Temperature and time interval for culture of postmortem neurons from adult rat cortex.
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.
Viel
J J
JJ
Department of Medical Microbiology and Immunology, Southern Illinois University School of Medicine, Springfield, Illinois 62794-9626, USA.
McManus
D Q
DQ
Cady
C
C
Evans
M S
MS
Brewer
G J
GJ
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002540:Cerebral Cortex / Q000473:pathology*; D003080:Cold Temperature; D005260:Female; D008564:Membrane Potentials / Q000502:physiology; D016900:Neurofilament Proteins / Q000378:metabolism; D009474:Neurons / Q000473:pathology* / Q000502:physiology; D011180:Postmortem Changes; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism
D016900:Neurofilament Proteins; D014404:Tubulin; C051774:neurofilament protein H