The glycosaminoglycans of Dupuytren's disease

The total and individual glycosaminoglycan (GAG) content at various stages of the Dupuytren disease process and in samples of normal palmar connective tissue (palmar dermis, palmar fascia and digital flexor tendon) from the hands of uninvolved age-matched controls have been assayed and compared. Morphological comparisons between the different tissues were made by histological examination of sections stained to demonstrate collagen fiber patterns and glycosaminoglycan distribution. Significant differences in the type and amount of GAG were found between the various manifestations of the disease process, i.e., nodules, cellular and fibrous bands, and between these and the normal palmar connective tissues. In the most actively proliferating cellular regions chondroitin sulfate levels were 11 times greater than those of the normal palmar connective tissues, whereas dermatan sulfate tissue levels showed a fourfold increase. On the other hand, tissue concentrations of hyaluronate were similar to those of normal palmar connective tissue. The relationship of these differences in GAG levels to the development and maturation of the normal palmar connective tissues and the Dupuytren's process is discussed.     

Light and electron microscopic observation of specific atrial granules using water-miscible resin as an embedding medium

A mixture of glycol methacrylate (GMA) and Quetol 523 was examined as an embedding medium for atrial tissue to be selectively stained for specific atrial granules. Semi-thin sections of rat atrial tissue embedded in this resin were stained with lead hematoxylin and observed under a light microscope. Atrial granules were found to be specifically stained blue black with lead hematoxylin. The same semithin sections stained with OsO4 vapor were examined electron microscopically and the atrial granules could be distinguised clearly from other cytoplasmic components. The GMA-Quetol 523 mixture is a useful embedding medium for studying the distribution of specific atrial granules by light and electron microscopy.     

The Glycosaminoglycans of Dupuytren's Disease

The total and individual glycosaminoglycan (GAG) content at various stages of the Dupuytren disease process and in samples of normal palmar connective tissue (palmar dermis, palmar fascia and digital flexor tendon) from the hands of uninvolved age-matched controls have been assayed and compared. Morphological comparisons between the different tissues were made by histological examination of sections stained to demonstrate collagen fiber patterns and glycosaminoglycan distribution.

Significant differences in the type and amount of GAG were found between the various manifestations of the disease process, i.e., nodules, cellular and fibrous bands, and between these and the normal palmar connective tissues.

In the most actively proliferating cellular regions chondroitin sulfate levels were 11 times greater than those of the normal palmar connective tissues, whereas dermatan sulfate tissue levels showed a fourfold increase. On the other hand, tissue concentrations of hyaluronate were similar to those of normal palmar connective tissue.

The relationship of these differences in GAG levels to the development and maturation of the normal palmar connective tissues and the Dupuytren's process is discussed.     

Characterization of proteoglycans and glycosaminoglycans in splenic AA amyloid induced in mink

Amyloidosis of the protein AA type is readily induced in mink using repeated injections of bacterial lipopolysaccharide (LPS). We have characterized splenic proteoglycans/glycosaminoglycans (PGs/GAGs) in mink during amyloidogenesis. Moderate to rich amounts of amyloid exhibiting green birefringence was demonstrated by polarization microscopy of the splenic section stained with Congo red in seven out of eight minks after 10 weeks of LPS-treatment, and a significant increase in the total amount of PGs and GAGs in AA amyloid spleens was observed (two to eight times that in unstimulated animals). Intact PGs as well as free GAGs were extracted, and heparan sulfate (HS) was the most abundant GAG in the amyloid as well as in the control spleens. The GAGs showing the most pronounced increase in the amyloid spleens was of the chondroitin sulfate/dermatan sulfate (CS/DS) type and these were extracted in the form of free GAG chains. We conclude that there is a selective enrichment of PGs/GAGs in extracted splenic amyloid in the mink, which confirms to previous observations in human amyloid as well as in other animal species, supporting their pathogenic significance in the formation of AA amyloid.     

