1. PRELIMINARY RESULTS:“HYPOTHERMIA PROTECTS AGAINST 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.
Recently, a report suggested that hypothermia to 30°C may provide substantial protection against
anesthesia induced neuroapoptosis in mouse pups (Creeley, CE. Anesth Analg (2010). 110: 442). Because
this degree of hypothermia is clinically unsafe, we elected to investigate whether cooling to 33°C could
produce protection against neuroapoptosis.
Methods
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 4 animals (2 unexposed and 2 exposed to
isoflurane) at 37°C. Unexposed animals showed a mean of 8 (SD 2.8) apoptotic cells per hemisphere
(cortex + caudate/putamen), while exposed animals showed 80 (SD 16.2) apoptotic cells (Figure 1).
Discussion
Isoflurane MAC for mouse pups is 2.7 vol% (Istaphanous, GK. Anesthesiology (2011). 114: 578).
Although the animals were exposed to only 27% of age adjusted MAC for isoflurane, the exposed mice
demonstrated an approximately tenfold increase in apoptotic cells in the brain. These findings replicate
findings previously reported in the literature. More importantly, these data validate our experimental
approach. Given the large difference in apoptotic cells between exposed and unexposed animals at 37°C,
we are well-positioned to detect reductions in neuroapoptosis at 33°C. At present,we are proceeding with
those experiments and anticipate that we will complete these within the next few months.
Acknowledgements
We are grateful to the ITHS and to the Anesthesiology Department at the University of Washington
for funding this investigation.
Figure 1
