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 37 degrees C to 33 degrees C or 30 degrees C on mediator release, including nitric oxide, interleukin-6, and tumor necrosis factor-alpha from lipopolysaccharide-stimulated microglia harvested from neonatal rats.


METHODS
Laboratory study.


METHODS
University medical school.


METHODS
Microglial cells isolated from primary cultures of rat brains.


METHODS
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.


RESULTS
At 30 degrees C and 33 degrees C, nitric oxide production stimulated by lipopolysaccharide decreased to 10 and 30% of control (37 degrees C), respectively, 24 hrs after the stimulation, and the decrease was sustained for 48 hrs. Interleukin-6 production at 30 degrees C and 33 degrees C 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 30 degrees C and 33 degrees C was depressed for only 6 hrs after stimulation, followed by subsequent elevation to concentrations similar to those at 37 degrees C. Microglial morphologic activation, showing changes from round to bipolar, reached a peak at 6 hrs in the 37 degrees C group, returning to round 12 hrs after lipopolysaccharide application. In 30 degrees C and 33 degrees C, the zenith was detected at 6 hrs, with activation remaining even 12 hrs after the stimulation, suggesting prolongation of the microglial response to lipopolysaccharide, 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.
Maekawa
Soichi
S
Department of Emergency Medicine, Ehime University, School of Medicine, Shigenobu, Ehime, Japan.
Aibiki
Mayuki
M
Si
Qiu-Shang
QS
Nakamura
Yoichi
Y
Shirakawa
Yoichi
Y
Kataoka
Kiyoshi
K
D000818:Animals; D000831:Animals, Newborn; D001930:Brain Injuries / Q000503:physiopathology*; D018929:Cell Culture Techniques; D007036:Hypothermia, Induced; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides; D017628:Microglia / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D051381:Rats; D013696:Temperature; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D015850:Interleukin-6; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide