RESUMO
BACKGROUND: Traumatic brain injury models are widely studied, especially through gene expression, either to further understand implied biological mechanisms or to assess the efficiency of potential therapies. A large number of biological pathways are affected in brain trauma models, whose elucidation might greatly benefit from transcriptomic studies. However the suitability of reference genes needed for quantitative RT-PCR experiments is missing for these models. RESULTS: We have compared five potential reference genes as well as total cDNA level monitored using Oligreen reagent in order to determine the best normalizing factors for quantitative RT-PCR expression studies in the early phase (0-48 h post-trauma (PT)) of a murine model of diffuse brain injury. The levels of 18S rRNA, and of transcripts of beta-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), beta-microtubulin and S100beta were determined in the injured brain region of traumatized mice sacrificed at 30 min, 3 h, 6 h, 12 h, 24 h and 48 h post-trauma. The stability of the reference genes candidates and of total cDNA was evaluated by three different methods, leading to the following rankings as normalization factors, from the most suitable to the less: by using geNorm VBA applet, we obtained the following sequence: cDNA(Oligreen); GAPDH > 18S rRNA > S100beta > beta-microtubulin > beta-actin; by using NormFinder Excel Spreadsheet, we obtained the following sequence: GAPDH > cDNA(Oligreen) > S100beta > 18S rRNA > beta-actin > beta-microtubulin; by using a Confidence-Interval calculation, we obtained the following sequence: cDNA(Oligreen) > 18S rRNA; GAPDH > S100beta > beta-microtubulin > beta-actin. CONCLUSION: This work suggests that Oligreen cDNA measurements, 18S rRNA and GAPDH or a combination of them may be used to efficiently normalize qRT-PCR gene expression in mouse brain trauma injury, and that beta-actin and beta-microtubulin should be avoided. The potential of total cDNA as measured by Oligreen as a first-intention normalizing factor with a broad field of applications is highlighted. Pros and cons of the three methods of normalization factors selection are discussed. A generic time- and cost-effective procedure for normalization factor validation is proposed.
Assuntos
Lesões Encefálicas/genética , Perfilação da Expressão Gênica/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Actinas/genética , Animais , DNA Complementar/análise , DNA Complementar/normas , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Camundongos , Modelos Animais , Fatores de Crescimento Neural/genética , RNA Ribossômico 18S/genética , Padrões de Referência , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/genética , Tubulina (Proteína)/genética , Estudos de Validação como AssuntoRESUMO
BACKGROUND: The metabolic response to head injury (HI) is characterized by a dysimmunity which may be a risk factor of a septic state. The use of immune enhancing diets (IEDs) could be a promising approach to improve immune functions. The aim of the study was to investigate the consequences of HI on lymphocyte function and to determine the effects of an enteral IED comparatively to a standard enteral nutrition. METHOD: A rat model of HI by fluid percussion was used. Twenty-five male Sprague-Dawley rats were randomized into 4 groups: rats receiving standard chow diet ad libitum (AL), rats sustaining HI and receiving standard chow diet and enteral saline (HI), rats receiving the enteral standard diet Sondalis HP (HIS), and rats receiving the IED Crucial (HIC). The two enteral diets were infused continuously during 4 days after the HI and were isocaloric, isonitrogenous and isovolumic. RESULTS: HI induced a thymus atrophy (HI vs. AL, P<0.05), and an impairment in lymphocyte CD25 receptor density responsiveness to stimulation. The IED blunted thymus atrophy and allowed to preserve the stimulation of blood and Peyer patches lymphocytes (HIC: Stimulated vs. Basal, P<0.05). CONCLUSION: IED seems more adapted for preserving lymphocyte function than standard diet in HI patients.
Assuntos
Traumatismos Craniocerebrais/imunologia , Traumatismos Craniocerebrais/terapia , Nutrição Enteral , Linfócitos/fisiologia , Sepse/prevenção & controle , Timo , Animais , Antioxidantes/administração & dosagem , Arginina/administração & dosagem , Traumatismos Craniocerebrais/complicações , Ácidos Graxos Ômega-3/administração & dosagem , Alimentos Formulados , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Sepse/etiologia , Sepse/terapia , Timo/citologia , Timo/imunologia , Timo/patologiaRESUMO
In this report, our findings highlighted the presence of a high level of calcium in the cortex following traumatic brain injury (TBI) in a rat model of fluid percussion-induced brain injury. This calcium increase represents a pitfall in the assessment of Ca2+-independent nitric oxide synthase (NOS) activity supposed to play a role in the secondary brain lesion following TBI. The so-called Ca2+-independent NOS activity measured in the injured cortex 72 h after TBI had the pharmacological profile of a Ca2+-dependent NOS and was therefore inhibited with a supplement of calcium chelator. The remaining activity was very low and iNOS protein was hardly immunodetected on the same sample used for NOS activity assay. The concentration of calcium chelator used in the assay should be revised and adjusted consequently to make sure that the calcium-free condition is achieved for the assay. Otherwise, the findings tend towards an overestimation of Ca2+-independent and underestimation of Ca2+-dependent NOS activities. The revised Ca2+-independent NOS activity assay was then tested, in relation with the amount of iNOS protein, in a model of LPS-induced neuroinflammation. Taken together, precautions should be taken when assessing the Ca2+-independent enzymatic activity in cerebral tissue after a brain insult.