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

Abstract

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.

Reducing synuclein accumulation improves neuronal survival after spinal cord injury

“…1C, red arrows). These neurons also exhibit other signs of post-injury degeneration, including FluoroJadeC® staining (Busch and Morgan, 2012), TUNEL staining (Shifman et al, 2008), and increased levels of activated caspases (Barreiro-Iglesias and Shifman, 2012; Hu et al, 2013), suggesting a mechanism of apoptosis. In contrast, another subset of giant RS neurons typically survives (e.g.…”

Activated caspase detection in living tissue combined with subsequent retrograde labeling, immunohistochemistry or in situ hybridization in whole-mounted lamprey brains

“…Here we describe a double-labeling protocol in whole-mounted lamprey brain for simultaneous detection of early stage apoptosis, using Fluorochrome-Labeled Inhibitors of Caspases (FLICA), and either mRNA, using in situ hybridization, or protein expression, using immunohistochemistry. To improve brain preservation, the working temperature during the FLICA stage was lowered from 37°C to 4°C (Barreiro-Iglesias and Shifman, 2012). Using this method, neurofilament protein was demonstrated by immunohistochemistry in neurons previously reacted by FLICA.…”

Expression of RBMX in the Light-Induced Damage of Rat Retina In Vivo

“…When translocated to the mitochondria, BAX oligomerization in the outer mitochondrial membrane leads to mitochondrial outer membrane permeabilization and reduction of membrane potential (Zhang and Saghatelian 2013). Then, release of cytochrome C along with Apaf-1, ATP, and pro-caspase 9 forms a complex known as the apoptosome and leads to the activation of caspase-9 (Adhami et al 2003;Barreiro-Iglesias and Shifman 2012;Berens and Tyler 2011;Cort et al 2012;Liu et al 2012;Naseer et al 2011). Next, the apoptosome can construct a Fig.…”