Electron microscopic demonstration of hyaluronidase sensible proteoglycans at the presynaptic area in mouse cerebellar cortex.

By application of electron cytochemical techniques to cerebellar tissue, the presence of proteoglycans was demonstrated at the axoplasmic matrix of mossy fiber endings. Blocks of glutaraldehyde (G) fixed mouse cerebellum were processed according to the following procedures: a) Some pieces of tissue were post-fixed in osmium tetroxide, dehydrated by ethanol and embedded in araldite. b) Other pieces were sectioned to 30 mum thick and then immersed in Alcian blue solution pH = 2.7 followed by osmium tetroxide fixation, dehydrated and embedded in araldite (GABOUL procedure). c) Parallel slices of (b) previous to Alcian blue immersion were washed and incubated in either methanol-HCl, neuraminidase, ribonuclease or testicular hyaluronidase with their respective controls. d) Other blocks of G fixed tissue without any other treatment and fixation were dehydrated and embedded in araldite. Ultrathin sections of a, b and c were doubly stained with uranyl acetate and lead citrate while ultrathin sections of (d) were stained with the osmium coordination compound Os-DMEDA. The electron microscopic study revealed at the presynaptic axoplasm of mossy fiber rosettes, the presence of a GABOUL and Os-DMEDA positive electron dense material surrounding synaptic vesicles and continuous with presynaptic dense projections. This material which coincides with cytonet distribution was resistant to neuraminidase and ribonuclease and sensible to hyaluronidase and carboxymethylation. These findings permit us to conclude that the axoplasmic material of mossy fiber endings is constituted by proteoglycans in which hyaluronic acid and chondroitin 4-and/or 6-sulphate are present. The probable importance of these proteoglycans in synaptic mechanisms is also discussed.     

大脑皮质轴—棘突触的超微结构

大脑皮质中的突触与年龄、学习、经验和记忆密切相关。目前,在大脑皮质的研究中,对听觉皮质区突触超微结构的报导远远少于视皮质区和其他一些皮质区,在国内尚未见报导。本文用4例猫大脑听觉皮质区材料,共观察到1526个突触。在这些突触中,轴-棘突触占绝大多数,为所观察的突触总数的61.1％。根据不同的指标又将轴-棘突触分为许多种类型的突触,并对各种类型突触进行了统计。此外,本文对各种类型突触的机能意义进行了讨论,为进一步研究和探讨大脑皮质的复杂活动提供一些实验数据和对照参数。     

眼组织的透射电镜制样方法

介绍一种眼组织透射电子显微镜标本制备方法。组织先用４％多聚甲醛－２．５％戊二醛混合固定液预固定,１％四氧化锇固定液后固定,丙酮逐级脱水,延长Ｅｐｏｎ８１２包埋液的浸透时间并包埋,用玻璃刀或钻石刀作超薄切片,醋酸主枸橼酸铅双重染色,Ｈ６００－Ⅳ型透射电子显微镜观察。此方法的优点在于眼组织各层超微结构保存良好,没有损伤及变性,我们对该方法的操作要点作了讨论。     

子宫颈微偏腺癌6例临床病理分析

目的探讨子宫颈微偏腺癌的形态学、组织化学及免疫表型特征。方法对6例子宫颈微偏腺癌组织学特征进行观察,并行黏液组化及免疫组化染色（S-P法）。结果6例均有子宫颈腺体显著增生,腺体腔缘面呈花边状、锯齿状或乳头状突入到腺管腔内,并有成角状外翻,腺体呈浸润性生长。黏液组织化学：AB（pH1．0、2．5）／PAS染色证实,腺体腔内为混合性黏液,主要含唾液酸黏液,硫酸黏液减少,中性黏液较多。免疫表型：CEA（5／6）阳性,CA125（6／6）阴性。vimentin、SMA浸润性腺体周围纤维母细胞／肌纤维母细胞（6／6）阳性。结论子宫颈微偏腺癌以其特殊的形态结构和细胞轻微的异型、AB／PAS阳性、CEA阳性及腺体周围反应性纤维母细胞／肌纤维母细胞增生为特征。     

Dissolution mechanism of zinc phosphate dental cement in acetic and lactic acid buffers

When zinc phosphate cement was immersed in 0.1 M acetic and lactic acid buffer solutions adjusted to pH 4.1, its dissolution rate was determined by chemical analysis. The eroded surface of the cement was examined by X-ray diffraction and SEM observation. The dissolution process of the cement immersed in the acetic acid buffer solution was mainly controlled by the diffusion of releasing species through the cement matrix and that of the cement immersed in the lactic acid buffer solution by the decomposition of the cement matrix at the surface.     

