ANATOMICAL STUDIES ON THE NEURONAL AND NON-NEURONAL CELLS IN THE CHICK THYMUS

Abstract

The thymus plays a fundamental role in the differentiation of blood-borne precursor cells into functionally mature T lymphocytes. It is known that thymic stromal cells, in conjunction with external influences, i.e., humoral factors and the neural input, provide the specific inductive microenvironment for the differentiation process. It was the endeavour of this study to further the current understanding of the nature of non-neuronal cells in the chick thymus and to provide a more comprehensive image of thymic innervation as a basis for understanding the relevance and possible sites of actions of an autonomic input to this gland. Using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry, the present study revealed a host of putative nitrergic non-neuronal cells located at the corticomedullary junction (CMJ) in the chick and rat thymus. Although light microscope study illustrated the heterogeneity of the labelled cells, the individual cell types could not be identified. Examination of the stained sections at ultrastructural level was therefore conducted and as the labelled cells were more abundant in the chick as compared to the rat, the former was selected for this study. Electron microscope examination revealed the distribution of NADPH-d reaction product in a varied cell population, namely, the undifferentiated, lymphoid, cystic, myoid, endocrine-like and some epithelial reticular cells. In addition, labelling was detected in macrophages and endothelial cells. In all the positive cells, the reaction product was essentially membrane bound with a widespread subcellular distribution. The presence of NADPH-d reaction product in the heterogeneous population suggested a putative role of nitric oxide (NO) in modulating the function of these cells. Besides non-neuronal cells, light microscope study of NADPH-d stained sections also revealed neuron-like cell bodies and fibres in the chick thymus. In addition to the above neuroactive chemical, nitric oxide synthase (NOS) immunoreaction product was also demonstrated in neuron-like cell bodies and fibres. The nitrergic neuron-like cell bodies were present in association with blood vessels in the interlobular connective tissue and at the CMJ. The cells were usually present singly or in pairs and rarely in aggregations of up to 6 cells. They were round to oval in shape with a large, oval nucleus which was invariably unstained. By staining adjacent sections with the two neuroactive chemicals, a partial colocalisation of NOS immuno- and NADPH-d reaction products was noted in the neuron-like cell bodies and fibres. NADPH-d stained stuctures were obviously more numerous than those positive for NOS. This suggested that either NADPH-d indicated the presence of other enzymes in addition to NOS or that the antibody against NOS used in this study did not fully recognise the isoform/s of NOS present in neuron-like cell bodies and fibres. Since the presence of neurons in the thymus has thus far yet to be unequivocally demonstrated, the NADPH-d positive neuron-like cell bodies were stained with neuronal marker, neuron specific enolase (NSE). This revealed a subpopulation of NADPH-d positive neuron-like cell bodies which were NSE positive. Double labelling techniques also demonstrated a one to one coexistence between NADPH-d and VIP in nerve fibres, both in the connective tissue and the cortex of the gland. Unlike the NADPH-d positive fibres, VIP positive ones were rarely seen in the medulla. Only a few neuron-like cell bodies stained for VIP. As in the case of the nitrergic cell bodies, these were located in association with blood vessels. While all VIP immunopositive cell bodies were also positive for NADPH-d, the reverse was not true. That nitrergic neuronal cell bodies and fibres expressed VIP suggested that NO and VIP may act as co-transmitters/modulators in regulating the blood flow to the gland. Other neuroactive chemicals like neuropeptide tyrosine (NPY), substance P (SP) and calcitonin gene-related peptide (CGRP) were also examined. NPY-, CGRP- and SP-immunoreaction was never observed in neuron-like cell bodies. These peptides, however, were widely distributed in nerve-like profiles. The distribution of CGRP- and SP- positive fibres was similar. They were found in the capsular and interlobular connective tissue, both in peri- and para-vascular position, and some of them gave off branches to supply the parenchyma. Solitary CGRP and SP fibres were also present in the cortical and the medullary parenchyma. NPY positive fibres, on the other hand, were restricted to JL perivascular distribution and were never seen to branch into the parenchyma. Solitary NPY positive fibres were never observed in the cortex or medulla. The precise distribution of individual neuropeptides suggested that they had specific roles in distinct thymic compartments. To further understand the putative role of neuropeptides and NO during development of_the thymus, ontogenetic expression of these neuroactive chemicals in the chick embryonic thymus