Postmortem interval and anesthetic neuroprotection in regenerating cortical neuron cultures used as a model for TNF neuroprotection from beta-amyloid toxicity.

摘要

The induction of an inflammatory response and release of cytokines such as TNF in the brain by microglia may be involved in the age-related etiology of Alzheimer's disease (AD). We hypothesize that with age there is increased ability of microglia to produce TNF and/or that age decreases the neuroprotective effect of TNF against beta-amyloid (Abeta) toxicity in neurons. Over the first twelve hours of exposure to 10 muM Abeta(1--40), microglia from embryonic and old rats increased TNF secretion, while microglia from middle-age rats did not. Low concentrations of TNF provided less neuroprotection from Abeta(1--40) for neurons from old rats compared to neurons from middle-aged rats. Toxic effects of enhanced TNF production, or reduced protective response of the aging neurons may in part explain the age-related pathogenesis of AD.; Studies of rat postmortem tissue are an important first step in developing similar models in man. A major difference between the animal model and human tissue collection is the use of anesthetics for humanitarian reasons. The neuroprotective role of the barbiturate anesthetic pentobarbital is well established under transient, focal ischemic conditions. When pentobarbital was administered prior to decapitation, a form of global ischemia, the survival of neurons harvested doubled versus non-anesthetic controls.; Frontal cortex sections were removed from adult rats immediately following sacrifice, at different postmortem intervals, and with the brain at 4°C or 22°C. Hypothermia increased the recovery interval four-fold. The amount of neurons recovered at each time interval was carefully quantified after determining the average number of neurons per mg of frontal cortex (160,000/mg). Cultured neurons that survived represented 40--75% of the viable cells, or 0.5--2.75% of those originally estimated to be present in the brain. Electrophysiology experiments show that neurons isolated 0 and 24 hours postmortem had average resting membrane potential of -48mV, voltage sensitive currents and action potentials. These experiments show that neurons cells can be reliably cultured up to 24 hr. postmortem under hypothermic conditions. These findings should encourage donation of human postmortem brain neurons for studies on ischemia, adult pharmacology and neurological disease.