The localization of primary efferent sympathetic neurons innervating the porcine thymus – a retrograde tracing study

The autonomic nervous system is a sophisticated and independent structure composed of two antagonistic (opposing) divisions (sympathetic and parasympathetic) that control many vital functions including: homeostasis maintenance, heart rate, blood circulation, secretion, etc. Thymus is one of the most important primary lymphoid organs playing a role in the developing of a juvenile’s immune system mainly by maturation, development, and migration of T-cells (T lymphocytes). In the last decades, several studies identifying sources of the thymic autonomic supply have been undertaken in humans and several laboratory rodents but not in higher mammals such as the pig. Therefore, in the present work, retrograde tracing technique of Fast Blue and DiI was used to investigate the sources of sympathetic efferent supply to the porcine thymus. After Fast Blue injection into the right lobe of the thymus, the presence of Fast Bluepositive neurons was found in the unilateral cranial cervical ganglion (82.8 ± 3.0% of total Fast Blue-positive neurons) as well as in the middle cervical ganglion (17.2 ± 3.0%). Injection of DiI resulted in the presence of retrograde tracer in neurons of the cranial cervical ganglion (80.4 ± 2.3% of total amount of DiI-labelled neurons), the middle cervical ganglion (18.4 ± 1.9%), and the cervicothoracic ganglion (1.2 ± 0.8%). The present report provides the first data describing in details the localization of primary efferent sympathetic neurons innervating the porcine thymus. Fast Blue, DiI, paravertebral ganglia, lymphoid organ, mammals A large majority of internal organs are supplied from both sympathetic and parasympathetic sources of the autonomic nervous system (ANS). Of key importance for autonomic reflexes are the interactions between biologically active substances that are stored and released from presynaptic neurons and receptors that are present in the effector cells (McCorry 2007). However, organ functional integrity and its defense line are dependent and regulated not only by ANS. Similar tasks are assigned to endocrine as well as immunological systems, in particular to lymphoid organs (Schultz and Grieder 1987). All three systems are structurally much expanded and use their own regulatory molecules (hormones, cytokines, and neurotransmitters/neuropeptides) for internal communication purposes. Therefore, numerous functional interactions between different regulatory cells are frequently observed (Gaillard 2002). These phenomena explain why many researchers concentrated their efforts to study the nervous supply to lymphoid organs and the influence of ANS on the activity of immunological cells (Bellinger and Lorton 2014). The thymus gland, belonging to primary lymphoid organs, has a specific place in the mammalian immunological system mainly due to very complicated cellular structure and multipotent functions. Using the histochemical technique, the expression of fluorescent of glyoxylic acid (noradrenergic neuron marker) or acetylcholinesterase (AChE; marker of cholinergic neurons) has been studied in the thymus of numerous mammals, including the mouse (Bulloch and Pomerantz 1984), the rat (al-Shawaf et al. 1991; Mićić et al. 1992), the rabbit (Felten et al. 1981), and humans (Topilko and Caillou 1985; Páldi-Haris et al. 1990). Additionally, primary antisera against major autonomic ACTA VET. BRNO 2017, 86: 117–122; https://doi.org/10.2754/avb201786020117 Address for correspondence: Dr. Anna Zacharko-Siembida Department of Animal Anatomy and Histology Faculty of Veterinary Medicine, University of Life Sciences Akademicka 12, 20-033, Lublin, Poland E-mail: [email protected] http://actavet.vfu.cz/ neurotransmitters as well as neuropeptides/neuropeptide receptors have been applied in immunohistochemical studies in rats (Gomariz et al. 1990; Kendall and al-Shawaf 1991; Bellinger et al. 1997), mice (Delgado et al. 1996; Mitchell et al. 1997), and humans (Lara-Marquez et al. 2000; Mignini et al. 2011). Experimental procedures like chemical sympathectomy with 6-hydroxydopamine (6-OHDA) or guanethidine (Kendall and al-Shawaf 1991), surgical denervation of the sympathetic chain, ablation of the vagus or recurrent laryngeal nerve (Mignini et al. 2010) allowed to determine