Temperature and time interval for culture of postmortem neurons from adult rat cortex

Abstract

For a model of neurological disease and ischemia, we extended recent work to culture adult postmortem rat brain neurons. Frontal cortex sections were removed from adult rats immediately following sacrifice and at different postmortem intervals and with the brain at either 22 degrees C or 4 degrees C. Brain could be stored four times longer at 4 degrees C between sacrifice and neuronal disaggregation to achieve the same 20% recovery of live cells from those plated compared to 22 degrees C. Each milligram of rat frontal cortex was estimated by the optical disector method to contain 160,000 neurons. When cells were isolated as rapidly as possible, 9% of the neurons originally present in the brain were viable. Various postmortem intervals from 2 to 24 hr resulted in a reduction from 6% to 3% of the cells originally present. After 5 days in culture, viable neurons were 23-42% of those isolated. Neuron-like cells 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 cells isolated 0 and 24 hr postmortem had neuronal electrical properties, including an average resting membrane potential of -48 mV, voltage-sensitive currents, and action potentials. Neuron-like cells were immunoreactive for neuron-specific enolase, neurofilament 200, glutamate, MAP2, and tau after 2 weeks in culture. These experiments show that neuron-like cells can be reliably cultured from adult rat cortex up to 6 hr postmortem when stored at 22 degrees C and up to 24 hr postmortem when stored at 4 degrees C. These findings should encourage donation of human postmortem brain neurons for studies on ischemia, adult pharmacology, and neurological disease.

Age‐ and concentration‐dependent neuroprotection and toxicity by TNF in cortical neurons from β‐amyloid

“…4), a situation opposite to what might be expected. Furthermore, the yield of isolated, viable hippocampal cells is independent of age (Brewer, 1997) and these cells have neuronal characteristics of action potentials (Evans et al, 1998; Viel et al, 2001). Additional experiments like that shown in Figure 7 to determine the expression of TNF receptors in vitro as a function of age could further elucidate age‐related mechanisms of toxicity and protection.…”

Increased intraneuronal resting [Ca2+] in adult Alzheimer’s disease mice

“…Adult cortical neurons were prepared as described previously (Brewer 1997;Viel et al 2001). Following anesthesia with pentobarbital, adult non-Tg (8-12 months), 3xTg-AD (8-12 months) and APP SWE (11-12 months) mice were decapitated, and the cortex was dissected out and placed in ice-cold Hibernate A medium (BrainBits, Sripngfield, IL, USA) supplemented with B27 and glutamine (Invitrogen, Carlsbad, CA, USA).…”

Cells in human postmortem brain tissue slices remain alive for several weeks in culture

“…The agonal state at death was assessed using the pH of the cerebrospinal fluid (16,17). During the postmortem delay, the bodies of the patients were transported to the autopsy room at ambient temperature, which has been shown to be compatible with neuronal survival after cardiac arrest in rodents (15,18).…”