José L. Ojeda

Neuronal body size correlates with the number of nucleoli and Cajal bodies, and with the organization of the splicing machinery in rat trigeminal ganglion neurons

Trigeminal ganglion neurons comprise three main cell body‐size types. This cell size heterogeneity provides an excellent neuronal model to study the cell size‐dependent organization and dynamics of the nucleoli, Cajal (coiled) bodies (CBs), and nuclear speckles of pre‐mRNA splicing factors, nuclear structures that play a key role in the normal neuronal physiology. We have analyzed the number of nucleoli and CBs and the structural and molecular organization of CBs and nuclear speckles in the three neuronal types by using immunofluorescence with antibodies that recognize nucleoli (fibrillarin), CBs (coilin), and nuclear speckles (snRNPs), confocal microscopy, and electron microscopy. Whereas the mean number of nucleoli per neuron decreases as a function of cell size, the number of CBs per cell significantly increases in large neurons in comparison with the small ones. In addition, large neurons have a higher proportion of CBs associated with the nucleolus. In all neuronal types, CBs concentrate coilin, fibrillarin, snRNPs, and the survival motor neuron protein (SMN). Immunostaining for snRNPs shows small speckle domains and extensive areas of diffuse nucleoplasmic signal in large neurons, in contrast with the large nuclear speckles found in small neurons. Furthermore, flow cytometric analysis shows that all neurons are in the range of diploid cells. These findings indicate that the fusion behavior of nucleoli, the formation of CBs and their relationships with the nucleolus, as well as the compartmentalization of the pre‐mRNA splicing machinery, is related to cell body size in the trigeminal ganglion neurons. Because transcriptional activity is a basic determinant mechanism of cell size in diploid cells, we suggest that our findings reflect a distinct transcription‐dependent organization of the nucleolus and splicing machinery in the three cell types of trigeminal ganglion neurons. J. Comp. Neurol. 430:250–263, 2001.

Nationwide and Long-Term Survey of Primary Glomerulonephritis in Japan as Observed in 1,850 Biopsied Cases

Primary chronic glomerulonephritis is the most common cause of end-stage renal failure in Japan. The incidence in dialysis patients in Japan is about four times higher than in the United States for reason which are unclear. We conducted a nationwide survey on the natural history and treatment of primary glomerulonephritis under a program project from the Ministry of Health and Welfare of Japan entitled ‘Progressive Chronic Renal Disease’. We analyzed patient characteristics, disease onset, clinical data, and histological findings in 1,850 patients with primary glomerulonephritis from 53 institutions in 1985 who underwent renal biopsy at least 5 years ago, and the follow-up study was carried out 8 years after registration. The incidence of diffuse-mesangial proliferative glomerulonephritis is 41.9%, that of minor glomerular abnormalities 17.5%, and that of focal-mesangial proliferative glomerulonephritis 13.0%. Of 1,045 biopsy specimens that were examined by immunofluorescence microscopy, 47.4% showed IgA nephropathy. Half of all cases with primary chronic glomerulonephritis were asymptomatic and were detected on routine health examination. The survival rates at 20 years from the apparent onset or earliest known renal abnormality are: focal glomerular sclerosis 49%, membranoproliferative glomerulonephritis 58%, diffuse-mesangial proliferative glomerulonephritis 66%, focal-proliferative glomerulonephritis 81%, membranous nephropathy 82%, minor glomerular abnormalities 94%, and IgA nephropathy 61%. In conclusion, a high incidence of IgA nephropathy and a better renal survival of membranous nephropathy are the features of primary chronic glomerulonephritis in Japan. This high incidence of IgA nephropathy together with its poor prognosis is probably the reason for the increased incidence of primary chronic glomerulonephritis in dialysis patients in Japan. In addition, the importance of routine health examination including urinalysis is demonstrated.

