Electron microscopic observations and X-ray microanalysis of a multinucleated giant cell.

Giant cells and macrophages play important roles in defence and in reparative functions of the body. This paper describes a giant cell and macrophage present in an inflammatory mass in the temporal bone. X-ray microanalysis performed at the ultrastructural level revealed the presence of a very high iron content in the electron dense precipitates observed in both types of cells. The high iron content is probably due to phagocytosed haemosiderin, a breakdown product of free haemoglobin since there was evidence of haemorrhage present in the biological sections. As the tissue was chemically fixed, it also demonstrates that there is still a place for electron probe microanalysis in tissues (including archived specimens) which have undergone chemical fixation.

Immunocytochemical localisation of substance P in vagal ganglion cells and pericellular arborisations in the monkey.

The quantitative cell count showed the presence of about 20,000 ganglion cells with associated satellite elements in the nodose ganglion in the monkey. Among these closely packed cells, at least one-third were substance P (SP) immunoreactive, ranging from faint or moderate to intense staining. Substance P immunoreactivity (SP-IR) was localised in the cell bodies and their long extending neurites. Ultrastructural study showed that SP-IR was not associated with any particular organelles or inclusions. A striking feature of the nodose ganglion was the occurrence of SP-positive pericellular arborisations associated with about 0.5% of the ganglion cells which were almost exclusively SP-negative. The pericellular arborisation displayed diverse morphological forms from a simple tortuous fibre to complex glomerular networks or plexuses encircling the soma of SP-negative ganglion cells. The varicose nerve fibres forming the pericellular arborisations appeared to terminate as 'boutons' contacting the soma of the ganglion cells. Electron microscopic study demonstrated the close spatial relation between the SP-IR profiles and the ganglion cell but there was no direct synaptic contact. In some instances, the SP-IR profiles containing agranular and dense-cored vesicles penetrated the cytoplasm of satellite cells, almost reaching the surface of the soma of the ganglion cell. The sources of origin of the nerve plexuses in the pericellular arborisation were either from the small and sparsely distributed jugular ganglion cells which were intensely SP-IR or from the intrinsic SP-IR nodose ganglion cells. The possibility that the efferent neurons in the dorsal motor nucleus of the vagus could also contribute to the pericellular arborisation was also considered. The function of the pericellular arborisations may be related to the modulation of the SP-deficient ganglion cells with which they associate through the release of SP and probably by way of the satellite cells.

Ultrastructure of murine cardiac ganglia in experimental Chagas' disease.

Albino mice, infected with Trypanosoma cruzi (Tulahuen strain) were sacrificed on days 7, 9, 12, 14, 16, 18, 21, 32 and 39 following infection. Transmission electron microscopic examination of the cardiac ganglia revealed no ultrastructural change at day 7. At day 9 there was peri- and intraganglionic monocytic infiltration but parasites were absent. Between days 12 and 16 there was intense monocytic infiltration, with intra-ganglionic presence of parasites within fibroblasts, monocytes and macrophages. None were seen within capsular cells, endothelial cells, Schwann cells, satellite cells and ganglion cells. The Schwann cells and satellite cells, however, showed phagocytic activity. Satellite cells were also reactive with proliferative pseudopodia which encircled neuronal processes. By day 18, parasites were absent in the ganglia. But monocytes were still present up to day 39, some of them still engulfing satellite cell and neuronal processes. Satellite cells continued to be reactive and Schwann cells phagocytic. Ganglion cells remained normal throughout the experiment. The results suggest that infection of Schwann cells, satellite cells and ganglion cells may depend upon the tissue tropism of the strain of the parasite used and its concentration in the inoculum. The results are consistent with the view that any parasympathetic dysfunction in experimental Chaga's disease in the mouse may be of a transient nature.

Ultrastructural changes of the nodose ganglion cells following an intraneural injection of Ricinus communis agglutinin-60 into the vagus nerve in hamsters.