Heparan sulfate proteoglycan in diffuse plaques of hippocampus but not of cerebellum in Alzheimer's disease brain.

Previous studies have shown the basement membrane form of heparan sulfate proteoglycan (HSPG) known as perlecan, co-localized to beta-amyloid protein (A beta)-containing amyloid deposits in brains of patients with Alzheimer's disease (AD) and Down's syndrome. Although HSPG was localized to diffuse A beta plaques in hippocampus, amygdala, and neocortex, it is not known whether they are present in diffuse A beta plaques in cerebellum. In the present study, Alcian blue staining and immunocytochemical techniques were used to determine whether highly sulfated glycosaminoglycans (GAGs) and/or HSPG (perlecan) were also present in diffuse A beta plaques of cerebellum. Tissues from cases of AD were examined for the co-localization of highly sulfated GAGs, HSPGs, and A beta in diffuse plaques in cerebellum in comparison with hippocampus. Consecutive serial sections of AD brain tissue were stained or immunostained with 1) the modified Bielschowsky stain; 2) a polyclonal antibody directed against synthetic A beta (1-40); 3) Congo red; 4) Alcian blue (pH 5.7) with varying concentrations of magnesium chloride for identification of sulfated and highly sulfated GAGs; and 5) polyclonal and monoclonal antibodies recognizing either the core protein or a specific GAG epitope on perlecan. All cases (7 of 7) of AD contained diffuse A beta plaques in the cerebellum as identified by positive Bielschowsky staining and A beta immunoreactivity. None of these cases demonstrated positive Alcian blue staining (at 0.3 and 0.7 mol/L MgCl2), HSPG, or HS GAG immunoreactivity in the same diffuse cerebellar plaques on adjacent serial sections. However, Alcian blue staining, HSPG, and/or HS GAG immunoreactivity were observed in blood vessel walls, choroid plexus, and within Purkinje cells, suggesting that the techniques used were reliable and specific. In cerebellum, all plaques containing amyloid cores that were Congo red-positive were also positive for highly sulfated GAGs (by Alcian blue staining at 0.7 mol/L MgCl2) and HSPG (both core protein and GAG chain) immunoreactivity. Even though HSPG immunoreactivity was not present in cerebellar diffuse plaques, all cases (4 of 4) examined demonstrated HSPG (both core protein and GAG chain) immunoreactivity in diffuse A beta plaques in hippocampus. Therefore, by Alcian blue staining and immunocytochemical methods, highly sulfated GAGs and HSPGs are not present in A beta diffuse plaques in cerebellum. Since previous studies indicate that the cerebellum contains relatively few amyloid-containing plaques in comparison with diffuse plaques, these studies suggest that HSPG may be an essential component needed for amyloid formation and/or persistence in brain as observed in cortical areas.(ABSTRACT TRUNCATED AT 400 WORDS)     

The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. III. Cholecystokinin

Summary Immunocytochemistry was used to examine the morphology and distribution of cholecystokinin-like immunoreactive neurons in the visual cortex of the developing and mature albino rat. In the adult, labelled neurons were observed in all cortical layers but were concentrated in layers II and III. The majority of these neurons exhibited morphological features characteristic of bitufted and multipolar non-pyramidal cells described in Golgi preparations.Cholecystokinin immunoreactivity was first observed on postnatal day 3 and was confined to a few immature non-pyramidal cells located in the subplate region. The number of stained cells increased markedly during the latter part of the first postnatal week and by day 8 they were present in all cortical layers. Their morphological maturation occurred gradually during the first three weeks of postnatal life with the exception of a period of pronounced growth during the latter part of the second postnatal week.     

