Critical interval of somal calcium transient after neurite transection determines B104 cell survival

Abstract

Nerve cells may survive or die after axonal or dendritic transection. After neurite transection near (<50μm) the cell body of Fura-2 loaded B104 neuroblastoma (rat brain derived) cells, the somal calcium concentration (SCC) undergoes a three-phase transient change: a rapid (0 to 0.15 min posttransection (PT)) rise phase followed by an early (0.15 to 1.5 min PT) rapid decay phase, succeeded by a late (1.5 to 60 min PT) slower decay phase that restores SCC to preinjury levels. The SCC in a critical interval (1.5 to 12.5 min PT) of the third transient phase correlates with cell fate, i.e., most transected cells that exclude dye (restore a barrier) and die have a significantly higher (p<0.005) SCC in this Critical Interval than transected cells that exclude dye and survive at 24h PT. Loading BAPTA (chelation of somal Ca 2+ ) before, but not after, the Critical Interval increases the percentage of cells that survive compared to cells transected without BAPTA loading. Furthermore, most transected cells that die, despite successful barrier restoration, exhibit characteristics consistent with apoptosis initiated during the Critical Interval of the SCC, including caspase activation and plasmalemmal phosphatidylserine translocation. These data suggest that decreased cell survival for injuries near the soma is due to Ca 2+ -initiated apoptosis during the Critical Interval of the third phase of the SCC transient.

A Model for Sealing Plasmalemmal Damage in Neurons and Other Eukaryotic Cells

“…We then added 3 kDa Texas Red dextran at 0 min PC (i.e., after neurite transection) to assess sealing. As reported previously (Yoo et al, 2003(Yoo et al, , 2004Nguyen et al, 2005), cells with transected neurites identified by their relationship to the score mark were always filled with fluorescein dextran (Fig. 1 B, E), whether or not they excluded Texas Red dextran.…”

“…As further confirmation of the validity of our single-dye assessment technique, when transection distance was not taken into account, the percentage of cells that sealed in the present study at 5, 20, and 60 min PC was not significantly ( p Ͼ 0.05, CMH 2 ) different than that in previous studies in which fluorescein dextran was used as an additional indicator of cells that were transected, but transection distance was not noted (Yoo et al, 2003;Nguyen et al, 2005). Hence, in all protocols described below, we used a single dye (Texas Red dextran) to assess dye exclusion as a measure of plasmalemmal sealing of transected B104 neurites.…”

“…As described previously (Nguyen et al, 2005), B104 cells were grown in 75 cm 2 vented cap flasks (BD Falcon; BD Biosciences) in a humidified incubator at 37°C in 5% CO 2 and in 4 ml of "cell growth medium: consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 (DMEM:F-12, HyClone) supplemented for growth with 10% heat-inactivated fetal bovine serum (FBS; HyClone) and 1% antibiotics (10,000 U of penicillin/ml and 10 mg/ml streptomycin; Sigma-Aldrich). The cell growth medium was changed every 2 d. Cultures were passaged at 80% confluency, and cells were then either subcultured in a vented cap flask or seeded at ϳ2000 cells/cm 2 in cell growth media on Petri dishes coated with poly-D-lysine (Sigma-Aldrich) to prevent cells from detaching during solution changes and/or neurite transection.…”

“…Ca 2ϩ influx through plasmalemmal disruptions initiates processes that lead to cell death, often via apoptosis (Schlaepfer and Bunge, 1973;Nguyen et al, 2005). However, such Ca 2ϩ influx also leads to competing processes that activate plasmalemmal sealing pathways , sometimes before the internal Ca 2ϩ concentration becomes toxic (Nguyen et al, 2005).…”

“…Therefore, identifying plasmalemmal sealing pathways in neurons may provide insights into sealing in other cell types. (Bottenstein and Sato, 1979) have often been used as a model system to study neuronal function in vitro (Toda et al, 1999;Tan et al, 2003;Yoo et al, 2003Yoo et al, , 2004Nguyen et al, 2005;Miller et al, 2006). B104 cells extend neurites that have properties typical of nerve axons such as generation of action potentials, smooth (but not rough) endoplasmic reticulum, release of neurotransmitter, and regeneration of severed neurites.…”