Differential effects of lowering culture temperature on mediator release from lipopolysaccharide-stimulated neonatal rat microglia

Objective: Therapeutic moderate hypothermia has the potential for neuronal protection against brain injury. Microglia, a type of immune-related cell in the brain, may play a certain role in neuronal damage subsequent to injury. We examined the effects of culture temperature changes from 37degreesC to 33degreesC or 30degreesC on mediator release, including nitric oxide, interleukin-6, and tumor necrosis factor-alpha from lipopolysaccharide-stimulated microglia harvested from neonatal rats. Design: Laboratory study. Setting: University medical school. Subjects: Microglial cells isolated from primary cultures of rat brains. Interventions: The production of nitric oxide was measured by a nitrite accumulation method in a culture medium, whereas cytokines, interleukin-6, and tumor necrosis factor-alpha were measured by enzyme-linked immunosorbent assay. Measurement and Main Results: At 30degreesC and 33degreesC, nitric oxide production stimulated by Iipopolysaccha ride decreased to 10 and 30% of control (37degreesC), respectively, 24 hrs after the stimulation, and the decrease was sustained for 48 hrs. Interleukin-6 production at 30degreesC and 33degreesC was also reduced to 30% of control 6 hrs after the activation. Such responses lasted throughout the study. However, tumor necrosis factor-alpha release at 30degreesC and 33degreesC was depressed for only 6 hrs after stimulation, followed by subsequent elevation to concentrations similar to those at 37degreesC. Microglial morphologic activation, showing changes from, round to bipolar, reached a peak at 6 hrs in the 37degreesC group, returning to round 12 hrs after lipopolysaccharide application. In 30degreesC and 33degreesC, the zenith was detected at 6 hrs, with activation remaining even 12 hrs after the stimulation, suggesting prolongation of the microglial response to lipopolysaccha ride, which was inconsistent with changes in tumor necrosis factor release. Conclusions: Decreasing culture temperature inhibits the production of nitric oxide and interleukin-6 from activated microglia. Differences were found in the degree or time course change between tumor necrosis factor-alpha and the other mediators. Also, the time course of morphologic changes in microglia was dependent on culture temperature. Further studies are required to define the mechanisms for such differences in mediator release from cooled microglia and also to clarify the inconsistency between morphologic change and its function in the cell.