Bruch''s membrane of the brachymorphic mouse

Bruch's membrane exists between the retinal pigment epithelium and the choriocapillary endothelium. Its structure is very complicated, having five sublayers containing basement membranes of retinal pigment epithelium and choriocapillary endothelium, outer and inner collagenous layers, and a central elastic layer. In the development of Bruch's membrane in normal mice, both basement membranes are created first. Secondarily, collagen fibers are accumulated in the space between these basement membranes and then form a collagenous layer. Finally, the elastic layer elaborated in the collagenous layer separates this into outer and inner collagenous layers. Brachymorphic mice have a disorder in the sulfation pathway, resulting in undersulfation. Consequently, in Bruch's membrane of brachymorphic mice, the expression of decorin, a small proteoglycan containing chondroitin sulfate and an indispensable component in collagen assembly, is at a very low level. It is clear that hypoplasia of the collagenous layer in Bruch's membrane of brachymorphic mice induces a disorder in the following formation of the elastic layer. These findings suggest that the formation of the collagenous layer, regulated with acidic glycoconjugates such as decorin, is important in the development of Bruch's membrane.     

Membrane ionic conductances in normal and denervated skeletal muscle of the rat during development

The development of membrane ionic conductances of rat extensor digitorum longus (EDL) muscle fibers was studiedin vitro using intracellular recordings. At 7–8 days after birth, the potassium conductance (GK) dominated the total membrane conductance while the chloride conductance (GC1) was very low. A rapid increased of GC1 towards adult values was observed after few days (12–14 day old rats), whereas GK did not decrease up to day 23. Denervation at 7–8 days after birth suppressed the maturation of the electrical parameters measured, and 15 days after the nerve crush, GC1 was just detectable. These results suggest that the maturation of the electrical properties, and in particular that of the resting chloride conductance in mammalian striated muscle fibers, occurs during the first weeks of postnatal life and is dependent on innervation.     

Effect of bilateral enucleation on the development of layers in the dorsal lateral geniculate nucleus

Previous results in tree shrew (Tupaia glis) have demonstrated that retinal afferents in the dorsal lateral geniculate nucleus segregate prenatally and that cell layers segregate postnatally (Brunso-Bechtold &Casagrande, 1980). In the present study, we examined the effect of bilateral enucleation at birth on the cytoarchitectural differentiation of these cell layers in the tree shrew lateral geniculate nucleus. The enucleated animals were killed at several postnatal ages up to maturity, their brains embedded in celloidin, and sections stained with thionin. The main result was that interlaminar spaces failed to form. Nevertheless, several cellular and cytoarchitectural features characteristic of normal layers were apparent; these included staining intensity, packing density and cell orientation.It can be concluded that the presence of retinal afferents appears necessary for the formation of interlaminar spaces in the lateral geniculate nucleus but not for the differentiation of all characteristics which define the layers.     

Postnatal development of neuropeptide Y- and calcitonin gene-related peptide-immunoreactive nerves in the rat urinary bladder

The postnatal development of neuropeptide Y- and calcitonin gene-related peptide-immunoreactive (NPY-IR and CGRP-IR) nerve fibers in the rat urinary bladder was investigated using whole-mount preparations and cryostat sections. In newborn and 3-day-old rats, many NPY-IR nerve fibers were observed in the subserous and muscle layers. Many NPY-IR nerve cell bodies clustered at branching points of the subserous nerve bundles. Within 4 weeks after birth, these cell bodies drastically decreased in number and spread along the bundles, although the number of NPY-IR nerve fibers increased moderately. In contrast, CGRP-IR nerve fibers in newborn and 3-day-old rats were less developed, and no CGRP-IR nerve cell body was observed in any rat. However, CGRP-IR nerve fiber distribution in the urinary tissues conspicuously increased within 4 weeks after birth. Especially, an increase of the infraepithelial fibers showing a meshwork appearance was prominent in the fundus and corpus of the bladder. The infra- and intraepithelial CGRP-IR nerve meshwork of the ventral wall was more dense than that of the trigone. At 4 weeks, NPY-IR and CGRP-IR nerves were similar to those of the adult rat (8–12 weeks old). The present study suggests a correlation between the development of the peripheral nervous system in the urinary bladder and maturation of micturition behavior in the rat.     

