Altersabhängigkeit des Toluidinblau-Dichroismus und der Anisotropie der Fasern in der Wachstumszone menschlicher Rippen

Zusammenfassung Die parallel zu den Säulenknorpelzellen in der Knorpelmatrix der Wachstumszone menschlicher Rippen verlaufenden Faserbündel untersuchten wir nach standardisierter Toluidinblau-Färbung absorptionsoptisch mit dem Leitz-Mikrospektrographen und polarisationsoptisch mit einem Berek- und einem Brace-Köhler-Kompensator. Bei 35 Autopsiefällen zwischen 0 und 22 Jahren wurde der Gangunterschied der Doppelbrechung an ungefärbten und an gefärbten Schnitten und der Dichroismus der Faserbündel an gefärbten Präparaten bestimmt. Der metachromatische Index wurde berechnet. Für alle Parameter ergab sich eine charakteristische altersabhängige Verteilung der Meßwerte, die darauf hinweist, daß die höchste molekulare und strukturelle Ordnung der Faserbündel bereits im 8. Lebensjahr erreicht wird. Der mit dem Lebensalter kintinuierlich zunehmende Gangunterschied kann durch den progredienten Wasserverlust der Fasern erklärt werden.

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Age-dependency of Toluidine-blue dichroism and birefringence of fibers within the costo-chondral junction of human ribs

Summary The chondral fibers running parallel to the chondrocyte columns within die costo-chondral junction of human ribs were investigated quantitatively following standardized Toluidine blue staining by using the Leitz-Mikrospektrograph for observing the absorption spectrum, and by employing both a Berek- and a Brace-Köhler compensator for polarization measurements. Autopsy material was obtained from the ribs of 35 children and adults at the age of 0 to 22 years. Refractivity was measured in stained and unstained paraffin sections, whereas dichroism and metachromatic index (MI) of the fibers were determined in stained specimens.All three parameters showed characteristic, quantitative age-dependent variations indicating that the highest degree of molecular and structural adjustment and alignment of the fibers is already being reached at the age of 8 years. The continuously higher refractivity of the fibers with increasing age and bone maturity may be explained by a progressive loss of water by the fibers.

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Die Ergebnisse dieser Arbeit sind Teil der Dissertationsschrift von Herrn Heuft

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Mit Unterstützung der Deutschen Forschungsgemeinschaft, AZ Bo 395/4/5     

Developmental study of neuropeptide Y-like immunoreactivity in the neurohypophysis and intermediate lobe of the rhesus monkey (Macaca mulatta)

Summary The purpose of this study was to examine the development and distribution of neuropeptide Y-immunoreactive fibers in the neurohypophysis of the rhesus monkey (Macaca mulatta) throughout life and the relationship of these fibers to the hypothalamo-hypophyseal portal vasculature. In rhesus monkeys, which varied in age from fetal life to 34 years, neuropeptide Y-immunoreactive fibers were present at all ages examined. In adult monkeys, varicose neuropeptide Y-labeled fibers were concentrated in the upper infundibular stem in association with capillary loops of the portal vasculature and the long portal vessels. Other fibers travelled down the infundibular stem and were distributed at the junction of the lower infundibular stem and infundibular process in the vicinity of the short portal vessels. In the infundibular process, neuropeptide Y-immunoreactive fibers were concentrated along the border of the intermediate lobe. Other stained fibers were sparsely distributed in the infundibular process and were often associated with small vessels. Neuropeptide Y-immunoreactivity was also located in a few fibers and cells of the intermediate lobe. Very few labeled fibers were seen in the fetal neurohypophysis, but their number increased gradually during the first postnatal year. At two years of age, a high density of stained fibers was observed, especially in the infundibular process. The number of axons in the infundibular process was lower at 12 years and continued to decline until 34 years of age. Neuropeptide Y may modulate hormone release at these sites and may also be released directly into vessels in the infundibular process. The close association of neuropeptide Y-labeled fibers with capillaries of the portal vasculature strongly suggests that neuropeptide Y is released into the portal blood of monkeys throughout life and may influence hormone secretion from the anterior pituitary gland.     

