1. PRELIMINARY RESULTS:“THE ROLE OF BAX IN ISOFLURANE INDUCED
NEUROAPOPTOSIS IN INFANCY”
Hikmatullah Arif, Leanne Cornell, David Jardine
Background
Exposure to anesthetics increases apoptosis during rapid brain growth in neonatal animals. These
findings have been replicated in a variety of laboratory animals from rodents to nonhuman primates.
Testing in early adulthood almost always reveals learning deficits. In human infants, the period of rapid
brain growth encompasses the first 12 months of life. Epidemiologic investigations of human infants
exposed to anesthesia during this time suggest that exposed infants have subtle developmental deficits,
although these findings are controversial.
The mechanism of anesthetic induced neurotoxicity remains unknown. We hypothesized that Bax, a
well-characterized proapoptotic protein plays an important role in the induction of neuroapoptosis after
exposure to isoflurane in infant mice. To test this hypothesis, we will subject a strain of Bax deficient
mouse pups to isoflurane anesthesia and quantitate the frequency of neuroapoptosis in Bax deficient mice
and Bax wild type mice.
Methods
Bax deficient transgenic mice were obtained from Jackson Labs (strain B6.129X1-Baxtm1Sjk/J). Bax
genotyping is performed by PCR. Bax wild type and Bax homozygous deficient mice are used for these
experiments. Mouse pups (P6) are exposed to either 0.75% isoflurane in room air or room air (no
anesthetic) for 240 minutes in a temperature controlled chamber at 35°C. Rectaltemperature is monitored
throughout the experiments. At the end of the experiment, the mice are sacrificed by intraperitoneal
pentobarbital injection. The mice undergo trans-cardiac perfusion with PBS followed by 4%
paraformaldehyde for 7 minutes each. After overnight fixation in 4% paraformaldehyde, the right
hemisphere of the brain is sectioned sagittally (50 µm slices). Every 10th
slice is collected for histology.
Staining is performed with an antibody to cleaved caspase-3 (Asp175) (antibody #9661, Cell Signaling).
Secondary staining is accomplished with a fluorescent goat anti-rabbit IgG (#35553, Thermo Scientific).
Images are collected using an automated microscope from the cortex and caudate/putamen areas of the
brain (average 12,000 images per brain hemisphere). Stereological counting procedures (optical
fractionator) are used to count apoptotic cells (Slidebook 5.5 software,Intelligent Imaging Innovations).
Results
The mouse pups (exposed and unexposed) remained active; although the exposed pups lose their
righting reflex. Rectaltemperature is maintained at 37°C.
At this time, we have collected complete data from 2 animals, 1 unexposed homozygous Bax
deficient mouse and 1 homozygous Bax wild type mouse exposed to isoflurane. The unexposed
homozygous Bax deficient mouse showed 3 apoptotic cells per hemisphere (cortex + caudate/putamen),
while the exposed homozygous Bax wild type mouse showed 59 apoptotic cells.
Discussion
These preliminary results show that this strain of mice responds to isoflurane exposure in a manner
that we would have predicted based on our experience with non-transgenic C57bl/6 mice (data not
shown). The homozygous Bax wild type mouse showed a level of neuroapoptosis after exposure to
isoflurane that is comparable to non-transgenic mice after isoflurane exposure. The unexposed
homozygous Bax deficient mouse also showed a level of neuroapoptosis that is comparable to unexposed
non-transgenic mice. In future experiments, we predict that after isoflurane exposure, Bax deficiency will
protect mouse pups from neuroapoptosis compared to the Bax wild type mice. We are currently
proceeding with experiments to fully test this hypothesis.
Acknowledgements
Funded by: Royalty Research Fund at the Univ. of Washington.