新生大鼠腰交感神经节凋亡细胞的电镜观察

目的 :观察出生后早期大鼠腰交感神经节细胞凋亡超微结构变化特点。方法 :大鼠按出生后 1d、1w、2w和3w分组 ,取L3、L4交感神经节常规超薄切片 ,铅轴染色 ,透射电镜观察。结果 :1d 2w组腰交感神经节内可见到明显的神经节神经细胞胞体凋亡和突起的退变 ,同时可观察到包绕上述突起的雪旺细胞也发生凋亡。早期凋亡细胞的超微结构特点为核染色质在核膜下的凝集。结论 :出生后早期部分腰交感神经节神经细胞因未能与其靶区建立联系而凋亡 ,随后包绕凋亡神经细胞突起的雪旺细胞也发生凋亡。     

Development of taste organs inRana temporaria

Summary The expression and distribution of several major extracellular matrix macromolecules were investigated at the epithelial-mesenchymal interface of the human fetal small intestine from 8 to 20 weeks of gestation. Localization of heparan sulfate proteoglycan, type-IV collagen and laminin, three basement membrane components, as well as fibronectin and tenascin, were assessed by indirect immunofluorescence staining on cryostat sections, and correlated to morphogenesis and epithelial cell differentiation. Basement membrane components and fibronectin were all detected as early as 8 weeks (a time when the epithelium is still stratified and does not express sucrase-isomaltase). Tenascin appeared only after short villi had developed (around 10 weeks) and was restricted to the connective tissue at the tip of villus rudiments. At 18 weeks, well-formed villi and crypts were apparent. The antibody against heparan sulfate proteoglycan stained exclusively the epithelial basement membrane. Anti-type-IV collagen and anti-laminin anti-bodies stained the epithelial basement membrane and also cellular and fibrillar structures in the lamina propria. Fibronectin was found uniformly distributed over the lamina propria except in the upper third position of the villus core. On the contrary tenascin was mainly localized in the stroma at the tip of the villi. Staining for tenascin was also detected at the epithelial-mesenchymal interface of the villus and in the mesenchyme immediately surrounding budding crypts. These results provide basic data concerning the development of the human gut, and suggest that extracellular matrix components could be involved in the remodelling process of the intestinal mucosa.     

Ultrastructural localization of glycosaminoglycans in human term placenta

Sulfated glycoconjugates were stained in normal human term placentas using Spicer's high-iron diamine (HID) method with thiocarbohydrazide and silver proteinate (TCH-SP) enhancement. Specific identification of glycosaminoglycans (GAG) was accomplished by digestion of the stained material with chondroitinase ABC or AC for removal of chondroitin sulfates and nitrous acid for removal of N-sulfated GAGs. The syncytiotrophoblast apical surface demonstrated moderate to intense staining with HID-TCH-SP, which was removed by prior digestion with the chondroitinases, but not by nitrous acid. The syncytiotrophoblast basal surface and endothelial cell surfaces lacked sulfate staining. A few cytoplasmic granules in syncytiotrophoblast cells demonstrated staining similar to the apical surface. Three layers of the basal lamina were identified in these preparations. The lamina lucida immediately beneath the syncytiotrophoblast and the majority of the lamina densa stained weakly or not at all, whereas the underlying lamina diffusa and stroma demonstrated moderate to intense staining. The majority of lamina diffusa staining was removed by chondroitinase ABC or AC; the remaining material was removed by nitrous acid digestion. Thus the syncytiotrophoblast surface contains a chondroitin sulfate and the basal lamina contains a mixture of intensely stained chondroitin sulfate and a weakly stained N-sulfated GAG.     

