Abstract
BACKGROUND: Anesthetics cause widespread apoptosis in the developing brain, resulting in neurocognitive abnormalities. However, it is unknown whether anesthesia-induced neurotoxicity occurs in humans because there is currently no modality to assess for neuronal apoptosis in vivo. The retina is unique in that it is the only portion of the central nervous system that can be directly visualized noninvasively. Thus, we aimed to determine whether isoflurane induces apoptosis in the developing retina. METHODS: CD-1 male mouse pups underwent 1-hour exposure to isoflurane (2%) or air. After exposure, activated caspase-3, caspase-9, and caspase-8 were quantified in the retina, cytochrome c release from retinal mitochondria was assessed, and the number and types of cells undergoing apoptosis were identified. Retinal uptake and the ability of fluorescent-labeled annexin V to bind to cells undergoing natural cell death and anesthesia-induced apoptosis in the retina were determined after systemic injection. RESULTS: Isoflurane activated the intrinsic apoptosis pathway in the inner nuclear layer (INL) and activated both the intrinsic and extrinsic pathways in the ganglion cell layer. Immunofluorescence demonstrated that bipolar and amacrine neurons within the INL underwent physiologic cell death in both cohorts and that amacrine cells were the likely targets of isoflurane-induced apoptosis. After injection, fluorescent-labeled annexin V was readily detected in the INL of both air-exposed and isoflurane-exposed mice and colocalized with activated caspase-3-positive cells. CONCLUSIONS: These findings indicate that isoflurane-induced neuronal apoptosis occurs in the developing retina and lays the groundwork for development of a noninvasive imaging technique to detect anesthesia-induced neurotoxicity in infants and children.