DNA cytochemistry in polytene chromosomes: electron contrasting agents for the ultrastructural detection of chromatin DNA after alkaline hydrolysis/methylation-acetylation.

Salivary glands from Chironomus tentans larvae were fixed in glutaraldehyde and either subjected to alkaline hydrolysis followed by methylation-acetylation, or dehydrated without these treatments as controls. Ultrathin sections from Durcupan-embedded samples were contrasted by means of uranyl acetate, ruthenium red, indium trichloride, or the complex indium (III)-hematoxylin. Electron microscopic observations revealed a general contrasting pattern in control sections, while after the hydrolytic and blocking procedure only chromatin from polytene chromosomes appeared selectively contrasted. The nucleolus, Balbiani ring granules and puff materials showed weak or no electron opacity. After toluidine blue staining of semithin sections, an orthochromatic blue colour was found in chromatin bands from treated samples. These results indicate that alkaline hydrolysis/methylation-acetylation followed by contrasting with cationic heavy compounds is a valuable procedure to visualize chromatin DNA in polytene chromosomes.     

Mucin histochemistry of heterotopic gastric mucosa of the upper esophagus in adults: possible pathogenic implications

The mucin profile of 24 endoscopic biopsies of heterotopic gastric mucosa (HGM) of the upper esophagus in adults and a control group of ten cases of Meckel's diverticula containing heterotopic gastric mucosa were studied with two combined histochemical methods: alcian blue pH 2.5/PAS and high iron diamine/alcian blue pH 2.5. The clinical and light microscopic features of the 24 HGM cases were also reviewed. In addition to overall secretion of neutral mucins by the 24 HGM cases, mucin histochemistry showed prominent secretion of acidic mucins in 19 of 24 HGM cases (79%), with sulphomucins in 11 of 24 HGM cases (45.8%). This mucin profile of HGM was unlike that of either normal gastric mucosa or heterotopic gastric mucosa in Meckel's diverticula. Moreover, a comparison between the mucin profile and clinical features of HGM and Barrett's esophagus showed certain similarities. The data suggest a physiopathologic link between HGM and Barrett's esophagus.     

Ultrastructural localization of antigenic substances inTrichinella spiralis

The in situ localization of antigenic substances inTrichinella spiralis muscle larvae was demonstrated at the subcellular level. Larvae recovered from mouse muscle were fixed with halfstrength Karnovsky fixative, dehydrated with alcohol, and embedded in LR White resin. Ultrathin sections were incubated with sera from infected Wistar rats and, subsequently, protein A-gold complex. The specificity of the immunostaining was confirmed by a control experiment. Positively immunostaining structures included the stichocyte granules, body cuticle, hindgut cuticle, hypodermis, hemolymph, glycogen aggregates, esophagusoccupying substance (EOS), midgut-occupying substance (MOS), brush border, cytoplasmic granules in the cord, intestinal gland cell granules, and discrete areas in the genital primordial cell. However, the esophageal cuticle, nucleus, nucleolus, mitochondria, rough endoplasmic reticulum, and muscle fibers were negative by immunostaining.     