Postnatal alterations of GABA receptor profiles in the rat superior colliculus

Midbrain sections taken from Sprague-Dawley rats of varying ages within the first four postnatal weeks were used to determine, immunocytochemically, putative changes of GABA(A) receptor beta2/3 subunits, GABA(B) receptor (R1a and R1b splice variants), and GABA(C) receptor rho1 subunit expression and distribution in the superficial, visual layers of the superior colliculus. Immunoreactivity for the GABA(A) receptor beta2/3 subunits was found in the superficial grey layer from birth. The labelling changed with age, with an overall continuous reduction in the number of cells labelled and a significant increase in the labelling intensity distribution (neuropil vs soma). Further analysis revealed an initial increase in the labelling intensity between postnatal days 0 and 7 in parallel with an overall reduction of labelled neurones. This was followed by a significant decrease in labelling intensity distribution between postnatal days 7 and 16, and a subsequent increase in intensity between postnatal days 16 and 28. The labelling profiles for GABA(B) receptors (R1a and R1b splice variants) and GABA(C) receptors (rho1 subunit) showed similar patterns. Both receptors could be found in the superficial layers of the superior colliculus from birth, and the intensity and distribution of labelling remained constant during the first postnatal month. However, the cell body count showed a significant decrease between postnatal days 7 and 16. These changes may be related to the time-point of eye opening, which occurred approximately two weeks after birth. For all three receptor types, the cell body count remained constant after postnatal day 16. By four weeks of age, there was no significant difference between the cell numbers obtained for the different receptors. Both GABA itself and neurofilament labelling were also obtained in the superficial superior colliculus at birth. Neurofilament, although found at birth, showed very little ordered arrangement until 16days after birth. When slices were double labelled for GABA(C) receptors and neurofilament, some overlap was observed. Double labelling for the presynaptic protein synaptophysin and GABA(C) receptors showed proximity in some places, indicative of a partly synaptic location of GABA(C) receptors. When GABA(C) and GABA(A) receptors were labelled simultaneously, some but not all neurones showed immunoreactivity for both receptor types.In conclusion, all three GABA receptor types were found to be present in the superior colliculus from birth, and all show some form of postnatal modification, with GABA(A) receptors demonstrating the most dramatic changes. However, GABA(B) and GABA(C) receptors are modified significantly around the onset of input-specific activity. Together, this points towards a contribution of the GABAergic system to processes of postnatal maturation in the superficial superior colliculus.     

小鼠面神经核的生后发育——形态计量研究

对30只生后不同年龄的小鼠脑干尼氏染色切片,用形态计量法对面神经核的体积、神经细胞数及胞体体积等作了定量研究。证明生后发育过程中,面神经核的体积逐渐增长而神经细胞数逐渐变少,到生后30天左右接近成年。神经细胞体体积生后逐渐增长,到两周左右接近成年。由面神经核体积、神经细胞体体积和神经细胞数算出胞体外成分的体积生后逐渐增长,到生后30天左右接近成年。胞体外成分包括神经纤维网、血管网和胶质细胞,这些成分的发展和成熟,标志着神经元间联系与功能的发展和完善。可见,小鼠面神经核在生后先是单个神经细胞的成熟,而后是神经元间的联系与功能的成熟。全面成熟期在生后一月左右。     

Maturation of taste buds on the soft palate of the postnatal rat

Taste bud distribution on the soft palate and within three types of tongue papillae (fungiform, foliate, and circumvallate) were examined histologically in the rat at different postnatal ages. After paraffin embedding, serial sections (10 microm) were made and stained by HE, and digitized images of each section were examined. The existence of a taste pore was used to identify mature taste buds. At birth, 53% (68 of 127 observed) of the taste buds on the soft palate, but only 14% (14 of 110 observed) within fungiform papillae, contained a taste pore. One week after birth, the number of mature taste buds increased rapidly, resulting in 90% of soft palate taste buds and 80% of fungiform taste buds containing taste pores. In contrast, no taste buds with pores were observed at birth within foliate and circumvallate papillae; however, at two weeks after birth 52% (71 of 132 observed) of the foliate and 68% (180 of 267 observed) of the circumvallate taste buds examined contained taste pores. These results suggest that taste buds within the soft palate play an important role in the detection of nutrients in the neonatal rat.     

