Methacarn as a whole brain fixative for gene and protein expression analyses of specific brain regions in rats

Abstract

-For molecular analysis in anatomically-specific brain regions for rodent studies, it is necessary to establish a fast and accurate procedure for tissue sampling to achieve high integrity and expression fidelity of extracted molecules. The present study was performed to examine suitability of whole brain fixation with methacarn and subsequent tissue sampling using punch-biopsy devices for gene expression analysis in rats. After fixation, each specific region, i.e., hippocampal dentate gyrus, corpus callosum, cingulate cortex or cerebellar vermis was collected, and the integrity and variability of expression data of extracted total RNAs and polypeptides were examined. Methacarn fixation, acetone fixation, and unfixed tissues were compared. Methacarn fixation resulted in high integrity of total RNAs sufficient for conducting global expression analysis and superior in terms of uniformity in the integrity among brain regions to that of acetone fixation. Extracted polypeptide after methacarn fixation revealed similar integrity to that without fixation or with acetone fixation. Methacarn fixation resulted in lower mRNA expression variability between samples than acetone fixation in microarray analysis. The fidelity of polypeptide expression was mostly equivalent between methacarn and acetone fixation in 2-dimensional differential in-gel electrophoresis, although the expression levels of a small number of polypeptides from acetonefixed tissues were affected. These results suggest that whole brain fixation with methacarn retains advantages for global analyses of mRNAs and polypeptides in rodent studies. Original ArticleThe Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.38, No.3, 431-443, 2013 Vol. 38 No. 3 431 throughput and use of fixed brain samples may be preferable for this purpose.Methacarn is a protein-precipitating and non-crosslinking organic solvent fixative (Mitchell et al., 1985;Puchtler et al., 1970), and fixation with this solution enables preservation of tissue morphology for histopathological assessment in paraffin-embedded tissues (Delfour et al., 2006;Srinivasan et al., 2002). Methacarn generally gives superior immunohistochemical results over aldehyde-based cross-linking fixatives (Banks, 1979;Orstavik et al., 1981;Rognum et al., 1980) because antigenicity is usually maintained (Mitchell et al., 1985). We previously found that methacarn fixation yields high quality DNA, RNA and protein even in paraffin-embedded sections for analyses of genomic DNA, expression microarrays, real-time RT-PCR and western blotting (Shibutani et al., 2000;Shibutani and Uneyama, 2002;Takagi et al., 2004;.For global assessment of genes and proteins in anatomically-specific brain regions in animal studies using rodents, both high integrity of extracted molecules and minimal inter-animal variability in the expression of extracted macromolecules are essential. Therefore, establishment of a procedure for quick and accurate tissue sampling and processing which can overcome these hurdles is necessary. For th...

Gene expression profiling of the hippocampal dentate gyrus in an adult toxicity study captures a variety of neurodevelopmental dysfunctions in rat models of hypothyroidism

“…For microarray and RT‐PCR analyses, whole brains were removed from euthanized animals ( n = 6/group) and fixed with a methacarn solution for 5 h at 4 °C with agitation (Akane et al ., ). Fixed brains were dehydrated in ethanol.…”

“…Fixed brains were dehydrated in ethanol. Tissue samples from the hippocampal dentate gyrus, parietal cerebral cortex, corpus callosum and cerebellar vermis were collected using the brain-matrix cast (Muromachi Kikai Co., Ltd., Tokyo, Japan) and punch-biopsy devices (Kai Industries Co., Ltd., Gifu, Japan) according to the previously described method ( Supplementary Figure 1; Akane et al, 2013).…”

“…To examine DNT, the functions and differentiation potentials of both neuronal and glial cell populations must be analyzed. We have recently established a high‐throughput tissue sampling method that enables us to identify RNA and polypeptide profiles simultaneously in different brain regions by applying a whole brain fixation method using methacarn fixative (Akane et al ., ).…”

Maternal exposure to 3,3’‐iminodipropionitrile targets late‐stage differentiation of hippocampal granule cell lineages to affect brain‐derived neurotrophic factor signaling and interneuron subpopulations in rat offspring

“…Expression of mRNA in the hippocampal dentate gyrus was examined using real-time reverse-transcription polymerase chain reaction (RT-PCR) in offspring on PND 21 and PND 77. Brain tissues were dissected and processed using methacarn solution as previously described (Akane et al, 2013b). In brief, 2-mm-thick coronal cerebral slices were prepared at the position of À3.0 mm from bregma.…”

Gene expression profile of brain regions reflecting aberrations in nervous system development targeting the process of neurite extension of rat offspring exposed developmentally to glycidol

“…It is also necessary to examine the effects of developmental neurotoxicity on neurogenesis, such as that in the cerebral subventricular zone and subgranular zone of the hippocampal dentate gyrus. We have recently established a high-throughput tissue sampling method that enables us to perform molecular analysis of RNAs and polypeptides in anatomically specific brain regions after whole brain fixation with methacarn (Akane et al, 2013b).…”