Virtually all the ganglion cells in the nodose ganglion in hamsters underwent rapid degeneration following an intraneural injection of RCA-60 into the vagus nerve in the cervical region. The earliest signs of neuronal degeneration were evident in animals which survived 5 days after the ricin application. A remarkable feature was the appearance of a variable number of granular dense bodies measuring 1-4 microns in diameter in the cytoplasm. They were composed of closely stacked cisternae which were continuous at the periphery with those of the rough endoplasmic reticulum. Associated with the membranous cisternae were large accumulations of glycogen. With longer survival time, these glycogen-membrane complexes appeared to disintegrate. Numerous vacuoles and neurofilaments accumulated in their vicinity. Satellite cells were activated between the 7th and 10th postoperative days. These penetrated deeply into the degenerating neurons dividing them into numerous fragments by their extensive cytoplasmic prolongations. The cytoplasmic fragments of the RCA-poisoned neurons eventually became necrotic and disintegrated in the satellite cells, suggesting a rapid mode of neuronophagia. The biosynthesis of acetylcholinesterase was inhibited by the ricin injected as shown by the drastic reduction of the enzyme activity in the rough endoplasmic reticulum and nuclear envelope. Some isolated ganglion cells apparently survived the RCA injection as shown by their occurrence in long surviving animals (30-90 days). A few of them displayed an enhanced density of their cytoplasm and neurites. It is postulated that this was induced by the RCA released from the RCA-poisoned neurons.

Ultrastructure of interstitial cells of Cajal at the gastro-oesophageal junction of the monkey (Macaca fascicularis).

The ultrastructure of the interstitial cells of Cajal (ICC) in the oesophagus of the monkey resembled that described in the oesophagus of other mammalian species but differed in their paucity and almost lack of smooth endoplasmic reticulum, caveolae and filaments. The plasmalemma of the ICC was in close contact (20- to 30-nm gaps) with that of smooth muscle cells. This may occasionally take the form of a desmosome, but gap junctions have not been observed. Vesiculated axon profiles, containing large granular or agranular vesicles were in close contact (20- to 30-nm gaps) with the plasmalemma of ICC. In a few vesiculated profiles a presynaptic density could be recognized. The intercalation of the ICC between the vesiculated axon profiles and the smooth muscle cells suggest a role in oesophageal motility. Between 3 and 21 days following bilateral vagotomy some ICC showed regressive changes such as increased electron density and shrinkage of the cytoplasm, crowding of the organelles and dissolution of the nuclear chromatin material. Axon profiles in the vicinity of the affected ICC contained glycogen granules suggesting injury. In late stages, the number of ICC and smooth muscle contacts was reduced. The results suggest that the vagus nerves exert a trophic influence on the ICC and that the intercellular relationships between ICC and smooth muscle cells possess a degree of plasticity. It is tentatively suggested that these vagal effects may be mediated via the oesophageal myenteric ganglia.

Immunocytochemical localization of CR3 complement receptors with OX-42 in amoeboid microglia in postnatal rats.

The present study described the labelling of amoeboid microglial cells in the postnatal rat brain with OX-42, an antibody that recognizes type 3 complement receptors CR3 in mononuclear phagocytes. Of the diverse morphological forms of amoeboid microglia present in the corpus callosum in early postnatal (2-5 days) rats, cells with a round regular outline, or showing short stout processes, were the most intensely stained. When traced from the main cell colony into the borderline zone with the cortex, the immunoreactivity of amoeboid microglia that assumed a ramified form was drastically reduced. Examination of materials from the late postnatal (8-12 days) age group showed that the majority of the OX-42 positive cells in the corpus callosum became oval, elongated and ramified. Immunoelectron microscopy confirmed the above observations, and also showed that the immunoreactivity in the round amoeboid microglia was localized in their plasma membrane, surface projections and invaginations, as well as in some of the subsurface vacuoles. The immunoreactivity was reduced in the oval cells, and diminished in the elongated or ramified form. It is proposed that the presence of CR3 membrane receptors in amoeboid microglial cells is related to their active role in endocytosis. These, however, diminish with the growth of the brain.

Neural degeneration and non-neuronal cellular reactions in the hypoglossal nucleus following an intraneural injection of toxic ricin.

The present study describes neuronal degeneration and its accompanying non-neuronal cellular reaction in the hypoglossal nucleus following an intraneural injection of Ricinus communis agglutinin-60 (RCA-60) into the hypoglossal nerve. The first noticeable structural changes were observed in neurons in hamsters killed 3 days after the RCA injection. Drastic alterations occurred in the period extending from the 5th to the 15th postoperative day. Two forms of neuronal degeneration were observed: light and dark types. In the light type, masses of free ribosomes were observed; other changes included the dilation of Golgi saccules and the presence of abnormal mitochondria. In the dark type of degeneration, the cells became condensed with vacuoles in their cytoplasm. Axon terminals presynaptic to the degenerating cells during this period appeared to be normal. A massive influx of mononuclear leucocytes by diapedesis occurred at the large venules. Some of the infiltrated cells were clearly lymphocytes, while others were monocytes which became indistinguishable from indigenous microglia once they were in the neuropil. Neural macrophages, most probably derived both from microglia and the infiltrated monocytes, were engaged in the phagocytosis of neuronal debris. A remarkable finding in the present study was the wide-spread occurrence of dark axon terminals in the neuropil in longer surviving animals (90 and 120 days). The structural alterations, e.g., clumping and swelling of some of the synaptic vesicles in the enhanced cytoplasmic density, suggest that these were undergoing atrophic changes resulting from the long period of dysfunction following the death of postsynaptic neurons induced by RCA.