Molecular weight estimation of sulfated glycosaminoglycans in human gingivae

The number-average molecular weights of human gingival epithelium and connective tissue glycosaminoglycans (GAG) have been determined. Radioactive labelled GAG were extracted from separated gingival epithelium and connective tissue following alkaline degradation of the tissue in the presence of tritiated sodium borohydride. They were identified by electrophoresis as heparan sulfate (HS), hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin 4-sulfate (ChS-4). Following densitometric quantitation of the sulfated GAG (HS, DS and ChS-4), the amount of radioactivity associated with each species was determined by liquid scintillation of each band staining positively for these GAG. The number-average molecular weights for each GAG were determined by end group analysis. The values obtained for each sulfated GAG indicated a degree of similarity in molecular weight distribution between the two tissue types ranging from 15,000 to 27,000.     

The surface evoked potential and parvalbumin-immunoreactivity in the somatosensory cortex of the developing rat

The development of the rat somatosensory system was followed electrophysiologically and immunohistochemically. In the surface evoked potential elicited in the primary somatosensory cortex by electrical stimulation of the whisker C3 follicle, a short-latency positive wave was first recorded on postnatal Day 2. A longlatency positive wave was recorded in some pups on postnatal Day 7 and in most pups on postnatal Day 8. On postnatal Day 10, a P/N complex appeared between the short- and long-latency positive waves. Parvalbumin, believed to appear with functional maturation, appeared mainly after postnatal Day 7 in Layer V in the underlying area, although a few weakly stained cells appeared on postnatal Day 5. On postnatal Day 10, weakly stained cells appeared in the area containing barrels; their staining increased with time. In this system, electrophysiological and immunohistochemical parameters changed by the 3rd postnatal week with the most marked changes occurring within 2 postnatal weeks. © 1995 John Wiley & Sons, Inc.     

Postnatal development of the rat organ of Corti

Summary The development of the rat organ of Corti was studied during the first postnatal weeks. The temporal and the spatial patterns of cochlear development were investigated between 4 and 24 days after birth by means of semi-thin sections at approx. ten equidistant positions along the entire cochlear duct. At all examined positions width, thickness and cross sectional area of basilar membrane, cross-sectional area of tectorial membrane, of cells of Hensen, Claudius and Boettcher and of the organ of Corti were quantitatively analyzed. The most conspicuous maturational changes occur between 8 and 12 days after birth. These are the detachment of the tectorial membrane, the first appearance of filaments within the basilar membrane, the formation of the tunnel of Corti and the opening of the inner spiral sulcus. Quantitative analysis revealed that structures of a given position along the cochlear duct do not develop synchronously. Width of the basilar membrane and cross-sectional area of the tectorial membrane are already mature at the onset of hearing (10–12 days after birth). Length, thickness and cross-sectional area of the basilar membrane as well as cross-sectional area of the organ of Corti and of the cells of Hensen, Claudius and Boettcher still develop after the onset of hearing (up to 20–24 days after birth). We suggest that basic cochlear function is established by structures which are mature before the onset of hearing. Cochlear structures which develop after the onset of hearing might be involved in this improvement during this period.     

The synthesis of glycosaminoglycans in aging rat liver. A brief note

The synthesis of glycosaminoglycans (GAG) was studied in liver slices from postnatal (9 days), young (140 days), adult (490 days) and senescent (940 days) rats. It was found that the rate of synthesis was highest in postnatal rat liver and decreased to about half in young rats with no further reduction in adult and senescent age groups. The specific radioactivity of the precursors of GAG synthesis did not change with age. The synthesis pattern of specific types of GAG in postnatal liver was characterized by a significant higher percentage of chondroitin sulfate and hyaluronic acid. In the following age classes the profile of specific GAG synthesis did not change significantly (heparin sulfate: chondroitin sulfate: hyaluronic acid: “keratin sulfate” = 84%:8.3%:1.5%:1.6%).