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Summary The histochemical reaction for myofibrillar (Ca-ATPase with and without acid pre-incubation was performed in samples of longissimus dorsi and gracilis muscles in neonatal (12–48 hr) piglets. Measurements of the two basic fibre types I and II show that type I fibres have a considerably greater diameter than type II, the ratio of D1: D2 being high, but the coefficients of variation are more pronounced in the case of type II fibres. Three basic types of primary bundles were seen: 1. Bundles with a single central type I fibre and other fibres of type II A. 2. Bundles with one central type I fibre with 1–2 adjacent II C fibres and II A fibres in the periphery. 3. Bundles with 2–3 fibres of type I and sometimes also 1–2 IIC fibres in the centre and the remaining fibres around them of type IIA. These bundle types are considered to represent consecutive stages in muscle differentiation. According to the histometric and type-differentiation trends the following criteria of advanced differentiation are formulated: a decreasing D1/D2 ratio, the occurence of the II C type fibre and the presence of more than one central type I fibre in the primary bundles. Zusammenfassung Morphologische und histochemische Typen-Differenzierung bei primaren und sekundaren fetalen Muskelfasern von neugeborenen Ferkeln Bei 12–48 Stunden alten Ferkeln wurden in Proben des M. longissimus dorsi und des M. gracilis die histochemischen Reaktionen fur myofibrillar Ca-ATPase mit und ohne Saure-Vorinkubation durchgefuhrt. Messungen an den beiden Fasertypen I und II ergaben, das der Durchmesser der Typ-I-Fasern betrachtlich groser war als derjenige der Typ-II-Fasern, so das daraus ein hoher Quotient D1: D2 resultiere. Die Variationskoeffizienten waren bei den Typ-II-Fasern groser. Es liesen sich 3 grundsatzliche Anordnungen von Primarbundeln unterscheiden: 1. Bundel mit einer einzigen zentralen Typ-I-Faser und einem Kreis von II-A-Fasern. 2. Bundel mit einer zentralen Typ-I-Faser mit 1–2 benachbarten II-C-Fasern und einem Kreis von II-A-Fasern. 3. Bundel mit 2–3 Typ-I-Fasern und manchmal auch mit 1–2 Typ-II-C-Fasern im Zentrum, umgeben von II-A-Fasern. Diese Bundeltypen werden als konsekutive Stadien der Muskeldifferenzierung betrachtet. Unter Berucksichtigung der morphologischen Veranderungen werden die folgenden Kriterien als Zeichen einer fortgeschrittenen Differenzierung postuliert: Abnehmender Quotient D1/D2; Auftreten von Typ-II-C-Fasern und Vorkommen von mehr als einer Typ-I-Faser im Primarbundel. Resume Differenciation morphologique et histochimique des types de fibres musculaires foetales primaires et secondaires chez des porcelets nouveaux-nes Les reactions histochimiques pour la Ca-ATPase myofibrillaire avec ou sans preincubation acide ont ete effectuees dans des echantillons de M. longissimus dorsi et M. gracilis chez des porcelets âges de 12–48 heures. Des mesures faites sur les deux types I et II de fibre ont montre que le diametre des types de fibres I etait nettement plus grand que celui des types de fibres II, si bien qu'il en resultait un quotient D1/D2 plus eleve. Les coefficients de variation des types de fibres II furent plus eleves. Il a ete possible de differencier 3 formes de base des faisceaux primaires: 1. Un faisceau avec une seule fibre de type I centrale et un cercle de fibres II A. 2. Un faisceau avec une seule fibre de type I centrale, avec une a deux fibres II C voisines et un cercle de fibres II A. 3. Un faisceau avec 2–3 fibres de type I et parfois egalement avec 1–2 fibres de type II C au centre entourees de fibres II A. Ces types de faisceaux sont consideres comme des stades consecutifs a la differenciation du muscle. En etudiant les modifications physiologiques, on a propose les criteres suivants pour designer une differenciation avancee: un quotient D1/D2 diminue; l'apparition de fibres du type II C et la presence de plus d'un type de fibre I dans un faisceau primaire. Resumen Diferenciacion morfologica e histoquimica de los tipos de fibras musculares fetales primarias y secundarias en los lechones recien nacidos En lechones de 12–48 horas de edad se efectuaron en muestras del m. larguisimo del dorso y del m. gracilis las reacciones histoquimicas para la Ca-ATPasa con y sin preincubacion acida. Las mediciones llevadas a cabo en ambos tipos de fibras I y II mostraron que el diametro de las fibras del tipo I son bastante mayores que los de las fibras del tipo II, resultando de aqui un cociente D1/D2 elevado. Los coeficientes de variacion eran mayores en las fibras del tipo II. Se pudieron diferenciar 3 disposiciones fundamentales de haces primarios: 1. Haz con una fibra central unica del tipo I y un circulo de fibras II A. 2. Haz con una fibra central del tipo I con 1 a 2 fibras II C vecinas y un circulo de fibras II A. 3. Haz con 2 a 3 fibras del tipo I y, a veces tambien, con 1 a 2 fibras del tipo II C en el centro, rodeadas de fibras II A. Estos tipos de haces se consideran como estadios consecutivos de la diferenciacion muscular. Considerando las modificaciones morfologicas, se proponen los criterios siguientes como signos de una diferenciacion avanzada: cociente D1/D2 decreciente; aparicion de fibras del tipo II C y presencia de mas de una fibra central del tipo I en el haz primario.