Ultrastructure of the chronically vagotomised atrial myocardium in the monkey (Macaca fascicularis).

The ultrastructure of the atrial myocardium in the monkey (Macaca fascicularis) was studied after bilateral cervical vagotomy and survival times of 100, 175 and 367 days. Changes were observed in the nucleus and the cytoplasm of the myocyte. Sequestered within the nuclei of the affected myocytes were cytoplasmic organelles and inclusions. In the late stages, there was a tendency towards condensation and margination of the heterochromatin. Changes in the cytoplasm included increased glycogen, mitochondrial degeneration and myofibrillar disorganisation and degeneration. There was increased collagen and mononuclear cell infiltration in the extracellular space in the later stages. This study has shown that the long term structural integrity of the atrial myocyte depends on an intact vagal innervation. The survival of the monkey after chronic bilateral vagotomy suggests that this nonhuman primate is a suitable model for functional studies of the parasympathetically denervated heart.

Neuroglial response to neuron injury. A study using intraneural injection of ricinus communis agglutinin-60.

The present study has shown the selective destruction of large ventral horn neurons in the lumbosacral cord segments following a single injection of RCA-60 into the sciatic nerve. The neurons appeared to undergo structural alteration beginning 3 days after the RCA application. In the postoperative period extending from 1 to 60 days, degeneration of neurons was progressive and irreversible and this elicited a rapid increase in the number of microglial cells. They were most numerous in the 7 days postoperative animals. The massive microglial cells penetrated the neuropil and appeared to strip off the axon terminals from the postsynaptic somata. Occasional axon terminals were phagocytosed by microglia. The numerous microglial cells often formed a multilayered 'barrier' encircling the somata of the RCA-poisoned neurons which eventually became totally disorganised. It is postulated that in the course of neuronal degeneration induced by RCA, microglial cells serve to prevent the leakage or diffusion of the toxic lectin from the neuronal somata into the neighbouring neuropil. They also function as scavenger cells in the removal of degenerating myelinated axons in the longer surviving rats. Oligodendrocytes do not appear to react actively to the degeneration process. However, astrocytes showed a significant increase in the 7 and 15 day postoperative rats and this coincided with the presence of mitotic astrocytes in the same period.

Ultrastructure of the pineal gland of the monkey, Macaca fascicularis, with special reference to the presence of synaptic junctions on pinealocytes.

The present study described the normal ultrastructure of the monkey pineal gland. The gland was composed of the principal pinealocytes, intramural neurons and glial cells. The nucleus of the pinealocytes was deeply infolded with evenly distributed chromatin materials. The abundant cytoplasm was rich in organelles including the well-developed Golgi apparatuses, multivesicular bodies, dense-cored vesicles and widely scattered free and polyribosomes. A variety of axon terminals was observed and the majority of them contained pleomorphic agranular vesicles with a few large dense-cored vesicles. A few terminals showed flattened vesicles or small dense cored vesicles. Some of the axon terminals formed synaptic contacts with the cell bodies of pinealocytes. These synapses were mainly concentrated in the posterior third of the gland. The occasional intramural neurons observed were postsynaptic to axon terminals containing round agranular vesicles. The sources of the nerve fibres and terminals forming synaptic junctions with pinealocytes and intramural neurons were discussed.

The dorsal motor nucleus of the vagus nerve of the hamster: ultrastructure of vagal neurons and their responses to vagotomy.

