Isolation of sensory neurons in vitro and generation of a conditional inducible transgenic mouse line against nerve growth factor (NGF) receptors to investigate the role of NGF in pain.

摘要

Nerve growth factor (NGF) is a known factor in the development of persistent pain, a condition which affects approx. 20% of the UK population. Current therapeutics available for pain exhibit limited effectiveness and therefore better targeted and more effective therapeutics are essential. Clinical trials using anti-NGF have been successful in consistently alleviating pain of patients suffering from chronic pain however the main mechanism of action of NGF is unknown. The main aim of this PhD was to investigate the role for TrkA and p75 receptors for NGF in the development of persistent pain, specifically targeted to primary sensory afferents. The first aim was to determine whether NGF acts directly or indirectly (via other cell types reported to express receptors for NGF) on sensory neurons in the development of pain. To address this we optimised and characterised a protocol for purifying sensory neuronal cultures from dorsal root ganglia (DRG) using magnetic assisted cell sorting (MACS), a method we found to reliably produce viable 95% pure neuronal cultures. The second major question surrounding the mechanism of NGF is whether NGF acts mainly through the TrkA or the p75 receptor, or do the two receptors work in synergy. In order to address this we used two different methods. First, we chose to use an approach using viral vectors, both lenti and adeno-associated virus, to introduce Cre recombinase into DRG neurons with floxed regions of the NTRK1 and NGFR genes to knockout expression of TrkA or p75 respectively. Secondly, we bred a new transgenic mouse lines for conditional knockout of TrkA or p75 under the control of tamoxifen by crossing the above mentioned floxed mouse lines with an Advillin CreERT2 transgenic mouse, where Cre activity is limited to sensory and sympathetic neurons.