Figure 5.

Hypothermia reduces astrocyte protective effect on neurons subject to OGD. Conditioned astrocyte media was produced by subjecting stable astrocyte cultures to varying durations OGD (selected at random) and then harvesting the supernatant. Panel (a) shows the experimental setup. At random, during OGD, the astrocytes were held at 33, 35 or 37 ºC for durations ranging from 30 to 240 min. The conditioned astrocyte media was added to neuronal cultures that were then subject to 2 h OGD at 37 ºC. After OGD, the neuron cultures were restored to normal oxygen and normal glucose-containing media; neuron survival was assayed with the MTT assay 22 h later. Panel (b): Using the MTT assay, neuron survival after OGD is shown. After standardizing all OD values to the No-OGD group, the data confirm the results in Figure 1 that 2 h OGD killed 80% of the neurons. Astrocyte-conditioned media (ACM) harvested after 2 h OGD at 37 ºC (labeled “37 – 120 min”) and placed on neuron cultures significantly protected neurons during subsequent neuronal OGD, with about 90% neuron survival. ACM prepared while hypothermia was applied to the astrocyte cultures during OGD suppressed this neuronal protective effect. ACM prepared during hypothermia to 33 ºC for 30, 60, 90 or 120 min (labeled “33 – 30 Min”, “33 – 60 Min”, “33 – 90 Min”, and “33 – 120 Min”) failed to protect neurons during neuronal OGD. Mild hypothermia at 35 ℃ for 120 or 240 min (labeled “35 – 120 Min” and 35 – 240 Min ”) blocked only about half of the ACM protective effect. These data suggest that hypothermia interferes with astrocyte protection of neurons in a temperature-dependent manner.