Stain Historadiography

Fresh bone specimens were dehydrated in acetone and embedded in Ward's Bio-Plastic. Sections 10-15μ thick were cut with a special bone-cutting microtome and subjected first to radiography and then stained with safranin-fast green. The radiograph was made with a Machlett AEG-50-A X-ray tube on a fine-grained, spectroscopic plate; which, after processing, was mounted (dry) on a glass slide adjacent to the stained specimen. By these means, it was possible to make a correlated study of the calcium-bearing parts of the tissue and determine accurately their relationship to the bone itself. The method serves well for investigations of growing bone and for bone that is undergoing pathological involution. It has been named “Stain Historadiography” because it combines a stained specimen with its radiograph.     

A Procedure for Preparing Undecalcified and Unembedded Bone Sections for Light Microscopy

We have developed a procedure for light microscopic investigation of undecalcified and unembeddedbone sections. Biopsy samples of human metatarsus and femur and rat femur were fixed in aldehydes and sectioned with a cutting machine equipped with a diamond saw blade. Free sections 100-150 μm thick, stained with toluidine blue and von Kossa, did not show artifacts following the cutting, and the spatial relations of mineralized and nonmineralized components remained intact. Compact and trabecular bone, bone marrow and all cell types appeared well preserved and easily recognizable. Our procedure provides a simple and rapid method for preparing bone sections which undergo no chemical treatment other than fixation. This method is a useful alternative to standard histological protocols for studying bone specimens.     

Notes on Technic: “Clearing” Steel Knife Epon Sections in a Polystyrene Film Sandwich

Serial sectioning epoxy embedments by steel knife permits rapid light microscope survey of large tissue volumes, and preselection of areas of interest for electron microscopy. Acetate film (Hollander 1970) and Turtox plastic slides (West 1972) have been suggested as substrates upon which the sections may be “cleared” with an added layer of cured epoxy. In our experience, these substrates are excessively adherent to Epon, and “cleared” sections thinner than 40-50 μm cannot be released from them reliably. The following method is suitable for processing Epon sections 10 or more microns thick.     

A new mitotic cycle marker

The entry into metaphase of Physarum nuclei becomes insensitive to cycloheximide 10 min earlier than to γ-radiation, i.e., the “CH-marker” occurs earlier in prophase than the “γ-marker”. Gamma-irradiation of plasmodia wich have passed the CH marker and even the γ-marker leads to further sensitivity to CH. Thus, the data reveal a “γ-CH marker” in the mitotic cycle of this organism.     

Infrared spectroscopic assessment of the inflammation-mediated osteoporosis (IMO) model applied to rabbit bone

A model of osteoporosis based on induced inflammation (IMO) was applied on rabbit bones. The structural heterogeneity and molecular complexity of bone significantly affect bone mechanical properties. A tool like Fourier transform infrared spectroscopy, able to analyze both the inorganic and organic phase simultaneously, could provide compositional information regarding cortical and trabecular sections under normal and osteoporotic conditions. In this study, we assessed the mineral/matrix ratio, carbonate and phosphate content and labile (i.e., non-apatitic) species contribution to bone mineral and collagen cross-linking patterns. Clear differences were observed between cortical and trabecular bone regarding mineral and carbonate content. Induced inflammation lowers the mineral/matrix ratio and increases the overall carbonate accumulation. Elevated concentrations of labile species were detected in osteoporotic samples, especially in the trabecular sections. Collagen cross-linking patterns were indirectly observed through the 1660/1690 cm^− 1 ratio in the amide I band and a positive correlation was found with the mineralization index. Principal component analysis (PCA) applied to female samples successfully clustered trabecular and osteoporotic cases. The important role played by the phosphate ions was confirmed by corresponding loadings plots. The results suggest that the application of the IMO model to rabbit bones effectively alters bone remodeling and forms an osteoporotic bone matrix with a dissimilar composition compared to the normal one.     

Staining and Histomorphometry of Microcracks in the Human Femoral Head

We developed staining techniques that permit identification and histomorphometric analysis of microcracks in the human femoral head 1) from thick, ground bone sections (100 μm) by prestaining with the Villanueva mineralized bone stain (MIBS), and 2) from plastic embedded, undecalcified thin bone sections (5-15 μm) by staining in gallocyanin chrome alum-Villanueva blood stain methods. Both methods represent a significant improvement in the stainability of the microcracks, cellular and tissue elements, and the simultaneous assessment of osteoid seams and tetracycline markers by histomorphometry. Shrinkage and other artifacts were minimized, which helped to clarify some of the uncertainties arising from artifacts resulting from some bone staining methods. Histomorphometric analyses of microcracks were conducted on thick, ground sections of subchondral and trabecular bone. Microcracks were more prevalent in the subchondral bone and osteochondral junction than in the more distant trabeculae. We have consistently localized microcrack areas in bone tissues prepared in these ways.     