Diaphragm muscle fatigue resistance during postnatal development.

Changes in the contractile and fatigue properties of the cat diaphragm muscle were examined during the first 6 wk of postnatal development. Both twitch contraction time and half-relaxation time decreased progressively with age. Correspondingly, the force-frequency curve was shifted to the left early in development compared with adults. The ratio of peak twitch force to maximum tetanic force decreased with age. Fatigue resistance of the diaphragm was highest at birth and then progressively decreased with age. At birth, most diaphragm muscle fibers stained darkly for myofibrillar adenosinetriphosphatase after alkaline preincubation and thus would be classified histochemically as type II. During subsequent postnatal development, the proportion of type I fibers (lightly stained for adenosinetriphosphatase) increased while the number of type II fibers declined. At birth, type I fibers were larger than type II fibers. The size of both fiber types increased with age, but the increase in cross-sectional area was greater for type II fibers. On the basis of fiber type proportions and mean cross-sectional areas, type I fibers contributed 15% of total muscle mass at birth and 25% in adults. Thus postnatal changes in diaphragm contractile and fatigue properties cannot be attributed to changes in the relative contribution of histochemically classified type I and II fibers. However, the possibility that these developmental changes in diaphragm contractile and fatigue properties correlated with the varying contractile protein composition of muscle fibers was discussed.     

A morphometric study of age-related changes in the elastic systems of the common carotid artery and internal carotid artery in humans

The present study aims to quantify the distribution of the elastic fiber system in the walls of the common carotid artery and the internal carotid artery in humans. In order to identify specific fibers, segments of carotid arteries were stained using the Verhoeff (for elastic fibers), Weigert (for elastic and elauninic fibers) and Weigert-oxone (for elauninic and oxytalanic fibers) methods. A histomorphometric study was undertaken in order to estimate the linear density (LV) of the fibrous components present in the elastic system and determine the total length of these components in each segment. Microscopic fields were sampled using a test system in which the colors and bands defined in the current study were superimposed on the histological preparations. Data were analyzed through linear regression using first-order rate equations and computer software. The internal carotid artery can be loosely defined as muscular in nature. In order to classify common carotid arteries, it is necessary to consider subject age. From 0 to 30 years of age, the LV of mature elastic fibers does not change significantly in either (left or right) common carotid artery, although a significant reduction in the LV of this type of fiber occurs in the right internal carotid artery. The most marked reduction in oxytalanic and elauninic fibers occurs between 0 and 10 years of age, becoming less intense between the ages of 10 and 30.     

Staining of Different Tissues in Thick Epoxy Resin-Impregnated Sections of Human Fetuses

Sections of undecalcified human fetuses, fixed in formaldehyde, embedded in the epoxy resin Biodur E 12 and cut on a diamond-wire saw were stained according to a slight modification of the method described by Laczko and Levai. The sections were immersed in a methylene blue/azure II solution at 90 C for at least 3 min and counterstained with a basic fuchsin solution at the same temperature. Differential staining was as follows: bone stained pinkish; cartilage, violet; collagen fibers, blue-violet; elastic fibers, red and muscle fibers, green-blue. Most other tissues were stained blue-violet against the transparent background of the embedding epoxy resin. Thanks to the distinct and differential staining of each tissue, contrast is sufficient for black and white as well as for color photography.     

Elastoid Changes in the Gingiva of Aged Humans

Elastotic changes were demonstrable in the gingivae of both dentulous and edentulous jaws obtained from both male and female humans varying in age from 62–92 years. Sections of gingivae from all the aged individuals exhibited numerable thick fibers that stained positive with iron hematoxylin or orcein. These positive staining fibers were found in the lamina propria radiating into the connective tissue papillae, coursing throughout the zona reticularis, as well as appearing as black amorphous masses. Pretreatment of sections with acid hydrolysis before staining by the two elastic tissue stains led to a loss of stainable fibers for elastin. In contrast the gingivae of a young adult did not contain elastic positive fibers as seen in the aged gingivae. The thick elastotic fibers found in the aged gingivae were argyrophilic in nature when the sections were impregnated with silver nitrate indicating that they were collagenous in nature. It is felt that the elastoid-like fibers in aging gingiva are another phase in the altering of collagen during the aging process.     