The expression of vimentin and the phosphorylated variant of high molecular weight neurofilament protein (NF-H) was studied in developing human fetal dorsal root ganglia and spinal cord. The technique used for examination of cryosections was double-label fluorescence with monoclonal antibodies. Both proteins were present in the nerve fibres inside the ganglia of 6- and 8-week-old embryos. During further development the expression of vimentin continued to increase in the satellite cells, but was found to be decreasing in the ganglion cells. Phosphorylated NF-H was found in the processes of ganglion cells, as well as in the perikarya at all developmental stages. In the spinal cord of 6- and 8-week-old embryos, phosphorylated NF-H protein was found in the longitudinal fibres of the marginal layer and in processes of the mantle zone; some of the fibres also contained vimentin. Later the co-expression of the two proteins ceased and vimentin was found only in glial and mesenchymal derivatives. Phosphorylated NF-H was located, at all developmental stages, in the axons of both white and grey matter, but not in the neuronal perikarya. The results indicate that phosphorylation of the NF-H in human dorsal root ganglia starts in the perikarya of the ganglion cells while in the ganglion cells of the spinal cord it takes place in the axons.

Expression of intermediate filament proteins was studied in human developing spinal cord using immunoperoxidase and double-label immunofluorescence methods with monoclonal antibodies to vimentin and glial fibrillary acidic protein (GFAP). Vimentin was found in the processes of radial glial cells in 6-week embryos, while GFAP appeared in vimentin-positive astroglial cells at 8-10 weeks. GFAP and vimentin were present in approximately equal amounts in differentiating astrocytes in 23-week spinal cord. In 30-week fetuses, astrocytes reacted strongly for GFAP, while both the reaction intensity and the number of vimentin-positive cells fluctuated predominantly in the grey matter. No clear-cut transition from vimentin to GFAP was noticed during the development of astrocytes. The majority of ependymal cells in 23-week fetuses contained vimentin but only a few of them reacted for GFAP. The expression of vimentin continued during the whole development of the ependymal layer, in contrast to the reactivity for GFAP which disappeared between the 30th week and term.

Luk8§ Z., L. Pivnik, I. Straka, J. Rampochova, L. Stl!panova, J. Kaman, J. Vitouch: Early Postnatal Differentiation of Muscle Fibres and the Occurrence of Myofibrillar Hypoplasia in Piglets. Acta vet. Brno, 51,1982: 31-45 The type-representation of muscle fibres affected by myofibrillar hypoplasia and an assessment of the degree of histochemical differentiation of primary muscle bundles were carried out on m. longissimus dorsi and m. gracilis of piglets sufferring from splayleg syndrome and healthy (piglets. Serial transverse sections with the detection of myosine Ca-A TPase with and without acid preincubation were used to establish the type of muscle fibres. It was found that myofibrillar hypoplasia (MFH) affects in our herds both piglets with splayleg syndrome and clinically healthy ones, including those form herds free from splayleg syndrome. No statistically signi­ ficant differences were found in the proportion of the size of primary and secon­ dary foetal fibres between the groups of animals studied. Histochemical differentia­ tion of primary bundles in the two groups includes the same stages of development, but structures with more advanced differentiation occurred more often in clinically healthy animals, i. e. those with II.C-type fibres present and with more than one type I fibre in the centre of primary bundles. Myofibrillar hypoplasia affects II fibres preferentially, and where differentiation had occurred H.C fibres were not usually affected by MFH. Nor was MFH found in intrafusal fibres. Splayleg syndrome, myofibrillar hypoplasia, muscle-fibre type differentiation, newborn piglen, histochemistry, histometry.