Selective histochemical staining of cartilage matrix by RivanolR

Rivanol^R, a fluorescent ethoxy derivative of acridine, interacts at different pH's with both glycosaminoglycans and proteins. The present study utilizes the specific interaction of Rivanol^R with acidic substances of the ground substance for histochemical studies of the cartilage matrix. This stain was applied to newborn mouse epiphyseal cartilages which were either unextracted or dissociatively extracted by graded concentrations of guanidinium chloride (GuHCl) from 0.5–3.0 M for four days at 25°C. Routinely prepared sections were then stained (0.1% solution) for two minutes at pH's ranging from 2.2–11.2. Stainability of the interterritorial matrix as well as the inner halo zone and outer corona zone of the lacunar matrix varied with pH. Whereas the interterritorial matrix decreased in stainability with rising pH, the halo and corona persisted in stainability up to pH 10.7. Dissociative extractions using GuHCl revealed the unextractable nature of the inner halo zone as well as the extractable nature of the corona above 1.0 M GuHCl concentration. Anionic sites on polyelectrolytes such as glycosaminoglycans are known to stoichiometrically bind many cationic dyes. The precise localization of stain-reactive glycosaminoglycans or proteoglycans in the region of the perichondrocytic matrix by Rivanol^R supports prior observations using other cationic stains. Our data demonstrates that Rivanol^R enables one to visualize the unique perichondrocytic matrix which may be interpreted to be both chemically and morphologically a “matrix within a matrix”.     

Responses of mucus-producing cells in gill disease of rainbow trout (Oncorhynchus mykiss).

This paper documents the responses of mucus-producing cells in the gills of rainbow trout (Oncorhynchus mykiss) throughout a naturally occurring outbreak of bacterial gill disease (BGD) and following exposure to experimentally induced high concentrations of ammonia and suspended solids. The responses were examined at three sites on the gill filament with three histochemical stains selected to identify the main types of mucous glycoproteins; these were periodic acid-Schiff (PAS), alcian blue pH 2.5 (AB2) and alcian blue pH 1.0 (AB1). In the BGD-infected fish, there was an increase in the numbers of PAS-positive and AB2-positive mucous cells and a corresponding decrease in AB1-positive cells. The greatest increase in mucus-producing cells occurred at the tips of the filaments, but the greatest relative change occurred at the mid-filamental (inter-lamellar) position. Fish exposed to high ammonia concentrations also had elevated numbers of mucus-producing cells, but there was no statistically significant change in fish exposed to high amounts of kaolin. The possible implications of these findings are discussed.     

A histochemical and immunohistochemical study of extra-ocular sebaceous carcinoma

A histochemical and immunohistochemical study of five cases of extra-ocular sebaceous carcinoma was performed using formalin-fixed and paraffin-embedded tissue specimens. Histochemically, the clear cells of sebaceous carcinomas were negative with periodic acid-Schiff and alcian blue staining. Immunohistochemically, the tumour cells of sebaceous carcinomas showed positive reactions for epithelial membrane antigen, human milk fat globules subclass 1, human milk fat globules subclass 2 and Leu M1, but did not express carcinoembryonic antigen, breast carcinoma associated antigen, S-100 protein, gross cystic disease fluid protein-15 or Dako M1. These histochemical and immunohistochemical findings were compared with those of other skin cancers which must be distinguished histopathologically from sebaceous carcinoma. We conclude that sebaceous carcinoma can be distinguished from eccrine porocarcinoma, malignant clear cell hidradenoma, extramammary Paget's disease, malignant trichilemmoma, squamous cell carcinoma and basal cell carcinoma by histochemical and immunohistochemical techniques using formalin-fixed and paraffin-embedded tissue specimens.     

Histological and mucin histochemical study of the small intestine of the Persian squirrel (Sciurus anomalus)

This article describes the histological and mucin histochemical properties of the small intestine of the Persian squirrel (Sciurus anomalus). This species is widely distributed in the Middle East and can be found as a companion animal. The histological studies revealed that the plicae circulares were not visible in the tunica mucosa. The maximum height and width of the villi were observed in the duodenum, which then decreased toward the ileum. The muscularis mucosa was scattered, whereas the tunica submucosa was composed of dense connective tissue. The lymphatic nodules were seen in the submucosa of the distal part of the jejunum and ileum, and Brunner’s glands were embedded in the initial portion of the duodenum. The tunica muscularis was significantly thicker in the ileum, and the circular muscle layer was thicker than the longitudinal muscle layer throughout the entire length of the small intestine. The mucin histochemistry, which was examined using the periodic acid-Schiff (PAS) and alcian blue (AB) (pH 1.0 and 2.5) and also PAS–AB (pH 2.5) and aldehyde fuchsin-AB (pH 2.5) techniques coupled with methylation and saponification reaction for some sections, showed that the small intestine mucous content included both carboxylated and sulfated acidic mucins with few neutral mucins. The results of this study contribute to the knowledge of the histological and histochemical characteristics of the gastrointestinal tracts of exotic mammals and provide data for comparison with other mammals.     