Staining of different tissues in thick epoxy resin-impregnated sections of human fetuses

Sections of undecalcified human fetuses, fixed in formaldehyde, embedded in the epoxy resin Biodur E 12 and cut on a diamond-wire saw were stained according to a slight modification of the method described by Laczkó and Lévai. The sections were immersed in a methylene blue/azure II solution at 90 C for at least 3 min and counterstained with a basic fuchsin solution at the same temperature. Differential staining was as follows: bone stained pinkish; cartilage, violet; collagen fibers, blue-violet; elastic fibers, red and muscle fibers, green-blue. Most other tissues were stained blue-violet against the transparent background of the embedding epoxy resin. Thanks to the distinct and differential staining of each tissue, contrast is sufficient for black and white as well as for color photography.     

Distribution of laminin and types IV and III collagen in fetal,infant and adult human spleens

Summary The immunohistochemical distribution of the basement membrane (BM) proteins, laminin and type IV collagen, and interstitial type III collagen was investigated in 12 fetal spleens at the 15th–38th gestational weeks (g.w.) and in spleens of 8 infants from term to 4 years. The results were compared with the distribution of the same proteins in adult human spleen. BM proteins were found to be abundantly present in the red pulp of all spleens during the whole of development. The content of type III collagen gradually decreased with advancing age and, in adult spleen, there were only occasional positively staining fibers in Billroth's cords. This finding indicates that the composition of reticular fibers in the red pulp of spleen is different from the reticular fibers elsewhere in lymphoreticular tissue. Early signs of ring fiber formation in the walls of venous sinuses were detectable at the 15th–19th g.w., although their more complete development occurred relatively late from the 36th g.w. onwards. Ring fibers contained both laminin and type IV collagen in all the investigated spleens. They never stained for type III collagen. The developing white pulp was positive for BM proteins, but showed no staining for type III collagen at the 15th g.w. At later ages, the white pulp stained similarly for both BM proteins and type III collagen.     

AN ELECTRON MICROSCOPE STUDY OF CORTICAL STRUCTURES OF OPALINA OBTRIGONOIDEA

The pellicular framework of Opalina obtrigonoidea consists of numerous longitudinal ribs parallel to the kineties. These ribs lie erect on the cell surface, and each is composed of striated longitudinal fibers. A membrane covers the ribs and the ectoplasm between them. Flagella, of conventional structure, emerge from the ectoplasm between the ribs. The two central fibers of each flagellum end at the cell surface; the nine peripheral fibers continue for about 400 mµ into the cell to form an open tubular kinetosome. From the anterolateral curvature of each kinetosome arise two rows of fibrils, each fibril oriented perpendicular to the cell surface and about 150 A in diameter. The two rows converge anteriorly and probably meet the next adjacent kinetosome. Minute granules or tubules, arranged in oblique rows and at least sometimes accompanied by very fine fibers, lie at the surface of the ectoplasm but show no detectable connection with the kinetosomes. The whole flagellar apparatus of Opalina thus bears a general resemblance to the infraciliature of some holotrich ciliates, but the degree of evolutionary relationship between them remains uncertain.     

NADPH-diaphorase expression in the meibomian glands of rat palpebra in postnatal development