Some Introductory Comments on Silver Staining

Silver staining has become a versatile method for the visualization of specific cell structures and products. The similarity of the impregnation “nuclei” of reduced silver staining to the silver “specks” or “nuclei” of the latent image in photography is noted. “Physical” development (reduction of ionic silver in solution) in silver staining as compared to “chemical” development (reduction of ionic silver remaining in a silver halide crystal) in photographic procedures is briefly discussed.     

Producing Frozen Sections of Calcified Bone

We describe a procedure for the rapid production and maintenance of fresh frozen bone biopsies which can be used for a variety of immunohistochemical techniques. Within 5 min of excision. tissue is placed in cold 5% polyvinyl alcohol, surrounded with 3% carboxymethylcel-lulose in a hand made aluminum foil embedding mold and frozen by immersion in an absolute ethanol/dry ice slurry at -70 C. The tissue block is attached to the specimen stub with cryocom-pound and installed in a -32 C cryostat whose tungsten carbide D profile knife is maintained at -70 C. Automatic controls are set at a slow cutting speed and the “sectioning window” is adjusted to fit the biopsy size. Knife angle, thickness gauge and antiroll bar are changed to produce a complete section. The block face is smoothly “papered” with a polyvinylpyrrolidone (PVP) impregnated Ross lens paper strip. A single section is cut and positioned on a sequentially numbered, acid cleaned, double dipped chrome-alum gelatin coated slide: adhesion is aided by “press-blotting” with bibulous paper. Sections are stored at -20 C or in a desiccator at room temperature. A brief fixation followed by removal of the water soluble PVP and lens paper generates fresh frozen bone sections suitable for further analysis.     

Combining theoretical and experimental approaches to understand the circadian clock

This review is intended as a summary of our work carried out as part of the German Research Association (DFG) Center Program on Circadian Rhythms. Over the last six years, our approach to understanding circadian systems combined theoretical and experimental tools, and Gonyaulax and Neurospora have proven ideal for these efforts. Both of these model organisms demonstrate that even simple circadian systems can have multiple light input pathways and more than one rhythm generator. They have both been used to elaborate basic circadian features in conjunction with formal models. The models introduce the “zeitnehmer,” i.e., a clock-regulated input pathway, to the conceptual framework of circadian systems, and proposes networks of individual feedbacks as the basis for circadian rhythmicity.     

The Pragmatic Approach to Masking

This paper advocates the use of a pragmatic approach to the problem of masking in real-life situations involving an abrupt change in the timing of sleep, i.e. shiftwork and “jet-lag” situations. Although “pure” chronobiological research has pointed to the importance of taking masking effects into account, the techniques that it has provided for doing so are extremely difficult to apply in real-life situations. The approach advocated here is based on Wever's pioneering work, and involves estimating the normative endogenous and exogenous components of the circadian rhythm in body temperature. These estimates are then used to: (a) simulate the results of shiftwork studies; and (b) to “remove” the exogenous component in “jet-lag” studies to allow analysis of the estimated endogenous component. The simulated curves obtained cross-correlated extremely highly with published night-shift temperature curves, while the “removal” of the exogenous component resulted in very similar findings to those obtained in temporal isolation studies. It is concluded that this pragmatic approach to masking may prove extremely useful in interpreting the results of field studies of shiftwork and “jet-lag”.     

The Lower Row Monkey Cage: An Overlooked Variable in Biomedical Research

A survey of 96 primatological articles revealed that cage location of research monkeys is rarely mentioned, although the environment of upper and lower row-housed animals markedly differs in terms of light quality, light intensity, and living dimension. Not accounting for these uncontrolled variables may increase variability of data and, consequently, the number of experimental animals needed to obtain statistically acceptable results. This study concluded that single-tier housing would be an important refinement of research methodology. Such housing would (a) enable all animals of a room to use the “arboreal ”dimension of their enclosure and retreat to “safe ”vantage points above the human “predator, ”(b) offer all animals access to uniform light, and (c) provide more favorable conditions for professional animal care.     

Stain Technology a Bone Stain for Osteoid Seams in Fresh, Unembedded, Mineralized Bone

Fresh, ground, mineralized bone sections 75-100 μ thick are stained 90 minutes or 48 hours in the Bone Stain, a preparation containing fast green FCF, orange G, basic fuchsin, and azure II. Surface stain is then removed by grinding under running water. Sections are washed in 0.1% zephiran chloride (benzalkonium chloride) or in 0.01% mild soap and again washed in tap water, followed with distilled water. Sections are next differentiated in 0.01% acetic acid in 95% methanol, dehydrated in 95% ethanol and 100% ethanol, cleared in alcohol:xylene 1:1, 1:4, 1:9 and 2 changes of xylol, and then mounted permanently in Eukitt's mounting media.