A study of fine structural changes in the cartilage-to-bone transition within the developing chick vertebra

Forty-eight chick embryos were killed at 9–16 days of incubation age. Tissue was obtained from the fourth or fifth cervical vertebra, immersed in Karnovsky's fluid, post-fixed in osmium tetroxide, dehydrated in ethanol, stained “en bloc” with uranyl acetate in ethanol, and embedded in Epon 812. Vertebrae were oriented for cross-sectional microtomy in cephalic to caudal sequence. Thin sections were stained with uranyl and lead salt solutions saturated with tribasic calcium triphosphate to prevent decalcification. Chondrocytes within the cartilaginous vertebral body occur in various stages of degeneration without orderly arrangement. Both reversible and irreversible stages are found at the cartilaginous resorption front. Electron-lucent osteoid and mineralization appear in the intercellular matrix at about 12.5 days. Rapidly invading blood vessels form a highly variable resorption front and irregular marrow cavity. Capillaries with accompanying cells border on the front, but else-where open capillaries allow blood elements to be in direct contact with cartilage. Chondroclasts are associated with small areas of calcified cartilage. At about 14 days trabeculae are formed at the resorption front by osteoblasts which deposit bone osteoid on uncalcified cartilaginous matrix. The matrix is eroded away. A free trabecula of bone without a core of calcified cartilaginous matrix remains. Basic differences between developmental growth processes in the epiphyseal plate and vertebral body may stem from the large amount of uncalcified cartilaginous matrix at the latter's resorption front.     

Placental intravillous accumulation of sulfated mucosubstances. A reevaluation of so-called hydropic degeneration of villi.

A histochemical study was performed on 46 placentas showing so-called hydropic degeneration of villi. The 46 cases included early abortuses (35 cases), incomplete moles (four cases), and complete hydatidiform moles (seven cases). All the specimens showed diffuse, positive staining of the distended villous stroma with alcian blue at pH 1.2 and 2.5 in the areas containing "hydropic changes." The alcian blue positivity was abolished following digestion with hyaluronidase. These findings indicate that so-called "hydropic degeneration of villi" represents an intravillous accumulation of strongly sulfated mucosubstances rather than the result of the accumulation of water. The intravillous accumulation of mucosubstances most likely represents a nonspecific stromal reaction of the connective tissue of the placenta to a variety of noxious stimuli. This finding may have some bearing on the interpretation of the physiologic mechanisms involved in placental villous distention, which has been largely centered in the past on the premise that the villous swelling was related to hemodynamic alterations caused by the accumulation of water.     

Substructure in rod photoreceptor membranes.

Frog retinae, fixed only in buffered glutaraldehyde, were embedded for sectioning in glutaraldehyde polymerized with urea. In suitably thin sections globular substructures were seen in negative contrast after ionic staining with uranyl acetate and lead citrate, or after staining with neutralized phosphotungstic acid. Efforts to extract at least some of the lipid from sections before ionic staining enhanced the visualization of the "globules". Exposure to KMnO4 solution, used as an oxidative section stain, also outlined globular substructure in negative contrast, but with the additional feature that positively stained surface "leaflets" associated with the aqueous compartment were well defined. Staining sections with OSO4 vapor resulted in positively stained membranes, but without any evident substructure. However, when sections which previously had been exposed to OSO4 vapor were secondarily stained with uranyl acetate and/or lead citrate, positively stained globular substructures then were revealed. The globular substructures always were centered in the hydrophobic core region of the disc membranes, and symmetrically spanned the full thickness of this layer. The diameter of individual particles approximated 50-55 A. Reasons are presented for the supposition that the evident globules incorporate at least hydrophobic components of rhodopsin molecules. Findings are discussed in relation to various models of disc membrane organization that have been proposed in recent years.     