The dorsal motor nucleus of the vagus nerve of the hamster was studied electron microscopically. Two types of neuron distinguished by their ultrastructural features were identified. Type I cells, which had an average dimension of 20 X 12 microns, were rich in cytoplasmic organelles. The smaller Type II cells (15 X 9 microns) had scanty cytoplasm and a deeply indented nucleus. Type I cells were labelled by horseradish peroxidase (HRP) following its injection into the vagus nerve, whereas Type II cells were not. Seven days after cervical vagotomy, Type I neurons underwent typical retrograde degeneration. By the fourteenth postoperative day, the density of the cells was greatly enhanced, an early sign of cell death. Type II cells were not affected by vagotomy. With the electron microscope, three types of axon terminals were identified in the neuropil of the DMN: (1) boutons containing round agranular vesicles, (2) boutons containing round and elongated dense-cored vesicles, and (3) boutons containing pleomorphic agranular vesicles, a few of which were dense-cored. All of them showed synaptic contacts with HRP-labelled dendrites. Occasional boutons containing pleomorphic vesicles showed degenerative changes after vagotomy. Moreover, a few of them were seen to contain HRP granules. The observations made in the present study strongly suggest that the Type I neurons send efferent fibres to the vagus nerve. The Type II cells which are not labelled by HRP nor affected by vagotomy, probably serve as interneurons, or they may be cells projecting to other areas of the brain.

Effects of bilateral vagotomy on the ultrastructure of the cardiac ganglia in the monkey (Macaca fascicularis).

This study describes the effects of bilateral vagotomy on the ultrastructure of the cardiac ganglia of the monkey (Macaca fascicularis). One to three days after bilateral vagotomy there is widespread glycogen accumulation in the cytoplasm of the principal cardiac neurons. This is associated with distension of the granular endoplasmic reticulum and the loss of ribosomes from the cisternae. Between five and ten days after operation, about 10% of the neuronal profiles show an overall increase in electron density and intense darkening of the dendrites. From twenty one to twenty eight days postoperatively, the majority of the neuronal profiles have pale cytoplasm with reduction in granular endoplasmic reticulum and polyribosomes. The plasma membrane of the neuron is ruffled over the major portion of its surface. The satellite cells, which are reactive throughout the course of the experiments, exhibit a phagocytic capacity at this stage by removing portions of the neuronal cytoplasm. Vacuolation of the neuronal cytoplasm to a variable degree occurs in a small number of profiles between five to twenty eight days. The results suggest that bilateral vagotomy causes a widespread disturbance in the metabolic activity of the cardiac neurons. This is followed by transneuronal degenerative changes that are of a prolonged nature.

Effects of vagotomy on the ultrastructure of the atrial myocardium in the monkey (Macaca fascicularis).

The ultrastructure of the atrial myocardium in the monkey (Macaca fascicularis) was studied after bilateral cervical vagotomy and survival times of 1, 3, 5, 7, 10, 21 and 28 days. During the first week after vagotomy, a few atrial cells showed a reduction in the sarcoplasm, crowding of the myofibrils, peripheral dispersion and reduced intercristal density of the mitochondria and increased sarcoplasmic reticulum and glycogen particles. In some profiles, there was increased electron density and granularity at the I bands and the intercalated discs. The number of such affected cells increased in the subsequent days such that by 21 to 28 days about 50% of the cells were estimated to be affected. During the latter stages further changes included, the degradation of the myofilaments and increased electron density, disorganisation and disintegration of the digital extensions at the intercalated discs. Throughout the experiments there was a leucocytic infiltration, more evident in the longer survival times.

Ultrastructural changes in the dorsal motor nucleus of monkey following bilateral cervical vagotomy.

The neurons of the dorsal motor nucleus (DMN) of the monkey (Macaca fascicularis) were of two main types: small (13 X 8 micron) and medium-sized (20 X 13 micron). The latter, which were the predominant form, contained a pale oval nucleus surrounded by organelle-rich cytoplasm. Between one and three long principal dendrites per section profile arose from each of the somata. Both axosomatic and axodendritic synapses were seen on these cells although the latter were more common. No structural changes were noted in the DMN 1-3 days after bilateral cervical vagotomy. Some of the dendrites of the medium-sized axotomized vagal neurons appeared darkened 5-10 days after the operation. With longer surviving intervals, i.e. 21 and 28 days after operation, darkened dendrites were more commonly seen and the cytoplasmic density of these dendrites was dramatically enhanced. Their mitochondria were pale and some of them also showed vesiculation. Both normal and degenerating axon terminals were seen to form synaptic contacts with the darkened dendrites. The degenerating axon terminals were characterized by the clumping of their round agranular vesicles. Both darkened dendrites and degenerating axon terminals were phagocytosed by hypertrophied astrocytes and activated microglial cells. Blood elements infiltrating into the DMN were a possible source for some of the neural macrophages. It was concluded from the present study that the dendrites of the vagal neurons were the first structures to degenerate in axotomy and these were subsequently removed by glial elements. Degenerating axon terminals on the darkened dendrites could represent endings of the central processes of peripheral vagal ganglion cells that had undergone transganglionic degeneration after damage to their peripheral processes.