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TITLE: PERIPHERAL MECHANISMS OF INFLAMMATORY PAIN: THE ROLE OF ION CHANNELS ACTIVATED BY EXTREME TEMPERATURES, TRPA1 AND TRPV2

DIRECTOR : PROF. DR. ALEXANDRU BABES

 

Project management:
Work plan. Objectives and activities.

Year Objective (Name) Associated activities Fulfilled
2007 1. Recording the activity of TRPV2 in DRG neurons in primary cultures: the response to thermal and chemical stimuli 1. Obtaining primary cultures of DRG neurons.
2. Optimizing the thermal stimulation protocol to heat DRG neurons to 52 oC.
3. Actual recordings using the patch clamp and calcium microfluorimetry techniques, identification of new TRPV2 antagonists, analysis of the results.
1. Fulfilled 100%

2. Fulfilled 100%

3. Fulfilled 100%

 

2007 2. Recording the activity of TRPA1 in DRG neurons in primary cultures: the response to thermal and chemical stimuli 1. Obtaining primary cultures of DRG neurons
2. Optimizing the thermal stimulation protocol to cool DRG neurons to 15 oC.
3. Actual recordings using the patch clamp and calcium microfluorimetry techniques, analysis of the results.
1. Fulfilled 100%

2. Fulfilled 100%

3. Fulfilled 100%

 

2008 1. Transient and stable transfection of HEK293 cells with TRPV2 and TRPA1. 1. Setting up HEK293 cell culture.
2. Transient transfection of HEK293 cells using the calcium phosphate method: cells will be co-transfected with TRPV2 (TRPA1) and GFP for identification of transfected cells.
3. Obtaining stably transfected cell lines.
1. Fulfilled 100%

2. Fulfilled 100%

3. Fulfilled 100%

 

2008 2. The study of the effect of some inflammatory mediators on TRPV2: bradykinin (BK), prostaglandin E2 (PGE2), NGF, etc… in cultured DRG neurons. 1. Setting up the experimental protocol for applying inflammatory mediators.
2. Recording the effects of BK, PGE2 and NGF and analysis of the results.
1. Fulfilled 100%

2. Fulfilled 100%

 

2008 3. The study of the effect of some inflammatory mediators on TRPA1: bradykinin (BK), prostaglandin E2 (PGE2), NGF, etc… in cultured DRG neurons. 1. Setting up the experimental protocol for applying inflammatory mediators.
2. Recording the effects of BK, PGE2 and NGF and analysis of the results.

1. Fulfilled 100%

2. Fulfilled 100%

 

2009 1. Identification of signaling pathways involved in the modulation of TRPV2 by inflammatory mediators. 1. Application of agonists and antagonists of receptors for inflammatory mediators and investigation of their effect on the modulation of TRPV2 in DRG neurons.
2. Application of agonists and antagonists of intracellular factors involved in the signaling pathways (PKA, PKC, PI3K, etc…): investigation of the effect of these agents on the modulation of TRPV2 expressed in HEK293 cells.
3. Analysis of the results.
1. Fulfilled 100%

2. Fulfilled 100%

3. Fulfilled 100%

 

2009 2. Identification of signaling pathways involved in the modulation of TRPA1 by inflammatory mediators. 1. Application of agonists and antagonists of receptors for inflammatory mediators and investigation of their effect on the modulation of TRPA1 in DRG neurons.
2. Application of agonists and antagonists of intracellular factors involved in the signaling pathways (PKA, PKC, PI3K, etc…): investigation of the effect of these agents on the modulation of TRPA1 expressed in HEK293 cells.
3. Analysis of the results.
1. Fulfilled 100%

2. Fulfilled 100%

3. Fulfilled 100%

 

2009 3. Introduction of the skin-nerve and guinea pig corneal recording techniques. 1. Setting up the experimental installations.
2. Applying the protocol for thermal and chemical stimulation of sensory cutaneous nerve endings.
1. Postponed for 2010. Work in progress.

2. Postponed for 2010. Work in progress.

 

2010 1. Recording of the electrical activity induced by TRPV2 and TRPA1 in sensory axons from the rat and guinea pig. 1. Application of TRPV2 (2-APB) and TRPA1 (cinnamaldehyde) agonists and monitorization of the resulting action potentials.
2. Validation of the results obtained on cellular models (DRG neurons and HEK293 cells) by applying inflammatory mediators on sensory nerve endings and identification of their effects on TRPV2 and TRPA1.
3. Analysis of the results.
1. Work in progress

2. Work in progress

 

2010 2. Introduction of the single-cell RT-PCR and messenger RNA interference techniques. 1. Optimization of experimental set-ups and work protocols.
2. Obtaining evidence regarding the expression of TRPV2 and TRPA1 in functionally identified neuronal sub-populations.
1. Fulfilled 100%

2. Fulfilled 100%

 

2010 3. The study of the effect of inhibition of TRPV2 and TRPA1 expression in DRG cultures using RNA interference methods (siRNA). 1. Incubation of primary DRG cultures with siRNA directed against TRPV2 and TRPA1.
2. Recording of the response of si-RNA-treated neurons to thermal stimuli and TRPV2 and TRPA1 agonists.
3. Analysis of the results.
1. Work in progress

2. Work in progress

3. Work in progress

 

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