preliminary sources of sympathetic and parasympathetic innervation to the rat thymus. A substantial progress in neuroanatomical studies on the thymus innervation has been achieved with the use of the retrograde tracing technique utilizing retrograde neuronal tracers such as Fast Blue (FB) (Kummer and Oberst 1993), Fluoro-Gold (FG) (Tollefson and Bulloch 1990), Diamidino Yellow (DY) (Dovas et al. 1998), or horseradish peroxidase (HRP) (Bulloch and Moore 1981). More recently, the central sympathetic centres projecting the rat thymus were transneuronally visualized with the use of the pseudorabies virus (Trotter et al. 2007). As mentioned above, so far most of researchers focused on the innervation of the rodents’ thymus whereas reports describing sources of the nerve supply to the thymus in large mammals are scarce. Therefore, in the present study, the localization of porcine thymus-projecting efferent neurons has been determined using two different neuronal tracers (Fast Blue and DiI). The choice of the pig as an experimental animal is justified as this animal species shows numerous anatomical, physiological, and genetic similarities to humans which makes it an excellent animal model for use in various scientific studies (Luo et al. 2012; Mair et al. 2014). Materials and Methods Experimental animals and anaesthesia All experimental procedures were carried out with the approval of the responsible Local Ethics Committee (number 1/2014) and were in agreement with the “Guide for the care and use laboratory animals” (NAP publication, revised in 2011). For retrograde tracing experiments, five crossbred Polish Landrace × Pietrain (n = 5) piglets (three weeks old, weighing ca. 10 kg) of both sexes were used. Each piglet was purchased from a different litter. The animals were sedated with an intramuscular injection (2 mg/kg b.w.) of azaperone (Stresnil®, Janssen Pharmaceutica N.V., Belgium). To induce a satisfactory level of a deep anaesthesia, intravenous injection (4–6.5 mg/kg b.w.) of propofol (Scanofol®, ScanVet, Gniezno, Poland) mixed with ketamine (2–6 mg/kg b.w.; Bioketan®; Vetoquinol Biowet, Gorzów Wielkopolski, Poland) and atropine (0.05 mg/kg b.w.; Atropinum Sulfuricum®, WZF, Polfa Warszawa, Poland) was given. Pain relief was achieved by a single intravenous injection (15–50 mg/kg b.w.) of metamizole sodium (Injectio Pyralgini®, Biowet Puławy, Poland). Injection of retrograde tracers Two different retrograde tracers were used. The first one was Fast Blue (FB; EMS-Chemie GmbH, Gross Umstadt, Germany) which is well soluble in water and labels cell cytoplasm. The second one was DiI (D-3911; Molecular Probes, Eugene, Oregon, USA) which shows a high affinity to lipophilic binding to neuronal membrane and stains the entire cell. Because DiI is poorly soluble in water, the tracer was dissolved in dimethyl sulphoxide (DMSO), which additionally improved the tracer penetration. In all experimental animals, a neck skin incision (approx. 4 cm) in the tracheal region was made, and the left and right lobes of the thymus were exposed. In all animals, the injection of a total of 50 ml of 5% FB into the right lobe of the thymus was done using the Hamilton syringe equipped with a 26 gauge needle. In each animal, 10 separate injections for 5 ml each, evenly distributed throughout the right lobe, were given. In order to minimalize the tracer leakage, after each injection the needle was kept for 1 min and carefully wiped out with a cotton bud. Finally, the right lobe of the thymus was gently removed and separated from the left lobe. Next, using a brand new Hamilton syringe equipped with a 26 gauge needle, the left lobe of the thymus was injected with a total of 50 ml of 5% DiI (10 separate injections of 5 ml each). The analogous tracer injection protocol as presented above for FB was applied. The skin wound was closed with surgical staples and the animals were treated with amoxicillin (Betamox®, ScanVet Gniezno, Poland; 15mg/kg b.w.) for the next four days. Tissue collecting and processing After a three weeks of post-operative period, the animals were deeply re-anaesthetised and transcardially perfused with 4% solution of buffered (pH = 7.2) paraformaldehyde. For further studies, the sympathetic bilateral cranial cervical ganglion (CCG), bilateral middle cervical ganglion (MCG), bilateral cervicothoracic ganglion 118