Anesthesia for Euthanasia Influences mRNA Expression in Healthy Mice and after Traumatic Brain Injury

Abstract

Tissue sampling for gene expression analysis is usually performed under general anesthesia. Anesthetics are known to modulate hemodynamics, receptor-mediated signaling cascades, and outcome parameters. The present study determined the influence of anesthetic paradigms typically used for euthanization and tissue sampling on cerebral mRNA expression in mice. Naïve mice and animals with acute traumatic brain injury induced by controlled cortical impact (CCI) were randomized to the following euthanasia protocols (n=10-11/group): no anesthesia (NA), 1 min of 4 vol% isoflurane in room air (ISO), 3 min of a combination of 5 mg/kg midazolam, 0.05 mg/kg fentanyl, and 0.5 mg/kg medetomidine intraperitoneally (COMB), or 3 min of 360 mg/kg chloral hydrate intraperitoneally (CH). mRNA expression of actin-1-related gene (Act1), FBJ murine osteosarcoma viral oncogene homolog B (FosB), tumor necrosis factor alpha (TNFα), heat shock protein beta-1 (HspB1), interleukin (IL)-6, tight junction protein 1 (ZO-1), IL-1ß, cyclophilin A, micro RNA 497 (miR497), and small cajal body-specific RNA 17 were determined by real-time polymerase chain reaction (PCR) in hippocampus samples. In naïve animals, Act1 expression was downregulated in the CH group compared with NA. FosB expression was downregulated in COMB and CH groups compared with NA. CCI reduced Act1 and FosB expression, whereas HspB1 and TNFα expression increased. After CCI, HspB1 expression was significantly higher in ISO, COMB, and CH groups, and TNFα expression was elevated in ISO and COMB groups. MiR497, IL-6, and IL-1ß were upregulated after CCI but not affected by anesthetics. Effects were independent of absolute mRNA copy numbers. The data demonstrate that a few minutes of anesthesia before tissue sampling are sufficient to induce immediate mRNA changes, which seem to predominate in the early-regulated gene cluster. Anesthesia-related effects on gene expression might explain limited reproduciblity of real-time PCR data between studies or research groups and should therefore be considered for quantitative PCR data.

Brief anesthesia by isoflurane alters plasma corticosterone levels distinctly in male and female rats: Implications for tissue collection methods

“…For example, the expression of the immediate early genes c-fos and c-jun were unchanged in the brain following 5 minutes of 2% isoflurane in male rats compared to unanesthetized rats [17]. Similarly, a 1-minute exposure to 4% isoflurane did not alter the expression of eight representative genes in the rat hippocampus [18]. The same isoflurane treatment also did not alter the expression of corticotrophin-releasing hormone, arginine vasopressin and oxytocin in the rat hypothalamus [2].…”

Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results

“…The use of anesthesia as a form of euthanasia is also a confounding factor that influences gene expression analysis [17]. Even a few minutes of general anesthesia seem to significantly alter cerebral mRNA expression levels.…”

Influence of Age on Cerebral Housekeeping Gene Expression for Normalization of Quantitative Polymerase Chain Reaction after Acute Brain Injury in Mice

“…Since it is to assume that a universal, absolute standard for normalization is unavailable for unbiased comparison of mRNA transcription, it is crucial to select the most suitable transcript to meet as closely as possible the criteria of invariant reference genes. Recently, there have been a number of reports demonstrating that classic reference genes can vary extensively and may be unsuitable for normalization purposes 15,16,48,[50][51][52][53][54] . In a murine model of diffuse TBI 18SrRNA levels, and ActB, GAPDH, B2M and S100B transcripts were determined in the injured brain region of traumatized mice.…”

“…The different results might possibly be explained by anesthesia related effects during organ sampling on gene expression, leading to limited reproducibility of real-time PCR data between studies or research groups 16 . This recent study demonstrated, that a few minutes of anesthesia before tissue sampling was sufficient to induce immediate mRNA changes 16 . In the study …”

“…However, the assumption of stable and constant expression of reference genes under various conditions or in different tissues is discussed controversially [14][15][16] . In a previous study Thal and coworkers tested six commonly applied control genes after experimental TBI in juvenile C57Bl6 mice 17 .…”