Caspase inhibitors reduce the apoptotic but not necrotic component of kainate injury in primary murine cortical neuronal cultures

Abstract

Excitotoxicity has been demonstrated to play a major role in ischemic neuronal injury. While the necrotic component of excitotoxicity has been well demonstrated, apoptosis has also been shown to play a role. We sought to quantitate and modulate the apoptotic component of kainate-induced injury. Experiments were performed in mouse primary cortical neuronal cultures after three or 10 days in vitro. Cell death was assessed by Hoechst/propidium iodide staining and cell counting. Apoptosis was further confirmed with inhibition by caspase inhibitors. Exposure of three-day old neurons to 100 microM kainate produced an injury in which 56% +/- 0.9% of cells showed apoptotic nuclei and 13.5% +/- 2.0% showed necrotic nuclei. After 10 days in vitro neurons were more easily injured by kainate, but the cell death had primarily necrotic characteristics. Inhibition of both caspases 1 and 3 significantly reduced the apoptotic injury of 3-day old neurons. Neither reduced the necrotic component. Inhibition of protein synthesis with cycloheximide was also effective in reducing the apoptotic injury without affecting the necrotic injury. Kainate injury causes both apoptosis and necrosis, with the injury depending on both the dose of kainate and the age of the culture. The apoptotic component can be selectively reduced by caspase inhibition or cycloheximide.

Development of Neonatal Murine Microglia In Vitro: Changes in Response to Lipopolysaccharide and Ischemia-Like Injury

“…Our results show that early-culture microglia are more vulnerable to stresses that simulate ischemia as well as to induction of apoptosis by loss of growth factors (SD). This decrease in vulnerability to injury with age in culture is similar to the response of oligodendrocytes (9) and contrasts with findings for neurons and astrocytes (18,19).…”

Anandamide-induced cell death in primary neuronal cultures: role of calpain and caspase pathways

“…It has been suggested that de novo protein synthesis may be essential for activation of caspase-regulated apoptosis. 32,55,56 Consistent with the studies using caspase inhibitors, inhibition of protein synthesis did not protect cortical neurons from the neurotoxic effects of anandamide. In contrast, parallel studies using treatment with etoposide, a classical method of inducing caspase-3-mediated neuronal apoptosis, showed cycloheximide blockade of apoptosis.…”

Recommendations from the INHAND Apoptosis/Necrosis Working Group

“…However in adult animals, most pathologists have been taught that the classic “dead red” neuron, which is the most common morphologic manifestation of neuronal death, is a necrotic cell (Figure 19A) (Morgan et al 2004). During the past 10 – 15 years a substantial amount of research has been done to characterize the mechanism of cell death resulting from various toxicants (e.g., domoic or kainic acid, TMT, lead) and pathologic conditions (e.g., seizures, cortical trauma), and it is becoming increasingly apparent that both necrosis and apoptosis can occur in response to the same type of insult, and with similar morphological features with H&E staining (Figure 19B) (Ettcheto et al 2015; Giordano et al 2008; Glassford et al 2002; Krajewska et al 2011; Martin et al 1998; Wang et al 2005; Zhang and Zhu 2011). Morphologically, one may have a sense for which process predominates based on cellular features (Figure 19C), the extent of neuronal loss/death, the amount of tissue damage, and the glial/inflammatory response, but it may be difficult to discern with certainty in most cases without special stains.…”