Enzyme and immunohistochemistry on undecalcified bone and bone marrow biopsies after embedding in plastic: A new embedding method for routine application

Summary A simplified method of low temperature methyl and butyl methacrylate embedding (up –20° to –15° C) is demonstrated using a proper redox system of benzoyl peroxide and aromatic amine. This method combines the morphological superiority of plastic-embedded bone tissue and bone marrow sections with the advantages of specific enzyme histochemical and immunochemical markers. The method permits good preservation of morphological details, the survival of antigenic determinants and the retention of enzyme activities. The specimens were fixed in 1.6% formaldehyde and 5% sucrose in 0.02 M phosphate buffer at pH 7.4, washed in 0.02 M phosphate buffer and 5% sucrose, dehydrated with acetone and impregnated with monomers of embedding medium. All these steps were carried out at +4° C. The method presented is especially suitable for enzyme histological and immunohistological diagnosis of primary and secondary bone tumours, soft tissue tumours, as well as myelo- and lymphoproliferative disorders of bone marrow biopsies. Examples are demonstrated with mono- and polyclonal antibodies and reaction products of hydrolytic enzymes.Dedicated to Prof. Dr. Gerhard Seifert on the occasion of his 70th birthday     

Ultrastructure and histochemistry of the medullary interstitial matrix of rat kidney.

Medullary tissue of the normal rat kidney was perfused with 3 percent glutaraldehyde (GA), incubated in 0.5 percent cetyl pyridinium chloride and postfixed in 1 percent OsO4. In comparison with the ordinary fixation with GA and OSO4, the medullary interstitium represented abundant matrical substance that is rich in acid mucopolysaccharides (AMPS) and morphologically represents a diffuse reticular structure consisting of 30 to 150 a thick microfibrils and granular structures of 300 to 500 A in diameter. When chondroitinase was applied before OsO4 treatment, the dense granes disappeared and the microfibrils were replaced by loosely textured 30 A thick microfilaments. After hyaluronidase treatment the microfibrils disappeared and most granules changed into a ring-shaped structure with an electronlucent central portion. These results suggest that the reticular structure consists of microfilaments of hyaluronates and amorphous masking substance of chondroitin sulfates. In the dense granule, hyaluronates become concentrated in the central portion and chondroitin sulfate in the peripheral zone. When perfused with a CPC-containing GA, the medullary interstitium was diffusely filled with a large amount of fine granular substances suggesting the presence of water soluble free AMPS filling the reticular space.     

Fine structure and fluoride resistant acid phosphatase activity of electron dense sinusoid terminals in the substantia gelatinosa Rolandi of the rat after dorsal root transection

Summary Fluoride resistant acid phosphatase (FRAP) activity of the rat substantia gelatinosa Rolandi is confined to electron dense sinusoid terminals under normal conditions. Transection of dorsal roots or removal of dorsal root ganglia results in a rapid degeneration of more than half of the electron dense sinusoid axon terminals. First signs of degeneration ensue 20 hours after surgery; at the 24 hours state osmiophilic degeneration bodies develop that are translocated into glial elements in the course of the second postoperative day. At the same time, light microscopically visible FRAP-activity of the Rolando substance disappears. Electron histochemical investigations reveal that decreased enzyme activity is due to degeneration of FRAP-positive terminals. It is concluded that FRAP-positive terminals, representing the majority of electron dense sinusoids in the Rolando substance, are dorsal root collaterals; the origin of non-degenerating FRAP-negative electron dense terminals remains unknown for the time being.     

Immunofluorescence on resin-embedded material

Some antigenic determinants can be preserved in tissues after plastic embedding. In the present study liver tissue was fixed with glutaraldehyde or paraformaldehyde, dehydrated with ethanol and toluene and embedded in araldite which was then polymerized at 37 degrees C. Immunofluorescence was performed on semi-thin sections etched with hydrogen peroxide. This procedure allows correlation with light microscopy (on the same stained semi-thin section) and with electron microscopy (on adjacent ultra-thin section). Good results were obtained with anti-nuclear, anti-mitochondrial, anti-microsomal and anti-alpha1-antitrypsin sera.