Araldite Sections Research Articles

Structural reorganization of the sciatic, peroneal, and tibial nerves during osteosynthesis of lower leg fracture and after fracture consolidation (an experimental study)

The study purpose was to identify histological changes in the peripheral nerves and determine functional significance of subclinical neuropathy in experimental simulation of lower leg fractures. We simulated a tibial shaft fracture in 24 dogs under anesthesia and performed osteosynthesis using an Ilizarov apparatus. After 7, 14, 20, 35–37, and 50 days of fixation using the apparatus and 30, 60–90, and 120 days after removal of the apparatus, the dogs were euthanised. Electromyography was performed before fracture and within the main phases of the experiment. Samples of the sciatic, peroneal, and tibial nerves were studied histologically using computer morphometry of semithin araldite sections. After 37 days of fixation, the amplitude of M-responses of the tibialis anterior and gastrocnemius muscles decreased by 70% compared to the baseline value, increased after removal of the apparatus, but not restored. There were no sighs of nerve damage by bone fragments or wires. The fraction of degenerating myelinated fibers in the studied nerves was less than 13%. Reorganization of the Remak bundles led to a temporary increase in the number density of nerve fibers. Along with axonal atrophy, the peroneal nerve was characterized by demyelination-remyelination, and the tibial nerve was characterized by hypermyelination. The dynamics of numerical density of endoneurial blood vessels was also different. Despite minor sharp degenerative changes in myelinated fibers, persistent axonal atrophy, demyelination, and retrograde changes were not compensated for up to 120 days after removal of the apparatus. Correlations between the morphometric parameters of myelinated fibers and the M-response amplitude indicated a causal role of subclinical neurological changes in functional outcomes of fractures.

Distribution of Clara cell secretory protein expression in the tracheobronchial airways of rhesus monkeys

Clara cell secretory protein (CCSP) is a protective lung protein that is believed to have antioxidant, immunomodulatory, and anticarcinogenic properties; to be present in all adult mammals; and to be well conserved in rodents, humans, and nonhuman primates. The rationale for this study is to define the distribution and abundance of CCSP in the airway epithelium and lavage fluid of the adult rhesus monkey and to provide information for evaluating CCSP as a marker of Clara cells and as a biomarker of lung health. Lung tissue and lavage fluid from 3-yr-old rhesus monkeys were examined using histopathology and immunohistochemistry. Proximal bronchi, midlevel bronchi, and terminal/respiratory bronchioles were compared for immunohistochemical localization of CCSP in three-dimensional whole mounts as well as in paraffin and Araldite sections. Immunoreactive CCSP was found in nonciliated cells throughout the airway epithelium. Proximal and midlevel airways had the highest labeling. CCSP decreased in distal airways, and respiratory bronchioles had little to no CCSP. CCSP in the most distal airways was in tall cuboidal cells adjacent to the pulmonary artery. Although a large number of cells were present in the terminal bronchioles that would be classified as Clara cells based on morphology (nonciliated cells with apical protrusions), only a small number stained positively for immunoreactive CCSP. Semiquantitative analysis of Western blots indicated that changes in lavage CCSP are consistent with, and may be predictive of, overall CCSP levels in the airway epithelium in this primate species that is phylogenetically similar to humans.

Wallerian Degeneration in the Optic Nerve of the Rabbit

Progressive anterograde axonal degeneration is known to follow after transection of the axon from the soma, which to some extent correlates with the passage of time after the lesion. However, the minimum time required for such changes to begin remains unresolved. In this study, 20 young adult rabbits of either sex underwent experimental monocular enucleation (left eye) under general anaesthesia. Left optic nerves from such animals were treated as experimental and those from either side of non-operated animals served as controls. Animals were sacrificed postoperatively at periods ranging from 12 h to 3 months. Brains were fixed with 10% formalin and Karnovsky fixatives by an intracardiac perfusion method. Light microscopy of 8-µm paraffin sections and 0.5-µm araldite sections from the optic nerves did not reveal any changes at 12 h. At 24 h, focal minute cavities appeared across the optic nerves. Those nerves from late postoperative stages revealed such cavities with increasing dimensions, disarray of fascicular organization, fragmentation, ovoid formation and finally dissolution of the myelin sheaths. There was an appreciable increase in the number, size and aggregation of glia cells. The debris of degeneration remained prominent even 3 months after enucleation. Electron microscopy revealed splitting of myelin, intramyelinic and periaxonal oedema and occurrence of amorphous and electron-dense materials in the degenerating nerve fibres. It was concluded that while the optic nerve showed degenerative changes as early as 24 h after enucleation, debris of degeneration was only partly removed even after 3 months.

