Multiple anesthetics have previously been reported to lead to caspase activation and neuronal cell death throughout the brain of postnatal day 7 animals1
. We report in an accompanying article in this issue that isoflurane decreases NPC proliferation (BrdU uptake) in vivo
in adults and neonates4
. This could be due to isoflurane mediated decreases in neuronal firing in neonatal and adult animals29
or to direct toxicity in neonates where cell death has been observed3
. Here we have attempted to determine whether this effect is independent of the actions of isoflurane on the surrounding brain by growing NPCs in an in vitro
environment. Cultures of hippocampal precursor cells were isolated from postnatal day 2 rats in order to maximize the contribution of cells from the DG and minimize the contribution from the sub-ventricular zone and other brain regions. Embryonic animals are often used for NPC cultures, however in rats the hippocampus is poorly defined or absent until birth making isolation nearly impossible. We are most interested in NPCs specifically from the dentate in order to correlate with in vivo
studies of this region and therefore obtained our cultures from postnatal day 2 animals.
Translating anesthetic studies from animals (let alone cell culture) to humans is at least flawed if not impossible. In order to minimize differences between and in vitro
and in vivo
models of anesthesia a comparable dose must be used. One can use an equal dose such as 1% isoflurane that is not clinically equivalent across species or one can choose to measure a clinical endpoint such as minimum alveolar concentration and use a dose that produces an equivalent endpoint between species. We have chosen to base our in vivo
studies on minimum alveolar concentration and therefore use the same dose for the in vitro
studies presented here. Fentanyl was used as a control in these studies because both GABAA
and N-methyl-D-aspartate receptors mediate signals that are known to influence proliferation and differentiation of NPCs13–16
and most anesthetic drugs act at one or both of these receptors29
. Opiates do not act at GABAA
or N-methyl-D-aspartate receptors and fentanyl is a commonly used opiate and was chosen as a control for that reason.
In the experiments presented here, with isoflurane as the sole anesthetic, we were unable to induce cell death, caspase activation, or LDH release in cultured NPCs. This suggests that NPCs do not undergo apoptosis or necrosis following isoflurane exposure, consistent with limited cell death being reported in the DG of the hippocampus (the source of these cultured cells) compared with other regions of the brain3
. Decreased proliferation 48 hours after isoflurane exposure combined with a decrease in the number of cells in S-phase and loss of the cell cycle regulator Ki-67 suggests the cells are either in growth phase arrest or have exited the cell cycle as would occur with differentiation. Cell cycle exit and differentiation is further suggested by the loss of the stem cell gene Sox2, and increased neuronal fate selection.
Culturing cells in different states can change the characteristics of those cells and how they respond to different signals or stresses and represents a potential limitation of this study. These experiments were performed on cells in two different states: floating as neurospheres or adherent to glass slides but undifferentiated. The results we observed, however, are similar in both cases. Growth inhibition, loss of Ki-67, and lack of caspase activation in floating neurospheres and decreased ratio of cells in S-phase and lack of nuclear cleaved caspase 3 in adherent cells, are all consistent with a lack of cell death and a decrease in proliferation. Similar effects on proliferation in vivo
are also reported in this issue4
Isoflurane facilitates opening of the GABAA
receptor, and has previously been shown to raise the intracellular calcium concentration in neurons and slice cultures by liberating it from endoplasmic reticulum30–33
receptor opening, and increased intracellular calcium decrease proliferation of precursor cells in sub ventricular zone13,34
and increase differentiation and selection of a neuronal fate in hippocampal progenitors of the DG14,16,35
. Isoflurane may lead to cell cycle exit and differentiation of hippocampal NPCs by facilitating GABAA
receptor opening and increasing intracellular calcium. Future studies are necessary to determine the exact mechanism of isoflurane mediated growth inhibition and increased neuronal differentiation we have observed in NPCs, but GABAA
receptor activation and changes in calcium concentration are likely targets.
Decreased proliferation and increased neuronal differentiation caused by isoflurane could lead to cognitive dysfunction in neonates by permanently disrupting the architecture of the hippocampus during a critical period of development or by depleting the pool of precursor cells present for the duration of the animals life. The same mechanism in an adult might not cause any cognitive dysfunction when the hippocampus is already fully developed and connected and can more easily accept an increased number of cells choosing to become neurons at any given time. The adult hippocampus routinely integrates new neurons into its circuitry with learning10,36
Elsewhere in this issue we report isoflurane-mediated changes in NPC proliferation in vivo4
and the present experiments demonstrate an effect of isoflurane on hippocampal neural precursor cells grown in culture, isolated from the DG demonstrating that this effect is on the cells themselves rather than being mediated by the surrounding tissue of the neonatal brain or specifically the DG. In addition to suggesting a possible mechanism for isoflurane-induced hippocampal dysfunction, the role of anesthesia in the field of neural stem cells and neural stem cell transplantation remains largely unexplored. The results reported here show that isoflurane may have a direct impact on the proliferation and fate selection of transplanted stem cells. Future studies on the effect of volatile and non-volatile anesthetics on NPCs are important for our understanding of how these drugs effect the biology of NPCs and eventually for choosing the appropriate anesthetic for trials of stem cell transplantation in animals and someday humans.
This model system can be used to efficiently screen many anesthetics that have been previously reported to cause neurodegeneration to determine if they have an effect on NPC proliferation and differentiation. This system may also be used to address questions of mechanism that are difficult to answer with in vivo studies. Understanding how anesthetics interact with the complex system of neurogenesis in the neonatal and adult brain may provide some insight into normal development and will guide our choice of drugs for future studies related to both anesthetic mediated cognitive dysfunction and transplantation of NPCs.