Long-term Fate Mapping to Assess the Impact of Postnatal Isoflurane Exposure on Hippocampal Progenitor Cell Productivity

Abstract

Background: Exposure to isoflurane increases apoptosis among postnatally generated hippocampal dentate granule cells. These neurons play important roles in cognition and behavior, so their permanent loss could explain deficits after surgical procedures.

Methods: To determine whether developmental anesthesia exposure leads to persistent deficits in granule cell numbers, a genetic fate-mapping approach to label a cohort of postnatally generated granule cells in Gli1-CreER::GFP bitransgenic mice was utilized. Green fluorescent protein (GFP) expression was induced on postnatal day 7 (P7) to fate map progenitor cells, and mice were exposed to 6 h of 1.5% isoflurane or room air 2 weeks later (P21). Brain structure was assessed immediately after anesthesia exposure (n = 7 controls and 8 anesthesia-treated mice) or after a 60-day recovery (n = 8 controls and 8 anesthesia-treated mice). A final group of C57BL/6 mice was exposed to isoflurane at P21 and examined using neurogenesis and cell death markers after a 14-day recovery (n = 10 controls and 16 anesthesia-treated mice).

Results: Isoflurane significantly increased apoptosis immediately after exposure, leading to cell death among 11% of GFP-labeled cells. Sixty days after isoflurane exposure, the number of GFP-expressing granule cells in treated animals was indistinguishable from control animals. Rates of neurogenesis were equivalent among groups at both 2 weeks and 2 months after treatment.

Conclusions: These findings suggest that the dentate gyrus can restore normal neuron numbers after a single, developmental exposure to isoflurane. The authors' results do not preclude the possibility that the affected population may exhibit more subtle structural or functional deficits. Nonetheless, the dentate appears to exhibit greater resiliency relative to nonneurogenic brain regions, which exhibit permanent neuron loss after isoflurane exposure.

Figure 1

A: Anesthetic exposure increases caspase-3 expression in two week-old dentate granule cells (DGC) immediately following exposure. Confocal maximum projections of hippocampal sections from representative 21-day-old mice exposed to 1.5% isoflurane (anesthesia) or room air (control) for six hours. Sections are immunostained for green fluorescent protein (GFP) and caspase-3, a marker of apoptotic cell death. Scale bar = 250 μm. B: Boxed regions in A are shown at higher resolution. Scale bar = 125 μm. C: Scatterplot shows the percentage of GFP-expressing cells in the dentate gyrus that were also immunoreactive for caspase-3 immediately following exposure. Each dot represents one animal. Boxes are means ± SEM. ***P<0.001, Student's t-test.

Figure 2

Green fluorescent protein (GFP) reveals the cellular morphology and integrity of dentate granule cells in control and anesthesia-exposed mice. Images show representative cells after a six-hour exposure to room air or isoflurane. Isoflurane dramatically increased caspase-3 immunoreactivity. Arrows denote double-labeled cells. In the anesthesia-treated animal, the two cells in the center of the image have short, aspiny dendrites projecting into the dentate molecular layer; morphological features of immature granule cells. Morphology suggests cellular disintegration. Scale bar = 20 μm.

Figure 3

Anesthesia-induced apoptotic cell death in two week-old dentate granule cells (DGC) does not lead to long-term diminution in neuronal density in this population. Confocal maximum projections show immunostaining for green fluorescent protein (GFP) in representative two month-old animals exposed to six hours of 1.5% isoflurane (anesthesia) or room air (control) on P21. No differences between control and anesthesia-treated mice were found. Scale bar = 250 μm. Scatterplot shows the density of GFP-expressing cells in the dentate granule cell body layer. Circles represent individual animal means, while squares reflect group means ± SEM. The two groups were statistically equivalent (P=0.915, Student's t-test).

Figure 4

Anesthesia-induced apoptotic cell death in two week-old dentate granule cells does not lead to alterations in neuronal proliferation in this population. Confocal maximum projections showing green fluorescent protein (GFP)+Ki67 (A) and GFP+Calretinin (B) immunostaining in the hippocampal granule cell layer. Double-labeled cells are denoted by arrows. Scale bar = 30 μm. Scatterplots show the percentage of GFP-expressing cells double-labeled with Ki67 (C) or calretinin (D). Circles represent individual animal means, while squares reflect group means ± SEM. The two groups were statistically equivalent for both Ki67 and calretinin.

Figure 5

Confocal maximum projections from P81 control and P21 anesthesia + 60 day mice showing green fluorescent protein (GFP)-expressing cells misplaced to either the dentate molecular layer (ML) or hilus. No differences in the frequency of ectopic cells were evident between groups, and the overwhelming majority of GFP-labeled cells were correctly localized to the granule cell body layer (DGC-L). Scale bar = 25 μm.

Figure 6

BrdU, Ki67, calretinin and caspase 3 labeling in P35 control (C) and P21 anesthesia + 14 day (Anes) mice. Scale bars = 100 μm (BrdU, calretinin) and 50 μm (Ki67 and caspase 3). Scatterplots shows the density of labeled cells in the dentate granule cell body layer for each corresponding image set. Circles represent individual animal means, while squares reflect group means ± SEM. The two groups were statistically equivalent for all measures (P>0.05, Student's t-test).