Functional morphology of the salivary gland of the snail, Helix pomatia: a histochemical and immunocytochemical study.

Functional morphology of Helix pomatia salivary gland cells was studied at light microscopic level by using different histochemical methods. Three cell types could be demonstrated in the salivary gland: mucocytes, granular and vacuolated cells. The distribution and the number of the different cell types were different in active and inactive snails. In active feeding animals, dilatated interlobular salivary ducts were observed, which were never present in inactive ones. In active animals an additional cell type, the cystic cell could also be observed. Periodic acid Schiff staining revealed both mucuos and serous elements in the salivary gland. Furthermore, hematoxyline-eosin staining indicated the occurrence of a cell layer with high mitotic activity in the acini. Applying immunohistochemical methods with monoclonal mouse anti-human Ki-67 clone, B56 and polyclonal rabbit anti-human Ki-67 antibodies, we also were able to demonstrate the occurrence of dividing cells in the salivary gland. Analysis of 1-2 microm semi-thin Araldite sections stained with toluidine-blue showed that the saliva can be released, in addition to possible exocytosis, by the lysis of cystic cells. Using an apoptosis kit, we could also establish that this process was due to rather an apoptotic than a necrotic mechanism. In the salivary gland of active snails, where an intensive salivation takes place, significantly more apoptotic cells occurred, if compared to that of inactive animals. It is suggested that programmed cell death may also be involved in the saliva release.

Peri-partum changes in the intraepithelial lymphocyte population of sheep interplacentomal endometrium.

Previous studies have shown that the proportion of gammadeltaTCR+ large granulated lymphocytes (LGLs) increased markedly during pregnancy and declined dramatically by 2 days after parturition in sheep interplacentomal uterine epithelium. In the present study, the distribution, dynamics and fate of these cells, just before, during and immediately after parturition are described. Interplacentomal tissues were collected at 140 days postcoitus (dpc), 148 dpc, during parturition, 1-2 hr postpartum, 1 day postpartum (dpp) and 3 dpp, and were studied using light and electron microscopy, and immuno histochemistry. Uterine washings were collected at 148 dpc and examined for the presence of LGLs. Semi-thin Araldite sections taken at different stages were used to quantify the intraepithelial LGLs, non-granulated lymphocytes (NGLs) and apoptotic cells, whereas frozen sections were used to quantify CD45R+, CD8+ and gammadeltaTCR+ intraepithelial lymphocytes (IELs). A dramatic decline in the proportion of IELs in the luminal epithelium during parturition was observed, mainly because of the decline in CD45R+, CD8+ and gammadeltaTCR+ IELs. There was also a significant decline in the number of granules/ LGL at parturition. This was accompanied by the presence of apoptotic cells of which some were LGLs. The proportions of IELs, LGLs and apoptotic cells markedly increased at 3 dpp. LGLs were found both in uterine washings at 148 dpc and in the uterine lumen at 3 dpp. Apoptosis of glandular epithelial cells was also evident at parturition and markedly increased at 1 dpp. Our results demonstrated that the dramatic decline in the proportion of gammadeltaTCR+ LGLs at parturition was because of de-granulation, apoptosis and migration of these cells into the uterine lumen.

Development of the human dispermic embryo.

In a recent CD-ROM, we portrayed the microstructure of the pre-implantation human embryo (Sathananthan et al., 1999), which was a multimedia production with computer colour-enhanced electron micrographs of mainly monospermic embryos. This disk portrays light and electron micrographs of over 250 tripronuclear (3PN), dispermic, human embryos during pre-implantation development, viewed in thick and thin Araldite sections, as well as appearances of whole embryos flat embedded in Araldite blocks visualized with the light microscope. The 100 figures were computerized (IBM TIFF format), edited and labelled using Adobe Photoshop 5. Some of the figures were coloured on computer. The early development of 3PN embryos overtly resembles that of normal embryos but there are important differences in their microstructure which are portrayed in this presentation. This is a multicentric study involving researchers from four IVF centres.

A new radiographic procedure for obtaining correction factors of 3H-beta-self-absorption for quantitative tritium autoradiography.

