Previous nerve tracing technique studies (HRP-WGA) from our lab have suggested that sensory nerves innervating the T2/3 facet joint in the rat may relay information to projection neurones like the spinothalamic tract in the superficial dorsal horn of the spinal cord. This study aims to use the immunohistochemical detection ofa protein product (fas) of the c-fos immediate early gene. The c­ fos can be used as a transynaptic marker for neuronal activity following noxious stimulation. The pattern of c-fos expression has been shown to correlate well with other anatomical studies, like HRP-WGA. To date no spinal data has been sourced which correlates spinal joint sensory nerves and their role as a source of pain in back pain sufferers. This proposed study has the potential to aid our clinical understanding of the nature of back pain, particularly that of joint origin.

Experiments will be performed on adult rats (400-S00g), anaesthetised, placed in a stereotaxic frame and surgical dissection performed to expose the facet joints. There are 4 joints from T1 to T4 and performing the same experiment 6 times per joint, this requires a total of 24 animals for this group. The joint space will be injected with SµL ofl.5% formalin in saline. The animal’s respiratory rate, hindplimb withdrawal reflex, mucous membrane and rectal temperature will be monitored at 15 minute intervals for the subsequent 2 hours under anaesthesia

The joint will then be washed with saline, the animal and perfused with saline followed by a fixative (paraformaldehyde) and the spinal cord removed. Sections will be cut at 40µm transversely.

The same procedure as outlined above will be performed except that the nerve to the joint will be cut at its most distal attachment (Tl to T4, at 6 animals per nerve leads to a total of24 animals). The nerve will be positioned vertically in a wax well (low melting point dental wax) to avoid surrounding tissue contamination, then bathed in SµL of 1.5% formalin in saline for 2 hour.

Control experiments will receive all of the steps outlined above including anaesthetic and surgery, however, no formalin injections will be delivered into the facet joint (N=4) or the nerve (N=4).

Using brightfield microscopy for cytoarchitectural boundaries and the location of c-fos reaction product, all central observations will be traced onto a spinal cord outline using the drawing tube.

Grant Value: $23,783
Chief Investigator: Dr Ray Hayek – Macquarie University
Status: Complete