Immunohistochemical localization of Vesicular Acetylcholine Transporters and Choline Acetyl Transferase Activity in Murine and Human Immune Tissues

Abstract

The cholinergic innervation of lymphoid organs, through autonomic nerves is not well understood. The aim was to localize the cholinergic nerve endings in immune tissues of Balb/C mice, Wistar rats and humans using antibodies of vesicular acetylcholine transporter proteins (VAChT) and choline acetyl transferase (ChAT) enzyme found in the nerve terminals of cholinergic system. Immune tissues were processed for Haematoxyline & Eosin staining and immunohistochemistry. Thin sections were labeled by primary antibodies, anti- VAChT, anti-ChAT and biotinylated anti-rabbit IgGs. Labeled StreptAvidin Biotin method was applied using Diaminobenzidene chromogen and by light microscopic visualization. Skeletal muscle was used as positive control. Photomicrographs were developed using a binocular light microscope equipped with a digital camera (Dino capture, Taiwan), which display the images in the monitor screen of a personal computer and images were analyzed depending on the intensity of immunostaining. Immunoreactive axon terminals of cholinergic neurons were counted. 0 (No staining), 1+ (focal staining, less than 3), 2+ (focal to diffuse staining, 4 to 6), 3+ (diffuse staining, 7 to 10). Immunoreactivity to VAChT antibody was high in capsule and red pulp while absent in white pulp of spleen. The capsular & septal regions of thymus showed 2+IR and the capsule & subcapsular areas of lymph nodes showed 1+IR to VAChT. The VAChT was not localized in liver and Peyer’s patches. The IR of ChAT was similar to the distribution of VAChT in spleen, lymph nodes, thymus and absent in Peyers’ patches. Also the IR of VAChT and ChAT in immune tissues of the three species were similar. The close proximity of the distribution of VAChT and ChAT in capsular and perivascular supporting framework of these tissues confirms that the immune tissues receive cholinergic innervation and is through capsular and perivascular supporting framework of these tissues.