A 3H-radiographic method based on the absorption of 3H-beta-particles by an overlying tissue section was established for obtaining correction factors of 3H-beta-absorption (c.f.s) on the cellular level for all kinds of sections in a simple and more precise way as possible with interferometry. Unlabelled paraffin or Araldite sections were mounted on a thin uniformly 3H-labelled section of resin, and autoradiographs were prepared. Grain densities of neuronal cell types and cell-free areas within and outside the paraffin or Araldite sections were evaluated in autoradiographs, where the exposure time or the thickness of the overlying histological section was varied. From these values c.f.s were calculated applying the Beer-Lambert law. It was shown that corresponding c.f.s determined with this new radiographic method correspond well with each other. However, they will only agree with those c.f.s obtained by interferometry, if relative c.f.s are compared. Since the c.f.s are quite sensitive to the section thickness, a new parameter phi was introduced, which helps to assess whether the microtome used works exactly. Generally, the method presented can be used on the cytological level as well as for whole areas in every other autoradiographic study.

Distribution of carbohydrates in the zona pellucida of human oocytes.

The purpose of the present study was to investigate the distribution pattern of carbohydrates in the zona pellucida of human oocytes using lectins and ruthenium red as histochemical probes. For lectin analyses, oocytes that failed to undergo fertilization following in vitro insemination were collected, washed, fixed with glutaraldehyde and embedded in araldite. For ruthenium red labelling, the oocytes were fixed with glutaraldehyde containing ruthenium red, post-fixed with OsO4 and embedded in araldite. Araldite sections (1 micron) were de-resined with sodium methoxide, rehydrated, labelled with ten different biotinylated lectins as probes and avidin-biotin-peroxidase complex as visualant, and examined under a light microscope. The zonae pellucidae of all oocytes studied exhibited a common lectin-binding pattern, expressed in intense binding of lectins from Concanavalia ensiformis (ConA), Lens culinaris (LCA), Ricinus communis (RCA-I), wheat germ agglutinin (WGA), and of succinylated WGA (S-WGA). Peanut lectin (PNA) bound to the zona pellucida only after neuraminidase treatment, whereas the lectins from Griffonia simplisifolia (GS-I), Dolichos biflorus (DBA), Ulex europhaeus (UEA-I) and soybean (SBA) did not bind at all. There was almost no binding of ruthenium red to the matrix of the zona pellucida. The results indicate that the human zona pellucida is characterized by normally exposed mannosyl, N-acetylglucosaminyl and beta-galactosyl residues. In addition, it contains masked beta Gal-(1-3)GalNAc sugar sequences that can be exposed only after removing terminal sialic acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical characterization of anionic constituents in oocyte-cumulus complex of rats.

Fertilization is a process that involves the recognition and adhesion of negatively charged spermatozoa to the oocyte investments. It is not known, however, if charge properties of the interacting gametes play a role in fertilization. The present study evaluates the content and distribution of anionic constituents in the oocyte-cumulus complex of rats. Polycationic colloidal gold (PCG), ruthenium red (RR) and wheat germ agglutinin (WGA) were used as cytochemical markers of anionic sites at the light (LM) and electron microscopical (EM) levels. Isolated oocyte-cumulus complexes were fixed with glutaraldehyde (GA) and OsO4 containing RR, or with GA without RR, and embedded in araldite or LR-gold. For LM, deresined, semi-thin, araldite-embedded sections were labelled with PCG intensified by silver, or with biotinylated lectins visualized by avidin-peroxidase. For EM, thin LR-gold sections were labelled with PCG, whereas RR labelling was examined in araldite sections. The zona pellucida (ZP) failed to bind any of the polycationic markers used, but intensely bound neutralized WGA. In contrast, cumulus cell membranes bound PCG but not RR, whereas the oolemma bound RR but not PCG. The results indicate that the ZP is practically devoid of negatively charged constituents, and tends to repel positively charged ligands possibly due to the presence of cationic determinants. The binding of PCG to cumulus cells probably reflects a high content of membrane-bound heparan sulphate, whereas the binding of RR to the oolema indicates the presence of membrane sialoglycoconjugates.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular localization of Mycobacterium leprae-specific phenolic glycolipid (PGL-I) antigen in human leprosy lesions and in M. leprae isolated from armadillo liver

