In Vivo Neuronal Injury in Painted Turtles Forcibly Submerged at 20°C

Painted turtles, Chrysemys picta , are regarded as the most anoxia‐tolerant air‐breathing vertebrates. However, if and when neuronal injury occurs during forced submergence is not known. In the present study, painted turtles were forcibly submerged for 36, 48, or 60 hours at 20°C and, after transcardial paraformaldehyde fixation, the extent of neuronal injury was assessed in 15 μm cerebrocortical sections using the following two histological methods: 1) staining with Fluoro‐Jade C, a marker that specifically labels degenerating neurons and 2) co‐immunolabeling of activated caspase‐3, a protein found in apoptotic cells, with NeuN, a nuclear protein expressed only in adult neurons. After 60 h of submergence at 20°C, plasma lactate was increased to 100 mmol l −1 . Fluoro‐Jade C and activated caspase‐3 staining were present in neurons throughout the medial and dorsal cortices, indicating apoptosis‐mediated neuronal injury resulting from long‐term anoxia. Despite this, several animals showed visible signs of life and required anesthesia prior to fixation. All of the animals’ hearts were visibly contracting at the time of sampling. Future studies should assess the extent of neuronal injury during anoxia in the cold, a more ecologically relevant condition, and the possibility that injured regions might undergo repair.