Osteoid seams stain either green to jade green or red to dark red, incompletely mineralized bone red or orange yellow, and the zone of demarcation light green. The walls of lacunae, canaliculae, feathered bone, procedural artifacts and periosteocyte lacunar low-density versions stain red.

The method helps in the differential diagnosis of certain metabolic bone diseases in human biopsy and autopsy material.     

Solochrome Cyanine R As An Indicator Dye of Bone Morphology

Sections of undemineralized bone embedded in a polyester resin and cut at 6 μ are stained for 10 min, without removal of the embedding matrix, in an aqueous solution composed of Solochrome cyanine R, 1 gin; glacial acetic acid, 2 ml; and distilled water, 98 ml. A pH about 2 is obtained by the acetic acid. The sections are washed and differentiated in tap water at 30 C, dehydrated in ascending alcohols, cleared and mounted in synthetic resin. “Young osteoid” stains light orange and, in the rest of an osteoid seam, two types of lamellae can be distinguished: one blue layer of ground substance or collagen and one orange layer of fibrillar collagen. The “calcification front” is sharply demarcated by its dark blue color.     

Sphenophyllum miravallis Vetter and Bowmanites cupulatus sp.n. from the “Illinger Flözzone” (“Heusweiler Schichten”, Lower Stephanian, Saar Basin, German Federal Republic)

This paper deals with a detailed study of Sphenophyllum miravallis Vetter, a member of the “Sphenophyllum thonii group”. New material from the Reisbach colliery, working the “Illinger Flözzone” of the “Heusweiler Schichten” (Lower Stephanian, Saar Basin, German Federal Republic), is described morphologically and anatomically, and the species is discussed. The new material enlarges the known range of variability of the normal aspect of the foliage, i.e. the foliage of the thinner branches. Thicker stems with their aberrant polymorphous foliage, and cellular details, are described for the first time. An emended diagnosis is given. Comparisons with other species are made.

The new species Bowmanites cupulatus is introduced to accommodate fructufications most probably belonging to Sphenophyllum miravallis.

S. crenulatum Knight ex Wagner is considered to be a heterotypic synonym of S. miravallis, the latter name having priority.     

A Rapid Stain-Clearing Method for Video Based Cytological Analysis of Cotton Megagametophytes

Optical “clearing” is a cost saving method for preparing large numbers of whole, dissected or thickly sectioned cytological specimens such as plant ovules and ovaries. Minimal labor is required and specimens retain three-dimensional integrity. Previous development of high contrast stain-clearing methods using hemalum to impart contrast has facilitated analysis and photography under brightfield illumination for small ovules. The deep stain intensity of hemalum, however, often precludes adequate light transmission and contrast within internal focal planes, limiting the applicability of hemalum-based stain-clearing to small specimens. Having encountered this problem for nucelli of cotton (Gossypium barbadense L.), which are roughly 300 μm thick at fertilization, we have developed a modified stain-clearing system. The two key features of these new methods are the use of azure, C, which allows the intensity of staining to be readily regulated, and contrast manipulation via video signal and image processing. Intensity of azure C stain was readily controlled by modifying the staining and/or dehydration media to produce relatively low contrast specimens. Analysis was facilitated by indirect viewing on a video monitor using adjustments of sensitivity, exposure, and contrast of the charge-coupled device (CCD) camera. Digital processing provided further enhancement. Acceptable images were obtained from virtually all specimens. These methods, which combine low contrast (high transmittance) specimens with high contrast imaging, should facilitate data acquisition on reproduction, thus the developmental and genetic characterization of reproductive mutants. Other applications, e.g., in pathology and embryology, are readily envisioned.     

A Tetrachrome Stain for Fresh, Mineralized Bone Sections, Useful in the Diagnosis of Bone Diseases

Fresh, unprocessed bone is ground to sections 75-100 μ thick, stained in an aqueous solution composed of fast green FCF, 0.1 gm; orange G, 2.0 gm; distilled water, 100.0 ml; and adjusted to pH 6.65, then in a mixture of 1 part alcoholic solution of 0.25% celestine blue B and 9 parts of alcoholic solution of 0.1% basic fuchsin. Surface stain is removed by grinding sections to 50 μ and washing them in 1% invert soap (Zephiran) to remove adherent debris. (Commercial detergents and alkaline soaps may interfere with chromophore groups of the dyes.) Wash in tap water; rinse in distilled water and differentiate in 1% acetic alcohol. Dehydrate in ascending alcohols, clear in xylene and mount permanently in a neutral, synthetic resin. Active osteoid seams stain dark to light green; resting osteoid seams, red to bright orange red; transitional osteoid seams, geenish-yellow, orange red to red; older, partly mineralized matrix, orange; new, partly mineralized matrix, red; osteocyte nuclei, red; osteoblasts and osteoclasts, greenish-blue to dark purple nuclei and green or light green cytoplasm. Hyper-trophic and differentiating cartilage cells are stained light pink and dark red respectively. The staining reactions are consistent; the solutions are stable.     

