2.1.1 Cell culture:
60 male Wistar rats (200–300g) were killed by stunning and cervical dislocation under Schedule 1 according to the United Kingdom Animals (Scientific Procedures) Act 1986. DRGs were removed from male Wistar rats and collected in cold (4°C) (HBSS; Gibco BRL). Using sterile scissors, the DRG were chopped into small fragments to increase surface area for enzyme digestion. Ganglia were incubated in (papain (1mg/ml) and L-cysteine (2mg/ml) in HBSS (Gibco BRL) for 15 min at 37°C. Ganglia were then replaced in (dispase (0.8%) and collagenase type F (1mg/ml,)) in HBSS for 22-30 min at 37°C, during the incubation a mechanical trituration was performed by using a sterile, fire-polished glass Pasteur pipette to enhance the disaggregation of neurons in the clamp. the cell suspension was centrifuged for 5 min at 1000g at 4°C this was then followed by the resuspension of the cell pellets with sterile HBSS to remove the enzyme. Cell pellets were resuspended in 1ml of feeding media, and the DRG neurons were plated onto the 16mm coverslips (Scientific Laboratory Supplies) coated with poly-D-lysine (0.1mg/ml, Sigma Aldrich) and laminin (20µg/ml, Sigma Aldrich). Cultures were maintained for 2d in feeding medium containing Ham’s F-12 supplemented with 10% horse serum (Sigma Aldrich), 2mM glutamine (Sigma Aldrich), 10ng/ml nerve growth factor (NGF) (Sigma Aldrich),and 1% penicillin/streptomycin (enzyme activity = 5,000units/ml penicillin and5,000µg/ml streptomycin, Gibco at 37°C in humidified air with 5% CO2.