The three‐dimensional microanatomy of the rabbit and human cornea. A chemical and mechanical microdissection‐SEM approach

The three‐dimensional (3D) microanatomy of the cornea is the major determinant of its optical and mechanical properties. Scanning electron microscopy (SEM) is the most commonly used method to obtain information on the overall 3D microanatomy of organs. However, SEM has not been successful in revealing the 3D microanatomy of the cornea, because the interior of the cornea is too compact to be explored by the electron beam. In this study, the 3D organisation of the cells and extracellular materials of human and rabbit corneas was examined after exposure by HCl and NaOH digestion, and by microdissection by the adhesive tape method. In the cornea of both species, all epithelial cells exhibited microplicae regardless of their location. This raises doubts about the tear film‐holding role assigned to the microplicae of the superficial cells. Human and rabbit corneas differed in the collagen fibre patterns of the epithelial basement membranes. The 3D organisation of the stromal lamellae was similar in both species. In humans and rabbits, the keratocytes showed similar 3D features. However, the surface of human keratocytes located near Descemets membrane exhibited small fenestrations that were not present in the rabbit keratocytes. The pattern of keratocyte innervation by the stromal neural plexus and 3D keratocyte microanatomy confirms that keratocytes form a large intercommunicating network within the corneal stroma. Two morphologically discrete subpopulations of keratocytes located at different stromal levels were identified in both human and rabbit corneas, suggesting that keratocytes are not functionally homogeneous. In addition, the density of the stromal neural plexus appeared to be greater in rabbits than in humans. Clear differences between human and rabbit corneas were observed in the collagen arrangement in Descemets membrane, which may reflect their different biomechanical requirements.

Cell death and phagocytosis in the neuroepithelium of the developing retina. A TEM and SEM study

Phagocytosis by neuroepithelial cells independent of the presence of pseudopodes is observed as the main mechanism of elimination of physiologically degenerating cells during the early development of the retina in the chick embryo.

Renal Corpuscle of the African Lungfish Protopterus dolloi: Structural and Histochemical Modifications During Aestivation

This work studies the structural and lectin‐binding modifications experienced by the renal corpuscle of the African lungfish Protopterus dolloi during aestivation. The kidney of the aestivating animals was studied by light‐ and electron‐microscopy, and by immunofluorescence methods. Upon aestivation, the renal corpuscles (RCs) undergo a marked size reduction, and all the structural RC components are affected. The parietal cells of Bowmans capsule lose their flattened appearance and adopt the organization of a stratified epithelium. The glomerular capillaries collapse. The podocytes approach each other. Concomitantly, the major processes between contiguous cells are lost, the rest of the major processes adopting a lamina‐like configuration. The foot processes lose their regular arrangement, the filtration slits are difficult to observe, and the subpodocyte space disappears. The glomerular basement membrane (GBM) thickens enormously, increases the amount of amorphous material and of collagen, and round inclusions formed by amorphous material and coiled fibrils appear. The mesangial cells compact and form a dense network embedded in the subendothelial lamina of the GBM. The endothelial cells show numerous irregularities, establishing abnormal interrelationships with the mesangial cells. These modifications are accompanied by changes in the expression of the carbohydrate moieties, as indicated by the modifications in lectin‐binding patterns. On the whole, these modifications thicken and compact the filtration barrier, thus reducing the filtration coefficient and allowing the organism to cope with dehydration. All these modifications are partially reversed during the first days of returning the animals to freshwater. The renal corpuscle appears to be a highly dynamic structure capable of modifying its architecture in response to environmental changes. Anat Rec, 291:1156‐1172, 2008.

The cerebellar arteries cortical patterns and vascularization of the cerebellar nuclei

This paper reports an anatomical study of the vascular supply in 60 human cerebelli from subjects of both sexes, aged between 20 and 60 years, who had died of various accidental causes. The origin, branching pattern and cortical and central distributions of the cerebellar arteries have been studied by gross dissection, vascular injection and corrosion procedures. Variations of the normal pattern of the cerebellar arteries in both origin and cortical distribution are frequently found, the anterior inferior and the posterior inferior cerebellar arteries being the arteries showing the highest number of variations. The anomalies most frequently found are duplication in the origin, abnormal origin, and the presence of hypoplastic vessels. The cerebellar nuclei were supplied by the rhomboidal artery, a collateral branch of the superior cerebellar artery. The formation of a precise segmentary vascular pattern into the dentate nucleus by branches arising from the rhomboidal artery is described. The results are discussed in the light of other anatomical studies.