Rapid restriction mapping of DNA cloned in lambda phage vectors

A protocol for the rapid restriction mapping of phage λ clones has been developed. Partial digestion products are selectively labelled at the right or left cohesive λ DNA termini by hybridisation with [³²P]oligonucleotides complementary to the single-stranded cos ends. After gel electrophoresis and autoradiography, the restriction map can be directly determined from the “ladder” of partial digestion products.     

Circadian control of migratory restlessness and the effects of exogenous melatonin in the brambling, Fringilla montifringilla

Circadian pacemakers control both “daytime” activity and nocturnal restlessness of migratory birds, and the daily rhythm of melatonin release from the pineal has been suggested to be involved in the control of migratory activity. To study the phase relations between the two activity components during entrainment and when free running, locomotor activity of bramblings (Fringilla montifringilla) was recorded continuously under a 12:12 “cool light” to “warm light” cycle (CL:WL, ca. 5000 K and ca. 2500 K, respectively) or blue light to red light cycle (BL:RL, maxima at 440 and 650 nm, respectively) at different irradiance ratios. Migratory activity was expressed primarily during the WL or RL phase of the light cycles. Under free-running conditions, the circadian periods τ correlated with the phase relations between day and night (migratory) activity components during preceding entrainment. Bramblings with migratory activity had significantly longer τ at constant light intensity than the same individuals without migratory activity. Birds with migratory activity reentrained faster after a 6h phase shift of the CL:WL cycle than birds without migratory activity. When exogenous melatonin was given in the drinking water (200 μg/mL 1% ethanol or 0.86 mM) to bramblings exposed to 12:12 CL:WL cycles with constant irradiance, the amounts of activity, which were initially higher during the WL phase of the light cycle, were suppressed to similar low levels during both light phases. The systematic changes in the amounts of activity during melatonin treatment were not correlated with consistent changes in entrainment status. The data support the hypothesis that changes in the amplitude and level of the daily melatonin cycle are involved in regulating migratory restlessness, by either allowing or inhibiting nocturnal activity. (Chronobiology International, 17(4), 471-488, 2000)     

Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction

Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone is a network of numerous osteocytes, whose specific function has remained an enigma. Here we describe a transgenic mouse model in which inducible and specific ablation of osteocytes is achieved in vivo through targeted expression of diphtheria toxin (DT) receptor. Following a single injection of DT, approximately 70%–80% of the osteocytes, but apparently no osteoblasts, were killed. Osteocyte-ablated mice exhibited fragile bone with intracortical porosity and microfractures, osteoblastic dysfunction, and trabecular bone loss with microstructural deterioration and adipose tissue proliferation in the marrow space, all of which are hallmarks of the aging skeleton. Strikingly, these “osteocyte-less” mice were resistant to unloading-induced bone loss, providing evidence for the role of osteocytes in mechanotransduction. Thus, osteocytes represent an attractive target for the development of diagnostics and therapeutics for bone diseases, such as osteoporosis.     

Period responses to Zeitgeber signals stabilize circadian clocks during entrainment

Circadian clocks with characteristic period (τ) can be entrained to light/dark (LD) cycles by means of (i) phase shifts which are due to D/L “dawn” and/or L/D “dusk” transitions, (ii) period changes associated with long-term light exposure, or (iii) by combinations of the above possibilities. Based on stability analysis of a model circadian clock it was predicted that nocturnal burrowing mammals would benefit less from period responses than their diurnal counterparts. The model further predicted that maximal stability of circadian clock is reached when the clock slightly changes both its phase and period in response to light stimuli. Analyses of empirical phase response curve (PRC) and period response curve (τRC) of some diurnal and nocturnal mammals revealed that PRCs of both diurnal and nocturnal mammals have similar waveform while τRCs of nocturnal mammals are of smaller amplitude than those of diurnal mammals. The shape of the τRC also changes with age and with increasing strength of light stimuli. During erratic fluctuations in light intensity under different weather conditions, the stability of phase of entrainment of circadian clocks appears to be achieved by an interplay between phase and period responses and the strength of light stimuli.