Rat pro-inflammatory cytokine and cytokine related mRNA quantification by real-time polymerase chain reaction using SYBR green.

BACKGROUND: Cytokine mRNA quantification is widely used to investigate cytokine profiles, particularly in small samples. Real-time polymerase chain reaction is currently the most reliable method of quantifying low-level transcripts such as cytokine and cytokine receptor mRNAs. This accurate technique allows the quantification of a larger pattern of cytokines than quantification at the protein level, which is limited to a smaller number of proteins. RESULTS: Although fluorogenic probes are considered more sensitive than fluorescent dyes, we have developed SYBR Green real-time RT-PCR protocols to assay pro-inflammatory cytokines (IL1a, IL1b and IL6, TNFa), cytokine receptors (IL1-r1, IL1-r2, IL6-r, TNF-r2) and related molecules (IL1-RA, SOCS3) mRNA in rats. This method enables normalisation against several housekeeping genes (beta-actin, GAPDH, CypA, HPRT) dependent on the specific experimental treatments and tissues using either standard curve, or comparative CT quantification method. PCR efficiency and sensitivity allow the assessment of; i) basal mRNA levels in many tissues and even decreases in mRNA levels, ii) mRNA levels from very small samples. CONCLUSION: Real-time RT-PCR is currently the best way to investigate cytokine networks. The investigations should be completed by the analysis of genes regulated by cytokines or involved in cytokine signalling, providing indirect information on cytokine protein expression.

Effects of a thermal injury on brain and blood nitric oxide (NO) content in the rat.

In the present study, the effects of a thermal injury on the nitric oxide (NO)-ergic system was investigated in freely moving rats. Using a voltammetric method allowing direct and in situ NO measurements, a significant decrease in cortical NO concentration was observed during the 24h following burning procedure. Since in the burning procedure halothane was employed, it was verified that this anaesthetic did not induce significant effect on cortical NO level. Experiments conducted in ex vivo conditions showed that blood NO and nitrites (NO(2)(-)) + nitrates (NO(3)(-)) concentrations increased strongly after burn injury while hypothalamic inducible NO-synthase (NOS(2)) mRNA level decreased significantly. A thermal injury was thus accompanied by a rapid impairment of the NO-ergic pathways, which might partly have been responsible for numerous changes occurring after burn injury.

Blood-brain barrier permeability after gamma whole-body irradiation: an in vivo microdialysis study.

The effects of total-body irradiation on the permeability of rat striatal blood-brain barrier (BBB) to [3H]alpha-aminoisobutyric acid (AIBA) and [14C]sucrose were investigated using the microdialysis technique. Seven days, 3 and 6 weeks, and 3, 5, and 8 months after gamma exposure at a dose of 4.5 Gy, no modification of the permeability to both [3H]AIBA and [14C]sucrose was observed. But, in the course of the initial syndrome, we observed a significant but transient increase in the BBB permeability to the two markers between 3 and 17 h after exposure. A secondary transient "opening" of the BBB to [14C]sucrose was noticed about 28 h following irradiation without the corresponding increase in BBB permeability to [3H]AIBA. On the contrary, the transport of [3H]AIBA through the BBB was decreased between 33 and 47 h postradiation. In conclusion, our experiments showed early modifications of BBB permeability after a moderate-dose whole-body exposure. Confirmation of these results with other tracers, in another experimental model or in humans, would have clinical applications for designing appropriate pharmacotherapy in radiotherapy and treatment of accidental overexposure.