In the current study, we aimed at investigating the presence of nitric oxide synthase (NOS) positive nerve fibers in rat meibomian glands (MGs) at various stages of development. There is good evidence to suggest that nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) is a surrogate for neuronal nitric oxide synthase (NOS). Sections of the central, upper eyelids of Wistar rats were processed histochemically for NADPH-d to investigate the presence and distribution of NOS-positive nerve fibers at the following time points: day 1 and weeks 1, 2 and 3 post partum, and in adult controls. At day 1, MG acini were lightly stained and located at a distance from the mucosal border. Vessels were accompanied by intensely stained NADPH-d positive nerve fibers. At the week 1 time point, both the vessels and the NADPH-d positive fibers were still present, but less numerous. MGs were now closer to the mucosa, so that the submucosa was thinner. The acini were mostly pale but occasionally darker. At week 3, there were fewer blood vessels in both the submucosa and within the septa. Darker acini were more common than lightly stained acini. NADPH-d positive dots were observed in the vicinity of the MGs. At the week 3 time point, MGs were adjacent to the mucosal border and stained more intensely than at earlier times; almost all acini were stained. The microscopic appearances were almost identical with those of adult palpebra. Submucosal and septal blood vessels and NADPH-d positive nerve fibers were less numerous. NADPH-d histochemical staining confirmed differences in the density of stained nerve fibers at different developmental stages. The greatest density of NADPH-d -positive nerve fibers occurred in 1-day-old rats whereas they were less numerous in adult rat eyelids. Nerves innervating MGs utilize nitric oxide (NO) as a neurotransmitter mostly in early developmental stages and this need thereafter decreases and stabilizes at 3 weeks postnatally.     

Determination in the system of the neuromuscular relations of the coronary arteries at stages in human postnatal development

By means of micromorphometric method in transversal sections of electively stained right and left coronary arteries the data, characterizing the area of the middle and external sheaths, amount of myelin fibers in nerves, accompanying the artery have been obtained in men of 8 age groups (from birth up to old age), who died from causes not connected with the cardiovascular pathology. The summary amount of all classes of myelin fibers increases synchronously with an increase of the muscle sheath area from newborn up to 35-45 years of age. After this age the amount of the myelin fibers decreases together with decreasing area of the middle sheath. Minimal values of these indices are observed in the old age (85-90 years). The external sheath area increases permanently from birth up to the old age. The correlative analysis reveals a positive and significant connection of various degree between the myelin fibers number and value of the middle arterial sheath in persons of juvenile, mature and elderly age: amount of the muscular layer is adequate to the myelin fibers amount. In other age groups the correlative connections are either small, or not significant. Alternative, corresponding to each age quantitative gradations of the nervous connections are determined by means of the peripheral substrate. Its role in the vessels investigated is performed, to a definite degree, by the muscle sheath.     

Histochemical fiber typing and staining intensity in cat and rat muscles.

In the gastrocnemius muscle of cat and rat, staining for oxidative enzymes differentiated three fiber types (A,B,C) and staining for adenosine triphosphate at pH 9.4 differentiated two fiber types (I, II) with a reliability of 90% and 98%, respectively. In cat 96% and in rat 90% of the fibers were typed identically after staining for nicotinamide adenine dinucleotidelinked lactic dehydrogenase (LDH) and succinic dehydrogenase (SDH). When differentiated by staining for LDH, A and B fibers were of type I. IN RAT, 80-90% OF ALL FIBERS WERE OF TYPE 22, COMPPRISING A, B and C fibers. Type I fibers stained for LDH intensely as did C fibers of type II, but stained intermediately for SDH. The degree of staining was measured by photometry. When fibers were stained for LDH, histograms of density showed three peaks corresponding to A, B and C fibers in cat, but only two peaks corresponding to A and C fibers in rat, In cat and rat, the densities of A, B and C fibers belonged to different populations. In soleus muscle of cat and rat stained for LDH, menadione-linked alpha-glycerophosphate dehydrogenase and adenosine triphosphatase at pH 9.4, the degree of staining differed from thatin any type of fiber in gastrocnemius muscle     

猫视神经年龄相关的形态学变化

对4只青年猫(1-3龄)和4只老年猫(10-13龄)视神经进行形态计量比较研究。取两个年龄组的颅内相应部分视神经进行横向连续切片,H.E染色于光镜下观察其基本结构;相邻切片进行结晶紫染色显示胶质细胞;神经丝蛋白(NF)免疫染色显示视神经纤维,胶质纤维酸性蛋白(GFAP)免疫染色显示星形胶质细胞(AS),对实验结果进行统计学分析并绘制纤维直径谱。与青年猫相比,老年猫视神经外膜厚度、直径、面积均显著增加,视神经纤维的密度和数量显著下降,且以视神经中央部纤维密度下降最显著;纤维直径谱分析结果显示,青、老年猫纤维直径分布范围相似,但老年猫的峰直径及纤维平均直径比青年猫的显著减小;另外,老年猫视神经束中的星形胶质细胞明显膨大,胶质细胞密度以及星形胶质细胞占胶质细胞总数的百分比均显著增加。结果表明:在衰老过程中视神经纤维出现明显的丢失现象,纤维平均直径显著减小使其对视觉信息的传导速度减慢,这可能是导致老年个体视觉分析速度下降的重要原因;老年个体视神经束内胶质细胞活动增强可能对维持视神经纤维形态、功能或延缓视神经进一步衰老起保护作用     

