Anesthesia for euthanasia influences mRNA expression in healthy mice and after traumatic brain injury.

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.

Single administration of tripeptide α-MSH(11-13) attenuates brain damage by reduced inflammation and apoptosis after experimental traumatic brain injury in mice.

Following traumatic brain injury (TBI) neuroinflammatory processes promote neuronal cell loss. Alpha-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with immunomodulatory properties, which may offer neuroprotection. Due to short half-life and pigmentary side-effects of α-MSH, the C-terminal tripeptide α-MSH(11-13) may be an anti-inflammatory alternative. The present study investigated the mRNA concentrations of the precursor hormone proopiomelanocortin (POMC) and of melanocortin receptors 1 and 4 (MC1R/MC4R) in naive mice and 15 min, 6, 12, 24, and 48 h after controlled cortical impact (CCI). Regulation of POMC and MC4R expression did not change after trauma, while MC1R levels increased over time with a 3-fold maximum at 12 h compared to naive brain tissue. The effect of α-MSH(11-13) on secondary lesion volume determined in cresyl violet stained sections (intraperitoneal injection 30 min after insult of 1 mg/kg α-MSH(11-13) or 0.9% NaCl) showed a considerable smaller trauma in α-MSH(11-13) injected mice. The expression of the inflammatory markers TNF-α and IL-1ß as well as the total amount of Iba-1 positive cells were not reduced. However, cell branch counting of Iba-1 positive cells revealed a reduced activation of microglia. Furthermore, tripeptide injection reduced neuronal apoptosis analyzed by cleaved caspase-3 and NeuN staining. Based on the results single α-MSH(11-13) administration offers a promising neuroprotective property by modulation of inflammation and prevention of apoptosis after traumatic brain injury.

Pioglitazone reduces secondary brain damage after experimental brain trauma by PPAR-γ-independent mechanisms.

Inflammatory and ischemic processes contribute to the development of secondary brain damage after mechanical brain injury. Recent data suggest that thiazolidinediones (TZDs), a class of drugs approved for the treatment of non-insulin-dependent diabetes mellitus, effectively reduces inflammation and brain lesion by stimulation of the peroxisome proliferator-activated receptor-γ (PPAR-γ). The present study investigates the influence of the TZD pioglitazone and rosiglitazone on inflammation and secondary brain damage after experimental traumatic brain injury (TBI). A controlled cortical impact (CCI) injury was induced in male C57BL/6 mice to investigate following endpoints: (1) mRNA expression of PPAR-γ and PPAR-γ target genes (LPL, GLT1, and IRAP/Lnpep), and inflammatory markers (TNF-α, IL-1ß, IL-6, and iNOS), at 15 min, 3 h, 6 h, 12 h, and 24 h post-trauma; (2) contusion volume, neurological function, and gene expression after 24 h in mice treated with pioglitazone (0.5 and 1 mg/kg) or rosiglitazone (5 and 10 mg/kg IP at 30 min post-trauma); and (3) the role of PPAR-γ to mediate protection was determined in animals treated with pioglitazone, the PPAR-γ inhibitor T0070907, and a combination of both. Inflammatory marker genes, but not PPAR-γ gene expression, was upregulated after trauma. Pioglitazone reduced the histological damage and inflammation in a dose-dependent fashion. In contrast, rosiglitazone failed to suppress inflammation and histological damage. PPAR-γ and PPAR-γ target gene expression was not induced by pioglitazone and rosiglitazone. In line with these results, pioglitazone-mediated protection was not reversed by T0070907. The results indicate that the neuroprotective effects of pioglitazone are not solely related to PPAR-γ-dependent mechanisms.

Selection of endogenous control genes for normalization of gene expression analysis after experimental brain trauma in mice.

Quantitative measurements of gene expression require correction for tissue sample size, RNA quantity, and reverse transcription efficiency. This can be achieved by normalization with control genes. The study was designed to identify candidates not altered after brain trauma. Male C57Bl/6 mice were anesthetized with isoflurane, and a pneumatic brain trauma was induced by controlled cortical impact (CCI) on the right parietal cortex. Brains were removed at 15 min, and 3, 6, 12 and 24 h after CCI and from naive animals (n = 6 each). Absolute copies of six control genes (beta-2-microglobin [B2M], cyclophilin A, beta-actin, hypoxanthine ribosyltransferase [HPRT], porphobilinogen deaminase [PBGD], and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) and one example target gene (iNOS) were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR; Lightcycler) in the traumatic focus and contralateral tissue. Control gene expression was stable until 12 h after CCI. At 24 h after CCI expression of B2M, cyclophilin A and HPRT remained stable in the contusion, while expression of beta-actin, GAPDH, and PBGD increased. Due to variations between animals (+/-85%), increases in beta-actin (+64%) and GAPDH (+59%) did not reach the level of significance. In non-contused tissue, expression of all genes dropped 24 h after CCI (range, -17% to -61%). Due to low variations between animals and stable expression after CCI, B2M and cyclophilin A seem to be suitable to serve as single normalizer. Normalization of the example target gene iNOS resulted in varying relative expression extending from onefold (PBDG) to 10-fold (HPRT). The results suggest that the knowledge of the temporal profile of control genes is essential to properly interpret results of mRNA quantification.