Metabolic activity of cultured rat brainstem, hippocampal and spinal cord slices.
The use of cultured brain slices has become an accepted technique for the ex vivo analysis of neural mechanisms, yet the viability of this preparation is not routinely measured. The tetrazolium dye 3-(4, 5-dimethlythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is reduced by active mitochondria to an insoluble purple precipitate which accumulates within living cells and is easily visualized with bright field or phase contrast microscopy. In this study, the MTT assay was used to assess the viability of cultured brainstem, hippocampal and spinal cord slices (150-300 micrometer) from 0 to 22 day-old neonatal rats at post-explant time points ranging from 2 to 29 days. After 2 weeks, 180-300 micrometer cultured slices from 4-13 day old rats remained 90-100% viable. Those from 0-1 day old rats had similar viability but displayed peripheral tissue outgrowth. Slices from older 18-22 day rats were no longer viable after 10-14 days. After 4 weeks, the thicker (300 micrometer) slices of hippocampus and spinal cord retained 75-89% viability, in contrast to the 50-74% viability of the brainstem. Thinner brainstem and hippocampal slices (150-220 micrometer) slices were less than 50% viable at 4 weeks. Morphologic characteristics of the brain regions gradually degenerated over the 4-week culture period. Slice viability was markedly influenced by tissue thickness, donor age and brain region. Use of the MTT assay provides an inexpensive and expeditious means to assess a significant functional parameter of regional slice viability under variable conditions and enhances the feasibility of this preparation for functional studies, such as those concerned with genetic and protein expression within circumscribed areas of the brain.
Connelly
C A
CA
Departments of Surgery/Transplantation Research, Cedars-Sinai Medical Center, Burns and Allen Research Institute and UCLA School of Medicine, 8635 W. 3rd Street, Suite 590W, 90048, Los Angeles, CA, USA. cconnell@ucla.edu
Chen
L C
LC
Colquhoun
S D
SD
D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D001933:Brain Stem / Q000166:cytology / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology*; D004396:Coloring Agents; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009924:Organ Culture Techniques; D051381:Rats; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism; D013778:Tetrazolium Salts; D013844:Thiazoles; D013997:Time Factors
D004396:Coloring Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue