures were clearly as vulnerable to isoflurane as in situ mouse neurons,8 suggesting the usefulness of cell cultures in anesthetic toxicity studies. Also, although we used 2 neuron-like cell lines, it is still possible that our findings are cell line specific. This issue can be resolved when additional human neuron-like cell lines are identified and are available. Finally, our results have relatively large standard deviations, although the corresponding standard errors of means are rather small. Large standard deviations can become a resource to obscure drug effects. However, LDH release data from cells exposed to 2% to 4% isoflurane, 6% sevoflurane, or 12% desflurane have relatively small standard deviations and the means under these volatile anesthetic exposure conditions are very similar to those under control conditions. These findings increase our confidence on the conclusion that volatile anesthetics at clinically relevant concentrations do not affect LDH release from human neuron-like cell cultures. Our Western blotting data have relatively large variability. This phenomenon can be attributed to variations in cell conditions (various sets of cells were used in the experiments) and blotting conditions in various experiments. To reduce the effects of variation in experimental conditions on final results, we normalized the data of caspase 3, synaptophysin, and drebrin by that of β-actin in the same samples and then normalized the results of samples treated with 2% to 4% isoflurane to those of the corresponding control. These normalization processes should have increased the possibility to detect isoflurane effects on the expression of caspase 3, synaptophysin, and drebrin proteins.
Supported by a grant (R01 GM065211 to ZZ) from the National Institutes of Health, Bethesda, MD, a grant from the International Anesthesia Research Society (2007 Frontiers in Anesthesia Research Award to ZZ), Cleveland, OH, and the Robert M. Epstein Professorship endowment, University of Virginia.
All 4 authors participated in study design. Drs. Lin and Feng conducted the study. Dr. Lin performed initial data analysis and drafted the Methods section. Dr. Zuo performed the final data analysis and prepared the manuscript.
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