Phenolic glycolipid (PGL-I), an antigen specific to Mycobacterium leprae, was localized subcellularly in M. leprae residing in human skin, in M. leprae isolated from armadillo liver ('isolated M. leprae') and outside M. leprae in human lepromatous skin. For a quantitative localization of PGL-I sites, specimens, including skin segments stored for 6 years in glutaraldehyde, were embedded in hydrophilic Lowicryl (K4M) resin for ultrathin sectioning. Ultracryosections and Araldite sections of comparable specimens were used for comparison of localization results. A monoclonal antibody (F 47-21-3) directed to antigenic oligosaccharide of PGL-I was employed as primary antibody in immunogold labelling of ultrathin sections. K4M-immunogold methods gave very satisfactory quantitative gold-labelling of PGL-I. The localization of PGL-I by this method partially corresponded with sites detectable in both ultracryosections and the qualititatively superior Araldite sections, but new sites were also localized. Cell walls in human M. leprae and in isolated M. leprae possessed many PGL-I sites, particularly in dividing organisms. PGL-I or its antigenic oligosaccharide was also found, to a lesser extent, in the bacterial cytoplasm. Capsules discernible around part of isolated M. leprae cells displayed heavy PGL-I labelling, sometimes clearly confined to a zone distant from the cell wall. Extrabacterial PGL-I in M. leprae-infected human skin was encountered (1) in phagolysosomes and cytoplasm proper of dermal macrophages containing M. leprae, and (2) intra- and extracellularly in epidermal areas where basal cells harboured M. leprae in untreated multibacillary patients.

Ultrastructural and histochemical changes in the murine zona pellucida during the final stages of oocyte maturation prior to ovulation.

The ultrastructural morphology of the mouse zona pellucida was studied in preovulatory follicles from the ovaries of immature mice treated with exogenous gonadotrophins. The ovaries were fixed in the presence of cetylpyridinium chloride, which precipitates carbohydrates, so that their loss during fixation and processing is substantially reduced. The semi-thin araldite sections obtained from osmicated material were viewed by conventional light microscopy, while the ultra-thin sections were examined by transmission electron microscopy. A parallel series of semi-thin sections of non-osmicated ovaries was deresined and subsequently stained with periodic acid Schiff (PAS). The morphological appearance of the zona pellucida in preovulatory oocytes changed during the final stages of oocyte maturation. A close correlation was observed between the ultrastructural appearance of the zona pellucida and that observed following PAS staining during the period studied. Real differences were observed in the location, density, and distribution of glycoconjugates within the zona pellucida during the final stages of oocyte maturation prior to and immediately following germinal vesicle breakdown. Similar changes in the zona were observed in adult females autopsied during proestrus and oestrus. The changes in the density and distribution of glycoconjugates are likely to have important consequences for fertilization by affecting sperm-zona binding and sperm-egg interactions.

Brain tumours in childhood in Bombay: I: Histopathology showing changing patterns; II: Tissue culture with light and electronmicroscopy, stressing ingestion & degradation of bacteria by glial cells in vitro.

The pathological pattern of 86 brain 'tumours' in childhood during the years 1981-85 (out of a total of 586 for all ages), showed a higher proportion of neoplasms and a much lower of tuberculomas compared to the preceding three decades. A large number of histologically unusual cases was revealed. Through tissue culture of brain tumours we carried out morphological, histochemical and fine structural study of the tumour cells in vitro. The abundant presence of lysosomal acid phosphatase, in outgrowing cells, correlated with the detection of lysosomal dense bodies and vacuoles in araldite sections, by light and electronmicroscopy. In view of the phagocytic propensity of schwann cells for M. leprae as the important factor in leprous neuritis, TC preparations of gliomas, (in addition to acoustic schwannomas and meningiomas), were inoculated with two mycobacteria, M. scrofulaceum and the ICRC bacillus. There was a pronounced intracytoplasmic uptake, i.e. endocytosis, of acid-fast bacilli by the growing cells of these tumours. This was confirmed by electronmicroscopy which showed intact and degrading bacilli in various stages, in such cells of a typical cerebral astrocytoma used as an illustrative case in this paper. Ingestion and Digestion appear to be an inherent property of growing tumour cells in vitro. Fine structural examination of in vitro growth of an unusual subependymal giant cell astrocytoma, not inoculated with bacilli, served as a control. Cells of both tumours showed copious autophagic activity and cytoskeletal features of developing microtubules and filaments.

