Neuronal counts and analysis following immunization A.

<p>Regions selected for neuronal counting (numbered 1 to 5), in the hippocampus (Hp), cerebellum (Cb) and brainstem (BS) are shown on a sagittal rodent brain section and in the grey matter on a coronal section of the spinal cord. B. EGFP-positive and total neuronal counts (calbindin+) in cerebellum, and (NeuN+) in two of the three spinal cord regions sampled. No differences were found between test and control mice. C. Representative images from region 5 of the brainstem demonstrating reduced numbers of EGFP+ neurons in the GAD65-immunized mice, compared with PBS-immunized controls, with some apparent reduction in the NeuN+ neurons. D. Counts obtained from the blinded analysis of all five brain stem regions. Although there was no significant reduction in total neurons, the EGFP+ cells showed a trend towards lower numbers in the brainstem (unpaired t test). E. To investigate further, the immunization and neuronal counting was repeated on three further GAD65 and PBS immunized mice, analyzing a larger number of neurons in the same regions of the brainstem with similar results. All neuronal counting was performed on coded sections by an independent investigator. Scale bar = 100 µm. F. To normalize between the results of D. and E., the EGFP-positive neurons were expressed as a percentage of the NeuN positive neurons for each slice.