青年猫和老年猫视神经超微结构观察比较

目的探讨青年猫和老年猫视神经年龄相关的形态学变化及可能造成的生理影响。方法取4只青年猫(2-3岁,2-2.5kg)和4只老年猫(10-13岁,2.5-3.5kg)颅内相对应部分视神经,制作横向半薄切片和超薄切片,半薄切片用甲苯胺蓝硼砂溶液染色,光镜观察、测量视神经的直径(不含外层神经膜);超薄切片标本用醋酸和柠檬酸铅染色,电镜观察、计数视神经纤维密度、测量视神经纤维外径D(含髓鞘)和内径d(不含髓鞘),按一定分级范围算出各种直径的纤维及各种d/D比值的纤维所占百分比,分别画出直方图,对实验结果进行统计学分析并绘制纤维直径谱。结果与青年猫相比,老年猫视神经直径显著增大(P0.05);纤维数量显著下降(P0.05)。纤维直径谱分析结果显示,青、老年猫纤维直径分布范围相似,但老年猫纤维的峰直径及纤维平均直径比青年猫的显著减小(P0.05),老年猫视神经纤维的d/D比值亦明显降低。另外,老年猫视神经中部分轴突肿胀,髓鞘疏松、结构紊乱,板层脱离、空泡化,有的轴索髓鞘溶解。结论在衰老过程中,老年猫视神经纤维丢失,纤维直径减小,d/D比值下降,以及纤维髓鞘的松散解体,这些变化均可能导致视神经纤维对视觉信息的传导速度减慢,是老年个体视觉分析速度下降的重要原因。     

Collagen and elastin fibers in human pulmonary alveolar walls

The morphology and morphometric data of collagen and elastin fibers in the pulmonary alveolar walls are presented. Specimens were obtained from postmortem lungs quick-frozen at specified transpulmonary pressures. Collagen was stained by silver, and elastin was stained by orcein. Photomicrographs were composed by computer. Young lungs typically show small collagen fibers that radiate from the "posts," whereas larger fiber bundles traverse the septum irrespective of capillary blood vessels. In older lungs, rings of collagen around the posts appear enlarged. Elastin bundles do not show obvious variation in pattern with age and inflation pressure. Statistical frequency distributions of the fiber width and curvature are both skewed, but the square root of the width and the cube root of the curvature have approximate normal distributions. Typically, for young lungs at transpulmonary pressure of 4 cmH2O, the mean of (width)1/2 (in micron1/2) for collagen fibers is 0.952 +/- 0.242 (SD), that of (curvature)1/3 (in micron-1/3) is 0.349 +/- 0.094. The corresponding values for elastin are 0.986 +/- 0.255 and 0.395 +/- 0.094.     

Age estimation from stages of union of the vertebral epiphyses of the ribs

This study attempts to fill a persistent gap in the literature by documenting the timing of epiphyseal union at the vertebral end of the ribs in a sample of modern Portuguese skeletons. The skeletal remains of 53 females and 45 males, between the ages of 11 and 30, were taken from the Lisbon documented skeletal collection. Individuals in the sample have been previously described as being representative of a middle‐to‐low socioeconomic segment of the early 20th century Lisbon population. Three anatomical locations were examined for epiphyseal union: the head, the articular tubercle and the nonarticular tubercle. The first epiphysis to show partial union is that of the nonarticular tubercle (females, 11–19 years; males, 11–19 years), followed by the epiphysis of the articular tubercle (females, 11–20 years; males, 16–20 years), and finally by the head epiphysis (females, 15–24 years; males, 16–22 years), which can still show incomplete epiphyseal closure at 25 and 24 years for females and males, respectively. A trend for earlier female maturation was observed, but the statistical tests only confirmed this result for some ribs and age groups. No directional asymmetry was found, but a significant fluctuating asymmetry was observed in all three epiphyses. A preliminary analysis showed that the asymmetric group of individuals in the study sample includes all the rural‐to‐urban migrants, relative to the symmetric group. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Age-independent constancy of mineral contents in human ribs