was also investigated. All the investigated neuroactive chemicals, i.e., NADPH-d, VIP, CGRP, SP and NPY, were first expressed in nerve-like profiles, predominantly perivascular in position, in the embryonic thymus during late ontogeny, i.e., embryonic day (E) 17-19. The thymic nerves showed a gradual increase from E 17 to E 21. Weakly stained NADPH-d and VIP positive neuron-like cell bodies were observed in 19- and 21-day-embryos. As nerve fibres were not detected in the embryonic thymus before E 17 /18, it is likely that early stages of morphogenesis, vasoregulation, precursor-cell chemotaxis and homing, and T-cell proliferation and differentiation in the thymic anlage were independant of the actions of the neurally released peptides. As previous work has detected the first mature T-lymphocytes in the blood circulation towards the end of the embryonic development, it is speculated that the existence of perivascular peptidergic and nitrergic nerves at this stage was required to facilitate the egress of the immunocompetent cells from the thymus through the thymic vasculature. Electron microscope investigation confirmed the light microscope observation of a rich innervation of the adult thymus. The neuron-like cells, however, remained elusive. The majority of nerve profiles were present in the interlobular connective tissue septa. Thick nerve bundles accompanied the major blood vessels supplying the gland and were generally invested by an epineurium. Near the capsular region, nerve bundles with up to 115 axons were identified. Deeper within the interlobular septa, fewer axons were present in each bundle. Based on the vesicles contained therein, axon boutons could be classified into three types. The most common type contained numerous small ( 40-60 nm) agranular vesicles and a few large (80-120 nm) dense-cored vesicles. The other types were those that contained either only small agranular ( 40-60 nm) or large dense-cored vesicles (80-120 nm). The majority of nerve profiles in the connective tissue were not associated with any cellular component but were surrounded by collagen fibrils. Nerve bundles in the parenchyma were also in general, surrounded by collagen fibres. A typical synaptic contact was not observed between axon profiles and parenchymal cells. In some instances however, vesiculated axon boutons, partially devoid of Schwann cell ensheathment, were observed in close apposition to parenchymal cells especially myoid cells. Previous study has shown that the thymic branch of the vagus nerve contains B fibres which are known to be preganglionic autonomic fibres and that vagal fibres are distributed chiefly to the CMJ in the thymus. The neuronal cell bodies observed in the interlobular connective tissue and at the CMJ could be postganglionic parasympathetic in nature. The axon profiles observed near the myoid cells may be postganglionic parasympathetic 'nerve endings' and their close apposition to these cells may represent neuro-effector sites. As the chicks did not survive bilateral vagotomy, unilateral vagotomy was performed to investigate the ultrastructural changes in the chick thymus at survival periods of three, seven and ten days. At three and seven days postoperation, degenerative changes were observed in several unymelinated axon profiles. These varied from granular disintegration of the axonal cytoskeleton to intense vacuolation of the degenerating axoplasm. Electron-dense myelin figures and clumping of small agranular vesicles occurred in many axon boutons. The large number of axon boutons with spherical or pleomorphic agranular vesicles appeared unchanged after left unilateral vagotomy. These might be preganglionic parasympathetic branches of the right cervical vagus or postganglionic parasympathetic fibres arising from the small ganglia in the periglandular connective tissue of the thymus or from the intrathymic ganglia distributed in the connective tissue septa and at the CMJ. An intrathymic neuronal cell body was found in the chick thymus seven days after vagotomy which showed no degenerative changes. The limited ultrastructural evidence does not preclude transneuronal changes in intrathymic neurons. Myoid and unexpectedly, cystic cells in various stages of degeneration were observed from the third to the tenth day after unilateral vagotomy. Degenerative changes in myoid cells appeared to peak at seven days and the number of degenerating myoid cells appeared to be reduced at ten days post-vagotomy. These changes ranged from swelling of the mitochondria to intense vacuolation of cytoplasm. Profusion of lipid droplets was observed in some myoid cells. Disorganisation and dissolution of the myofibrils was common. Fragments of myoid cells were phagocytosed by reticular epithelial and cystic cells. Three days after vagotomy, cystic cells showed an increase in electron-density and the appearance of myelinated figures and lamellated bodies in their cytoplasm. At seven days after operation swollen cisternae of rough endoplasmic reticulum were apparent in some cystic cells. Dying cystic cells could be seen by ten days post operation.