Abstract
The study of a bidirectional communication between the nervous and immune system has been examined extensively in rodents and primates. These studies suggest that various types of stressors, including psychological stressors, may alter immune function. Our laboratory has shown the presence of noradrenergic sympathetic nerve fibers in specific compartments of both primary and secondary lymphoid organs. These nerve fibers directly contact lymphocytes and macrophages, as well as vascular and trabecular Smooth muscle. Furthermore, noradrenergic denervation with chemical sympathectomy results in altered measures of immunity.
Studies utilizing stressors and experimental manipulation of the immune system are not applicable to the study of neural-immune interactions in cetaceans. Most information about the status of the immune system of cetaceans comes from the evaluation of blood samples. Through a collaborative effort with the Canadian Fish and Marine Mammal Management Office, we were able to obtain immune tissue from beluga whales taken by the native Inuit during a sanctioned hunt on Hudson Bay. The tissue was obtained after a relatively short post-mortem interval, permitting proper fixation for processing the tissue for immunocytochemistry. We were able to localize nerve fibers and their target immune cells, permitting comparison with the extensive data al ready obtained in rodents and primates.
The immune organs (spleen, thymus, pseudopancreas and lymph nodes) of the beluga whale (Delphinapteras leucas), were obtained approximately 1-2 hours after the animal's death, cut into I cm3 pieces and placed in 10 ml of 4% parafon-naldehyde. After two hours in the fixative, the tissue was microwaved It low power for a maximum of 20 minutes after bringing the temperature of the fixative in the microwave to 115°F This permits excellent penetration of fixative. After microwave fixation, the tissue remained in tile fixative for another hour, after which it was placed overnight in 30% sucrose. Twenty-four hours later the tissue was frozen on dry ice and stored in liquid nitrogen.
Pieces of beluga spleen were cut at 40gm on a sliding microtome and processed for tyrosinc hydroxylase (TH) and IgM immunocytochemistry. The Chemicon anti-rabbit TH at a 1:6000 dilution proved to give the cleanest staining, with donkey anti-rabbit IgG-SP-biotin as the second antibody diluted out to 1:3000. Rabbit IgM provided good staining at 1:2500.
Our preliminary results demonstrate both immune compartmentation and neural innervation of the beluga spleen. The spleen is compartmentalized into red pulp and white pulp, with extensive zones of white pulp. The white pulp is organized around a central arteriolar system, as it is in rodents and primates. The tyrosine hydroxylase antibody provided excellent staining of nerve fibers in the spleen. These fibers are associated with the smooth muscle of trabeculae and the vasculature. The central arteriolar system of the white pulp is densely innervated. Some nerve fibers branch from the central arteriolar system into the parenchyma of the periarteriolar lymphatic sheath, whose cellular composition has not yet been ascertained, although T cells probably are clustered here, as they are in other mammals. Additional nerve fibers were found along a zone that we have tentatively identified as a marginal sinus. IgM positive cells were present in both white pulp and red pulp. The IgM cells in red pulp were scattered among other cellular elements; in white pulp, some dense clusters of IgM-positive cells were present, suggesting the presence of follicles.
We will continue to modify and improve the staining to permit visualization of T lymphoctyes and macrophages, and to obtain double labeling of TH and various immune cells in addition to the IgM staining already achieved. After light level studies we will examine these neural-immune relationships at the EM level to explore the possibility of direct contacts between the terminals and lymphocytes, as seen in rodent spleen. While this has only been looked at in beluga spleen, we will also stain beluga thymus, lymph nodes, and pseudopancreas for innervation patterns and compartmentation of these immune organs. This study has great applicability for investigation in cetaceans that have beached themselves. One working hypothesis for strandings is that the animal undergoes specific stressors that may lead to an induced state of immunosuppression, which in turn may lead to infections that result in a stranding. Histochemistry and immunocytochemistry examination of immune organs of beached animals may reveal altered organization and compartmentation of the various immune organs after the animal has beached itself. It would be ideal to compare neural-immune interactions in animals that are stranded, that die in captivity or that are drowned in fishing nets with the hope of shedding some light on the causes for the strandings that take place every year and how to best care for cetaceans in captivity.