On age relationships of mineral contents in human bones, the contents of the sixth rib and a piece of its compact bone were determined by inductively coupled plasma atomic emission spectrometry (ICPS). The ribs were resected from 21 subjects (14 men and 7 women) who died in age ranging from 65 to 93 yr. There were no age-dependent decreases in Ca and P contents of the ribs in the age range on ICPS. It was found that there were no age-dependent decreases in Ca and P in compact bones of ribs.     

Ultrastructure of Babesia major vermicules from the tick Haemaphysalis punctata as demonstrated by negative staining.

The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph of Haemaphysalis punctata adults and negatively stained with phosphotungstic acid. Most of the organelles observed were typical of those found in apicomplexan parasites. These were the apical complex with the polar ring and the ribs, micronemes and subpellicular microtubules. The number of ribs was 27 or 28. The outer membrane of the pellicle was composed of a large number of fibrils running along the length of the parasite. The inner membrane had large numbers of irregularly scattered holes. A cytoplasmic organelle similar to the granular body described in Theileria annulata ookinetes was seen for the first time in a B. major vermicule.     

Diaphragm capillarity and oxidative capacity during postnatal development.

In the cat diaphragm, fiber capillarity, cross-sectional area, and succinate dehydrogenase (SDH) activity were measured across the first 6 wk of postnatal development. Fibers were classified as type I, IIa, IIb, or IIc on the basis of staining for myofibrillar adenosinetriphosphatase (ATPase). Capillaries were identified in sections stained for ATPase at pH 4.2. Fiber cross-sectional areas and SDH activities were quantified using an image-processing system. During postnatal development, the proportions of type I fibers increased while type II fibers decreased. At birth, all type II fibers were IIc. From the 1st to the 2nd postnatal wk, the proportion of type IIc fibers decreased while the numbers of IIa and IIb increased. Thereafter the proportion of type IIb fibers continued to increase while the number of IIa steadily declined. At birth, capillarity, cross-sectional areas, and SDH activities of type I and II fibers were low compared with other postnatal age groups. Fiber cross-sectional areas increased progressively with age. The number of capillaries surrounding type I and II fibers increased markedly by the 2nd wk and then continued to increase at a slower rate. The number of capillaries per fiber area reached a peak by the 2nd wk and then declined as fiber cross-sectional area increased. Postnatal changes in capillarity depended on fiber type, being greatest in IIb. SDH activities of type I and II fibers were initially low during the first 2 postnatal wk and then peaked by the 3rd wk. After the 6th wk, fiber SDH activities decreased to adult values. Among the type II fibers, IIb showed the greatest change in SDH activity during early postnatal development.     

Dorsal rib histology of dinosaurs and a crocodylomorph from western Portugal: Skeletochronological implications on age determination and life history traits

Bone histology is an important tool for uncovering life history traits of extinct animals, particularly those that lack modern analogs, such as the non-avian dinosaurs. In most studies, histological analyses preferentially focus on long bones for understanding growth rates and determining age. Here we show, by analyzing ornithischians (a stegosaur and an ornithopod), saurischians (a sauropod and a theropod), and a crocodile, rib histology is a suitable alternative. The estimated age for all sampled taxa ranges between 14 to 17 years for Lourinhanosaurus antunesi and 27 to 31 years estimated for Draconyx loureiroi. The theropod Baryonyx was skeletally mature around 23–25 years of age but showed unfused neurocentral sutures, a paedomorphic feature possibly related to aquatic locomotion. Our results show that ribs can contain a nearly complete growth record, and reveal important information about individual age, point of sexual maturity, and, in some cases, sex. Because ribs are more available than long bones, this method opens new possibilities for studying rare and incomplete fossils, including holotypes.