Ultrastructural localization of lysozyme in human colon eosinophils using the protein A-gold technique: effects of processing on probe distribution.

The distribution of lysozyme (muramidase) within eosinophil leukocytes situated in the lamina propria of human colon was studied by immunoelectron microscopy using a range of standard techniques. Tissue processed in a variety of glutaraldehyde- or paraformaldehyde-based fixatives was partially dehydrated and embedded in the acrylic resin LR White. Tissue thus treated showed lysozyme in pale cytoplasmic granules and the matrix of specific granules, but not in their crystalloids. Trypsinization of sections had little effect on this result, and tissues fixed in glutaraldehyde and embedded in Araldite showed a low level of reactivity with a similar distribution. After etching LR or Araldite sections with sodium metaperiodate, the pale granules and specific granule matrices became negative for lysozyme and the crystalloids became positive. Because crystalloids also were labeled with normal rabbit immunoglobulin after etching, this apparent redistribution of label could be due to nonspecific binding rather than exposure of masked epitope.

The threshold concentration of dietary ethanol necessary to produce toxic effects on hippocampal cells and synapses in the mouse

A nutritionally complete liquid diet containing 0, 3, 5, or 7% ethanol was fed to C57 mice for 4 months and killed after a further 4 months on a standard food pellet diet. The number of pyramidal cells in Araldite sections of the hippocampus was not significantly reduced in any group relative to the zero alcohol intake group. Synaptic counts in the area occupied by the pyramidal cell basal dendrites showed the 7% alcohol group to have a reduced number of synaptic contacts, with no convincing differences between the other groups relative to the zero alcohol group. All measurements made collectively could not be used to distinguish 3 or 5% ethanol consuming animals from 0% ethanol consuming animals. Significant differences in body weight and dry brain weight were detected in the comparison of the group treated with 7% ethanol and 4-month recovery with the group receiving the 0% ethanol dose and 4-month recovery. It is concluded that maintaining mice on a diet containing not more than 5% ethanol has no adverse long-term structural effects on hippocampal pyramidal cells or synapses.

Age-related depletion of the cell population in the human trabecular meshwork.

The number of cells in the human trabecular meshwork was assessed for counting meshwork cell nuclei in semithin Araldite sections cut in the meridonal plane. The counts of nuclei decreased with increasing age and the decrease was associated with all regions of the meshwork. The appropriate regression equation and a mean length for meshwork nuclei was used to estimate cell numbers. At 20 years of age the estimated cell number for the whole meshwork was 763,000 which decreased to 403,000 cells by 80 years with a loss rate of 6,000 cells per year.

Changes in Atrial Biopsies in Chronic Rheumatic Heart Disease II: Muscle Fibre Reaction

Myocardial fibres were studied in the right atrial biopsies from 32 patients with chronic rheumatic heart disease (RHD). Paraffin and, particularly, araldite sections showed many muscle fibres well preserved, and others with large hyperchromatic nuclei, or with depleted myofibrils and increased mitochondria. With the SDH and ATPase reactions, there was no type difference in the myocardial fibres, and the former showed the reaction predominantly in the centre while the latter showed it mainly at the periphery of the fibres. At electronmicroscopy, fibres with intact myofibrils were found close to "degenerating" fibres with variable degrees of myofibrillar and myofilament disorganisation and loss, mitochondrial proliferation, occasionally with degeneration of cristae, and accumulation of lipofuscin in varying amounts, and irregularly tortuous or loosened intercellular junctions. This study has revealed more severe muscle changes than expected, even in the clinically less affected right atrial chamber in chronic RHD. It is speculated that this might be due to subclinical involvement of the tricuspid valve, known to be frequent in Indian patients, and the resulting "back pressure" on the right atrium might lead to changes in its myofibres.

Fibronectin production by synovial intimal cells.