The conus valves of the adult gilthead seabream (Sparus auratus)

The conus (bulbo‐ventricular) valves of teleosts perform a key function in the control of blood backflow during ventricular diastole. However, the structural characteristics of these valves are almost unknown. This paper presents a systematic anatomical, histological and structural study of the conus valves of the adult gilthead seabream (Sparus auratus). S. auratus shows two major left and right valves consisting of the leaflet and the supporting sinus. Each valvar leaflet can be divided into a stout proximal body and a flap‐like distal region. The proximal body is structured into three layers: a luminal fibrosa, a dense cellular core and a parietal fibrosa. The luminal fibrosa is a collagenous structure extending the entire length of the leaflet, while the parietal fibrosa is restricted to the most proximal area. The dense cellular core consists of fibroblastic cells and a matrix rich in glycoconjugates, collagen and elastin. The histochemical and structural data suggest that the luminal fibrosa bears most of the force associated with valvar closure, while the cellular core acts as a cushion dampening vibrations and absorbing the elastic recoil. The sinus wall is a fibrous layer which shows proximal–distal differences in thickness. It also shows compositional differences that can be related to mechanical function. We describe the presence of a fibrous cylinder formed by the sinus wall, the fibrous interleaflet triangles and the fibrous layer that covers the inner surface of the conus myocardium. This fibrous cylinder constitutes the structural nexus between the ventricle, the conus and the bulbus arteriosus, provides support for the conus valves and separates the valvar complex from the surrounding tissues. The structure of the conus valves in S. auratus is different from that found in other vertebrates. Anatomical similarities between the conus valves and the mammalian arterial valves are emphasized. Each phyletic group appears to have developed specific structures in order to perform similar functions.

Polycystic kidney disease induced by corticoids. A quantitative and qualitative analysis of cell populations in the tubular cysts

The sequential changes of cell morphology and the ratio distribution of the different types of cells which exist in tubular cysts induced by methylprednisolone acetate have been studied by light, transmission and scanning electron microscopy. We have also studied the blood levels of sodium and potassium by flame photometry. In both control and cystic ducts, at the level of the outer cortex, the first intercalated cells (IC) were not observed until the 4th postnatal day. Some intermediate cell configurations were observed during the 3rd postnatal day, suggesting that some primitive principal cells (PC) are transformed into IC. Development of IC seems to be independent of both the effects of corticoids and the blood levels of potassium. The ratio distribution and the types of IC observed throughout the period studied was similar in both normal and cystic ducts. The type of IC characterized by the presence of a huge apical process, which has gone previously undescribed with either TEM or SEM, was observed in both control and cystic ducts. We propose to name these cells as cells with surface pattern type V. During the period of regression of the tubular cysts dead and migrating cells were observed closely associated with cilia of the PC. Both types of cells do not seem to represent, based in their localization and frequency, abnormal cell types of the cyst wall. Our results support the hypothesis that renal cysts are giant collecting ducts which conserve both the morphology and the function of the epithelium.

A Technique for Fluorescence Microscopy in Semithin Sections

We describe here a procedure to improve contrast and resolution in fluorescence microscopy of sectioned tissues. Tissue fragments were fixed in ethanol-glacial acetic acid, embedded in diethylene glycol distearate, and semithin sectioned. This method maintains tissue antigenicity while preserving the structure of cells and tissues. The thinness of the sections eliminates scattered and emitted light from tissue structures outside the plane of focus. The procedure is simple and quick, and works excellently with fluorescein-conjugated lectins and antibodies.

Endocardial cell polarity during the looping of the heart in the chick embryo.

Abstract In order to investigate whether endocardial cells (EC) in chick embryo hearts have polarity of shape and structure, the position of the Golgi apparatus, silver impregnated, and the shape of the cells were studied using light and TEM and SEM microscopy. The EC have a prominent Golgi apparatus located lateral to the nucleus and show a statistically significant polarization. The pattern of endocardial cell polarity was mainly observed in the bulbus cordis and in the primitive atrium from stage 11+ and in the primitive ventricle at stages 9+ and 10+. The EC are elongated in shape, show the morphological characteristics of migrating cells, and are arranged with the long axis perpendicular to the blood flow. It is suggested that the polarity of endocardial cells is associated with cell emigration. The possible morphogenetic role of EC polarity is discussed.