Measurement of Hippocampal Levels of Cellular Second Messengers Following In Situ Freezing

Abstract

The in situ freezing technique has been widely used to fix labile metabolites and cellular second messengers in cerebral cortex. In this study, we isolated specific brain regions at 0 degree C from coronal sections of frozen heads following in situ brain freezing and measured regional concentrations of labile metabolites and cellular messengers. These levels in the cortex were compared with those in cortical punches obtained at freezing temperature (less than -40 degrees C) from the same in situ frozen brains and those of cortex dissected from decapitated animals. In both isoflurane- and pentobarbital-anesthetized animals, we observed that the levels of lactate, free fatty acids, inositol 1,4,5-trisphosphate, and diacylglycerol, as well as the proportion of protein kinase C associated with the membrane fraction, were similar in cortical punches taken at freezing temperature and those dissected at 0 degree C. However, with animals decapitated at room temperature, cortical and hippocampal levels of lactate, free fatty acids, and inositol 1,4,5-trisphosphate and the proportion of membrane protein kinase C were significantly higher than those of corresponding brain regions isolated at 0 degree C from in situ frozen brains (p < 0.05). These results indicate that dissection of cortex and hippocampus at 0 degree C following in situ freezing will eliminate decapitation-induced production of artifacts and changes in the levels of cellular second messengers such as inositol 1,4,5-trisphosphate, diacylglycerol, and protein kinase C. The present technique, used in conjunction with in situ freezing, will fix cellular second messengers and labile metabolites in several regions of brain and may facilitate accurate characterization of molecular and cellular mechanisms underlying CNS function.

Regional Concentrations of Cyclic Nucleotides After Experimental Brain Injury

“…The experimental animals (n = 6, for each time point) were subjected to in situ brain freezing at 5 and 20 min after injury (Tzigaret et al, 1993;Prasad et al, 1994b). The control animals were also subjected to in situ freezing at corresponding times after craniotomy.…”

“…Regions approximately 5 mm long and 2 mm wide were removed from the left parietal cortex 6 mm lateral to the sagittal suture (injury-site cortex), from the contralateral right cortex 6 mm lateral to the sagittal suture, and left and right hippocampi were dissected from in situ frozen brains at -5 to 0°C. These regions were immediately frozen in liquid N2 as described previously (Tzigaret et al, 1993;Prasad et al, 1994b).…”

“…Measurements of Lactate, Glucose, cAMP, and cGMP Lactate, glucose, cAMP, and cGMP from the frozen brain regions were extracted into 1 mL of 1 M trichloroacetic acid (Tzigaret et al, 1993;Prasad et al, 1994b). Two hundred microliters of the protein-free extract was neutralized with 5 M potassium carbonate and used for lactate and glucose measurements (extract 1).…”

Effect of Difluoromethylornithine Treatment on Regional Ornithine Decarboxylase Activity and Edema Formation after Experimental Brain Injury

“…For measurements of brain ODC, 54 rats were subjected to in situ freezing and dissection of the brain as previously described (Tzigaret et al, 1993) (« = 6 at each time point, total 48 rats and 6 control rats). At 30 min, 3, 6, and 24 h after injury, the 6 animals from each group were anesthetized and their brains were frozen in situ by continuously pouring liquid N2 through a styrofoam funnel over the skulls.…”

Theoretical local freezing times of small rodent brains submerged in situ in liquid nitrogen

“…In situ freezing is a standard procedure used to terminate metabolism and diffusion (6,7,8). Since the freezing procedure is a relatively slow process, even in liquid nitrogen, the regional concentration of a given substance may have time to change considerably before a sufficiently low temperature has been reached (2, 9).…”