Agar freezing solution for long-term cryopreservation of brain slices from non-hibernating animals

Abstract

Our previous studies have shown that function of ionotropic glutamate receptors such as AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) and NMDA (N-methyl-D-aspartate) was impaired after long-term cryopreservation of brain slices at -10°C within 30-50 days. To elucidate the reasons for cryodamage to AMPA- and NMDA-dependent mechanisms, artificial cerebrospinal fluid (aCSF) solutions that contain agar at different concentrations (33, 44, and 50%) were used for cryopreservation of the rat olfactory cortex slices. After cryopreservation, the slices were warmed to 37°C and the amplitudes changes of AMPA and NMDA potentials, which reflected the activities of the AMPA and NMDA mechanisms, were evaluated; the results were compared with those obtained before cryopreservation. It was found that AMPA and NMDA potentials changed differently depending on the concentration of agar in artificial cerebrospinal fluid. In solutions with 33% agar, the amplitude of AMPA potentials increased by 60%, whereas, in contrast, the amplitude of NMDA potentials was equal to the values before cryopreservation. At agar concentration of 44% in the solution, the AMPA and NMDA amplitudes were increased by 70% and 80%, respectively. A complete recovery of the activities of AMPA and NMDA mechanisms was obtained after cryopreservation in a freezing solution with an agar concentration of 50%. Under these conditions, the amplitudes of the AMPA and NMDA potentials corresponded to those seen before cryopreservation. Thus, the results obtained indicate that agar added to the artificial cerebrospinal fluid solution is a cryoprotectant that protects AMPA- and NMDA-dependent mechanisms from cryoinjury. The freezing solution (artificial cerebrospinal fluid and agar) developed by us for cryopreservation of brain explants of non-hibernating animals will be used to create a cryobank of nervous tissue.