The distribution of fibronectin in the inflamed synovium has been described previously using immunohistochemical methods. Under favourable conditions, it is possible to demonstrate apparent cytoplasmic staining of the intimal cell layer. We have further investigated the localisation of fibronectin in the synovial intimal cells using higher resolution techniques with peroxidase-antiperoxidase staining and high power light microscopy of semi-thin Araldite sections and immunoelectron microscopy using a protein A-gold technique. Synovia from 11 mechanical/traumatic, or osteoarthritic joints; 12 seropositive rheumatoid arthritis and nine cases from other joint diseases made a total of 32 cases examined in semi-thin sections, while six rheumatoid and two osteoarthritis synovia were studied by immunoelectron microscopy. Fibronectin was demonstrated in individual cells of the synovial intimal layer in 22 out of 32 samples examined by the light microscope method, and electron microscopy of adjacent sections showed that the positively staining cells were type B synoviocytes. Immunoelectron microscopy confirmed the presence of fibronectin within the rough endoplasmic reticulum of type B synoviocytes in all but one of the eight samples examined. The results provide evidence that the type B synoviocyte is responsible for fibronectin production.

Morphometry study on pre and post-hatching nerve cell bodies of lumbar spinal ganglia of Gallus domesticus

A cytomorphometric study was performed in lumbar spinal ganglia neurons of Gallus domesticus on the 10th and 18th incubation days and 8th, 35th, 61st, and 120th post-hatching days. The absolute volume of nucleus and relative volume of cytoplasm were respectively estimated by the Bach caryometric method and by point-counting volumetry, carried out in 0.5 micrometer thick araldite sections. The relative volume, the surface-to-volume ratio and the total surface of RER, SER, mitochondria, dense bodies, Golgi complex and the relative volume of hyaloplasm inside and outside the Nissl bodies were estimated from electronmicrographs by the Weibel et al. method. The conclusions were: a) there was an increase of the cell volume and a decrease of the nucleo-cytoplasmic ratio, particularly between the first two ages; b) the relative volumes of RER and SER change inversely with respect to each other: the RER increases before hatching, decreasing progressively afterwards; the changes of relative volume of dense bodies are similar to those of the RER, and the mitochondria show relatively small variations concerning the same parameter; c) the relative volume of hyaloplasm inside the Nissl bodies decreases while those outside increases; d) the surface-to-volume ratio drops sharply for all organelles from the 10th to the 18th day of incubation; after hatching, a tendency to increase is observed; e) the membrane surface-to-cytoplasmic volume ratio decreases for all organelles from the 10th to the 18th day of incubation; after hatching, this ratio increases slightly for mitochondria and Golgi complex, sharply for SER, dropping for dense bodies. The RER values alternate regularly.

Endocrine control of galactogen synthesis in the albumen gland of the slug, Limax maximus

An in vitro method for culturing Limax maximus albumen glands is described in which the biosynthetic activity of the slug albumen gland was monitored by measuring the incorporation of [ 14C]glucose into galactogen. Homogenates of the central nervous system were shown to cause a 3.5- to 12-fold increase in galactogen synthesis in albumen gland explants as compared to controls. The major sources of the galactogen-synthesis stimulating factor (Gal-SF) were found to be the cerebral ganglia and their surrounding connective tissue. Gal-SF was demonstrated to be peptidase sensitive and heat labile suggesting that it is probably a polypeptide. Autoradiographs of semithin araldite sections supported the incorporation data: in albumen gland explants cultured with cerebral ganglion homogenate considerably more label was found over secretory granules than in control-cultured explants. The possible cellular source of Gal-SF is discussed in relation to its possible origin in other investigated pulmonates.

Aging in the rat medial nucleus of the trapezoid body I. Light microscopy

The medial nucleus of the trapezoid body (MTB), a cell group in the superior olivary complex, was examined in an age-graded series of rats for neuron loss, changes in the giant synaptic endings (chalices of Held) on MTB neurons, and accumulation of age pigment. Neuron counts were done on protargol-stained paraffin sections of MTBs from a series of 17 rats aged 2–3, 6, 18, and 24 months. Between 2–3 and 24 months a 34% decrease in the mean number of MTB neurons was observed. Significant loss ( p < 0.05) was first evident in the early portion of the life span, between 2–3 and 6 months. In thionin-stained sections, there was no change with aging in the proportions of three MTB neuron types: principal cells (∼82%), elongate cells (∼15%), and stellate cells (∼3%). In young adult rats, 25–26% of all MTB neurons were associated with identifiable chalices of Held in protargol stained sections. This ratio did not vary significantly with aging. Age pigment accumulation in the MTB was examined in 2 μm Araldite sections stained with toluidine blue. Age pigment deposits were larger and more numerous in the MTBs of old animals, but not extensive as has been described previously in many other parts of the nervous system. This study is the first to report neuron loss in an animal brainstem nucleus.