Use of Fluorochrome-Labeled Inhibitors of Caspases to Detect Neuronal Apoptosis in the Whole-Mounted Lamprey Brain after Spinal Cord Injury

Apoptosis is a major feature in neural development and important in traumatic diseases. The presence of active caspases is a widely accepted marker of apoptosis. We report here the development of a method to study neuronal apoptotic death in whole-mounted brain preparations using fluorochrome-labeled inhibitors of caspases (FLICA). As a model we used axotomy-induced retrograde neuronal death in the CNS of larval sea lampreys. Once inside the cell, the FLICA reagents bind covalently to active caspases causing apoptotic cells to fluoresce, whereas nonapoptotic cells remain unstained. The fluorescent probe, the poly caspase inhibitor FAM-VAD-FMK, was applied to whole-mounted brain preparations of larval sea lampreys 2 weeks after a complete spinal cord (SC) transection. Specific labeling occurred only in identifiable spinal-projecting neurons of the brainstem previously shown to undergo apoptotic neuronal death at later times after SC transection. These neurons also exhibited intense labeling 2 weeks after a complete SC transection when a specific caspase-8 inhibitor (FAM-LETD-FMK) served as the probe. In this study we show that FLICA reagents can be used to detect specific activated caspases in identified neurons of the whole-mounted lamprey brain. Our results suggest that axotomy may cause neuronal apoptosis by activation of the extrinsic apoptotic pathway. 1. Introduction Studies in the basic neurosciences are heavily reliant upon rat and mouse models (for a review see [1]). Seventy-five percent of current research efforts are directed to rat mouse, and human brains, which represent 0.0001% of the nervous systems on the planet [1]. In recent decades, an increased number of studies have shown the usefulness of nonmammalian models for understanding developmental, pathological, and regenerative processes of the nervous system. Lampreys and fishes, for example, have proven to be valuable animal models for studying successful regeneration in the mature central nervous system (CNS) [2–5]. Lampreys occupy a key position close to the root of the vertebrate phylogenetic tree [6] and are thought to have existed largely unchanged for more than 500 million years, which makes them important animals from the standpoint of molecular evolution [7–10]. The unique evolutionary position of lampreys as early-evolved vertebrates, the sequencing of the sea lamprey (Petromyzon marinus L.) genome, and the adaptation and optimization of many established molecular biology and histochemistry techniques for use in this species have made it an emerging nonmammalian model organism