Dynamic Recording of Cell Death in the In Vitro Dorsal Vagal Nucleus of Rats in Response to Metabolic Arrest

Abstract

Müller, Michael and Klaus Ballanyi. Dynamic recording of cell death in the in vitro dorsal vagal nucleus of rats in response to metabolic arrest. J Neurophysiol 89: 551-561, 2003. 10.1152/jn.00559.2002. Anoxic/ischemic neuronal death is usually assessed in cell cultures or in vivo within a time window of 24 h to several days using the nucleic acid stain propidium iodide or histological techniques. Accordingly, there is limited information on the time course of such neuronal death. We loaded acute rat brain stem slices with propidium iodide for dynamic fluorometric recording of metabolic arrest-related cell death in the dorsal vagal nucleus. This model was chosen because dorsal vagal neurons show a graded response to metabolic inhibition: anoxia and aglycemia cause a sustained hyperpolarization, whereas ischemia induces a glutamate-mediated, irreversible depolarization. We found that the number of propidium iodide-labeled cells increased from 27% to 43% of total cell count within 1-7 h after preparation of slices. Compared with these untreated control slices, cyanide-induced anoxia (30 min) or aglycemia (1 h) did not cause further cell death, whereas 3-h aglycemia destroyed an additional 13% of cells. Ischemia (1 h) due to cyanide plus iodoacetate immediately labeled an additional 20% of cells, and an additional 48% of cells were destroyed within the following 3 h of postischemia. Continuous recording of propidium iodide fluorescence showed that loss of membrane integrity started within 25 min after onset of the ischemic depolarization and the concomitant intracellular Ca 2ϩ rise. The results show that propidium iodide can be used to monitor cell death in acute brain slices. Our findings suggest that pronounced cell death occurs within a period of 1-4 h after onset of metabolic arrest and is apparently due to necrotic/oncotic mechanisms.

Optical and pharmacological tools to investigate the role of mitochondria during oxidative stress and neurodegeneration

“…Hence it simply could be a local shortage of ATP that inhibits mitochondrial movements, since ATP is required to drive the molecular motors along microtubules. Also high local concentrations of Ca 2+ , either resulting from e.g., glutamate-mediated Ca 2+ influx or released from dysfunctioning mitochondria (Kulik and Ballanyi, 1998;Müller and Ballanyi, 2003) could be responsible for the arrest of mitochondrial movements by impairing cytoskeletal integrity (van Rossum and Hanisch, 1999).…”

“…The mobilized Ca 2+ stores then can enhance the generation of free radicals, which secondarily cause lipid peroxidation (Gunasekar et al, 1996). CN − also causes the release of Ca 2+ and cytochrome c from mitochondria (Liu et al, 2003;Müller and Ballanyi, 2003). Depending on the cell type and degree of insult, the application of CN − can lead to apoptosis or necrosis.…”

“…Activation of K ATP channels by CN − was found in dorsal vagal neurons, where K ATP channels were activated independent of ATP depletionobviously as a direct consequence of impaired mitochondrial metabolism or mitochondrial depolarization (Müller et al, 2002). However, a loss of membrane potential or massive Ca 2+ influx did not occur in these cells even during prolonged CN − induced chemical anoxia (Müller and Ballanyi, 2003). CN − has also been shown to increase voltage-dependent Na + currents in CA1 hippocampal cells (Hammarström and Gage, 1998).…”

Optical assessment of motoneuron function in a “twenty-four-hour” acute spinal cord slice model from fetal rats

“…This fluorescing dye binds to nucleic acids and responds with increased fluorescence emission upon loss of membrane integrity (Arndt-Jovin and Jovin, 1989). The method for using propidium iodide as a dead cell marker in acute brain slices is described elsewhere (Muller and Ballanyi, 2003). In brief, propidium iodide was excited at 535 nm and fluorescence emission was measured beyond 590 nm, using the Omega Opticals XF34 filter set (exciter: 535 nm bandpass, dichroic mirror: 570 nm, emitter: 590 nm longpass) in the TILL Photonics imaging system.…”

“…To study whether glutamate increased overall cell death within 24 h after 1 h treatment, slices were loaded with propidium iodide (Laake et al, 1999;Muller and Ballanyi, 2003). The number of dead cells in control slices after t 0 (5.3 ± 0.8; At these time points, slices were transferred to the recording chamber and two consecutive glutamate (300 M) pulses were applied at an interval of 5-8 min.…”