Effects of anesthesia on immunohistochemical detection of phosphorylated extracellular signal-regulated kinase in cerebral cortex

During attempts to examine the phosphorylation status of extracellular signal-regulated kinase (ERK) in cerebral cortex immunohistochemically, we determined whether deep anesthesia for euthanasia disturbs the phosphorylation status of ERK, because the anesthesia might influence activity-dependent phosphorylation of ERK. We compared effects of short (2 and 5 min) and long (>10 min) anesthesia by pentobarbital on the immunoreactivity for phosphorylated ERK in the visual and entorhinal cortices of rat. The long anesthesia drastically reduced the density of phosphorylated ERK immunopositive cells to about 15% of the short anesthesia condition. The reduction was observed in all cortical regions. We found no significant difference in pERK immunoreactivity between 2 and 5 min groups. A reduction of similar degree was induced by long anesthesia with isoflurane. Even if a similar duration of anesthesia is given, the immunohistochemical results possibly contain a variation due to the individual difference in the sensitivity to the anesthetics. We demonstrated that the variation of pERK immunopositive cell density in the visual cortex was significantly reduced by normalizing the values to the density in the nonvisual area in the entorhinal cortex, thus enabling us to detect differences between animals under different visual conditions with higher sensitivity. Therefore, the variation could be reduced by calculating the ratio of immunoreactivity in the area of interest to that in other cortical area as reference.