Fig. 6.

Detection of cytokine mRNA expression by in situ hybridization. The figure demonstrates numbers of acetylcholine receptor (AChR)-reactive mRNA expressing cells for the cytokines: interferon (IFN)-γ, tumour necrosis factor (TNF)-α, interleukin (IL)-4, IL-10 and transforming growth factor (TGF)-β per 10⁵ splenocytes by in situ hybridization technique. Splenocyte suspensions were prepared from non-denervated, denervated and sham-operated female Lewis rats after immunization with AChR + complete Freund's adjuvant (CFA). Two hundred-µl aliquots of suspensions containing 4 × 10⁵ splenocytes were plated into round-bottomed microtitre plates in triplicate. Ten µl aliquots of AChR were added into appropriate wells at the final concentration of 10 µg/ml. After culture for 24 h, the cells were washed, counted and applied onto restricted areas of electronically charged glass slides. Synthetic oligonucleotide probes were labelled using [³⁵S]-deoxyadenosine-5′-α-(thio)-triphosphate with terminal deoxynucleotidyl transferase. Cells were hybridized with 10⁶ counts per minute of labelled probe per 100 µl of hybridization mixture. After emulsion autoradiography, development and fixation, the coded slides were examined by dark field microscopy for positive cells containing more than 15 grains per cell in a star-like distribution. A control probe used in parallel with the cytokine probe on cells from each rat revealed no positive cells (for details see Materials and methods). At each time-point six animals were included in each group. Mean and standard deviations are shown. Stars denote statistical difference between denervated rats (solid bars) and non-denervated controls [open bars; experimental autoimmune myasthenia gravis (EAMG)] (*P< 0·01, **P > 0·001, ***P > 0·0001). There was no statistical difference between sham-operated rats (hatched bars) and non-denervated controls (open bars).