LukU Z., Kaman J., Pivnik L.: Histochemical Characteristics of the Splayleg Syndrome in Newborn Piglets. Acta vet., Bmo, 47,1978: 51-66. Histological examinations of the muscles extensor carpi radialis, pectoralis super­ ficialis, triceps brachii, gracilis, adductor, biceps femoris, and longissimus dorsi were carried out in 9 piglets with "splay1eg" syndrome associated with myofibri11ar hypoplasia, and in four piglets without clinical symptoms from the same breed, all of them aged 1-3 days. In· addition to routine staining, histochemical reactions were performed for CaI+-ATPase (PH 9.4), NADH-tetrazolium reductase, acid phosphatase, non-specific esterase, cholinesterase, glycogen and lipids. Non-affected extrafusal fibres showed two reaction intensities for CaI+-ATPase: an intensive reaction in fibres located mostly on the periphery of primary bundles, and a fainter reaction in fibres located closer to the centre demonstrated the inci­ pient type di1ferentiation of the fibres. Such a distinction was only possible by means of the NADH-tetrazolium reductase reaction. The localization was evidently mitochondrial and intensive reaction was also to be observed on the periphery of some fibres located in the centre of primary bundles. Reactions for glycogen and lipids were generally equal in .all fibres. Azo-dye reactions with alpha-naphthylacetate and histochemical de­ tection of acetylcholinesterase demonstrated regularly distributed and normally shaped motor- end plates. Intrafusal fibres showed an intensive, mostly uniform reaction for NADH-tetrazolium reductase, and a strong gradual reaction for Ca2+-ATPase. In deficient regions fibre type di1ferentiation was less pronounced but it is the second population of fetal myofibres that seems to be affected by MHF. Glycogen was accumulated in spaces defficient in myofibrils. Neither size and shape nor distribution of motor end-plates were altered. No deficiency of myo­ fibrils and no changes of the reactions under study were observed in intrafusal fibres. Splayleg syndrome, myofibrillar hypoplasia, congenital reversible myofibrillar dys­ maturity, newborn piglets, muscle fibre type differentiation, neuromuscular unit, his­ tochemistry. As in many countries with advanced pig breeding, typical causes of the losses of newbom pig­ lets in Czechoslovakia in the last years are partly due to so called "splayleg" syndrome connected clinically with the weakness of leg muscles and morphologically above all with an insufficient, retarded development of myofibrils in muscle fibres of some skeletal muscles. At present, the

Cholinesterase distribution in trigeminal ganglion was studied in rabbits by the thiocholine method. The presence of acetylcholinesterase conditions either a) a diffusive cytoplasmatic reaction of varying intensity — from very weak to very strong — or b) an unevenly intensively distributed reaction pronounced on the periphery of ganglion cells. — The histochemical localization of biogenic monoamines was studied with the aid of the fluorescent method according to Falck and Hillarp. Green-yellow varicose terminals were found in the neighbourhood of blood vessels, between myelinized fibres, and, finally, in close vicinity of ganglion cells with which they probably form synaptic contacts.

Distribution of cholinesterase-containing nerve fibres was studied in 15 human dental pulps by the thiocholine method. Falk's fluorescent method was used to demonstrate catecholamines (8 dental pulps). Cholinesterases were localized partly in the subodontoblastic plexus sending out fine branches towards odontoblasts, and partly in the nerve fibres attached to the blood vessel walls. These fibres in contrast to those of the subodontoblastic plexus were finer and showed fine varicosities. Monoaminergic terminals were localized mainly along blood vessel walls, however, some fibres having no relation to the blood vessels were also found. Cholinesterase-containing nerve fibres in the periphery of the pulp are considered to be sensitive nerve fibres originating from n.V. Distribution of cholinesterase-containing nerve fibres and monoaminergic terminals along the blood vessel walls indicates that the blood vessels in the human dental pulp might be under both parasympathetic and sympathetic control.

The presence of 1% agar in the fixation and substrate solutions for the histochemical demonstration of thiamine pyrophosphatase (4.4 mM TPP; 3.6 mM Pb2+; 0.025 Tris-maleate buffer, pH 7.2) clearly facilitates the localization of the enzyme in Golgi apparatus in cold microtome sections prepared from unfixed specimens.

The thiocholine method for the histochemical detection of cholinesterases according to Karnovsky-Roots was adapted for unfixed cryostat sections by addition of the agar solution to the incubation mixture and by using the semipermeable membrane interposed between the section and the incubation medium. The procedure prevents the leakage of the enzyme activity of the section and is suitable for tissues where the cholinesterase activity is low.

Three monospecific monoclonal antibodies (BA16, BA17 and A53-B/A2) recognizing different epitopes of the human keratin 19 were used to determine tissue distribution of this 40 kDa keratin polypeptide. Immunohistochemical methods revealed four different staining patterns among normal human epithelial tissues: firstly, complete negativity of the epidermis, sebaceous glands, hepatocytes and other tissues; secondly, homogeneous positivity as seen for example in the gall bladder and urinary bladder epithelium, endometrium and many other epithelia; thirdly, a mosaic of positive and negative cells among mammary gland luminal cells, prostate epithelia and some other epithelia and fourthly, a more complex heterogeneous pattern found in non-keratinizing squamous epithelia and hair follicles with generally the basal layer being the most strongly or sometimes exclusively stained. The pattern seen in non-keratinizing squamous epithelia varied considerably according to the fixation method and the antibody used as well as among different donors and in different areas of the same organ. The other three staining patterns were on the other hand nearly identical with all three antibodies on both frozen sections and sections of methacarn-fixed paraffin-embedded tissues. Our results provide evidence for differential expression of the human keratin 19 at the single cell level, an observation which could be exploited in the study of epithelial differentiation and pathology.