Sympathetic innervation of human and porcine spleens: implications for between species variation in function

Abstract

Background The vagus nerve affects innate immune responses by activating spleen-projecting sympathetic neurons, which modulate leukocyte function. Recent basic and clinical research investigating vagus nerve stimulation to engage the cholinergic anti-inflammatory pathway (CAP) has shown promising therapeutic results for a variety of inflammatory diseases. Abundant sympathetic innervation occurs in rodent spleens, and use of these species has dominated mechanistic research investigating the CAP. However, previous neuroanatomical studies of human spleen found a more restricted pattern of innervation compared to rodents. Therefore, our primary goal was to establish the full extent of sympathetic innervation of human spleens using donor tissue with the shortest procurement to fixation time. Parallel studies of porcine spleen, a large animal model, were performed as a positive control and for comparison. Methods Human and porcine spleen tissue were fixed immediately after harvest and prepared for immunohistochemistry. Human heart and porcine spleen were stained in conjunction as positive controls. Several immunohistochemical protocols were compared for best results. Tissue was stained for tyrosine hydroxylase (TH), a noradrenergic marker, using VIP purple chromogen. Consecutive tissue slices were stained for neuropeptide Y (NPY), which often co-localizes with TH, or double-labelled for TH and CD3, a T cell marker. High-magnification images and full scans of the tissue were obtained and analyzed for qualitative differences between species. Results TH had dominant perivascular localization in human spleen, with negligible innervation of parenchyma, but such nerves were abundant throughout ventricular myocardium. In marked contrast, noradrenergic innervation was abundant in all regions of porcine spleen, with red pulp having more nerves than white pulp. NPY stain results were consistent with this pattern. In human spleen, noradrenergic nerves only ran close to T cells at the boundary of the periarterial lymphatic sheath and arteries. In porcine spleen, noradrenergic nerves were closely associated with T cells in both white and red pulp as well as other leukocytes in red pulp. Conclusion Sympathetic innervation of the spleen varies between species in both distribution and abundance, with humans and pigs being at opposite extremes. This has important implications for sympathetic regulation of neuroimmune interactions in the spleen of different species and focused targeting of the CAP in humans.

Bidirectional crosstalk between the peripheral nervous system and lymphoid tissues/organs

“…However, one limitation of our study was that, by using NF-H as a general neuronal marker, we did not identify the types of nerve fibers inside the spleen. This type of nerve-immune interaction (demonstrated by high-resolution microscopic imaging) inside the spleen has been observed in some studies by other investigators ( 72 , 114 – 116 ). For example, Kirkland et al.…”

“…This type of nerve-immune interaction (demonstrated by high-resolution microscopic imaging) inside the spleen has been observed in some studies by other investigators (72,(114)(115)(116). For example, Kirkland et al have shown the close association of TH + sympathetic fibers with CD3 + T cells in the human and porcine spleen (72). In addition, Murray et al have also demonstrated close associations between TH + (sympathetic)/ ChAT + (parasympathetic) nerve fibers with CD3 + T cells in the murine spleen (73).…”

“…However, a few limitations to the above studies should be mentioned. The first is that, in some investigations, only sparse neurons/nerve fibers/nerve endings have been identified in the spleen ( 59 , 72 ). Second, even though the major nerves (after entering the spleen through hilum) have been shown, some studies have not identified fine nerve fibers inside the spleen ( 64 ).…”

“…For example, Kirkland et al. have shown the close association of TH + sympathetic fibers with CD3 + T cells in the human and porcine spleen ( 72 ). In addition, Murray et al.…”

Regulation of Bone by Mechanical Loading, Sex Hormones, and Nerves: Integration of Such Regulatory Complexity and Implications for Bone Loss during Space Flight and Post-Menopausal Osteoporosis

“…The findings with the spleen discussed by Wulf and Tom [225] is interesting as like bone, tissues of the immune system are disseminated throughout the body, operate in different environments, and are innervated [226][227][228][229]. The spleen [230,231], the lymph nodes [232], the gut mucosal immune system [233,234], the thymus [235], and the bone marrow [236] are all innervated, and the innervation plays a regulatory role. Innervation of tissues, such as the spleen, exhibit some speciesspecific innervation differences [231], and in rodents, the integrity of some immune system components, but not all, can decline with age [235].…”