Techniques to Render Dendritic Spines Visible in the Microscope.

A tiny detail visible on certain neurons at the limit of resolution in light microscopy went in 130 years of neuroscience research through a dazzling career from suspicious staining artifact to what we recognize today as a complex postsynaptic molecular machine: the dendritic spine.This chapter deals with techniques to make spines visible. The original technique, Golgi silver staining, is still being used today. Electron microscopy and automated field ion beam scanning electron microscopy are ultrahigh resolution techniques, albeit specialized. Other methods are intracellular injection, uptake of dyes, and recently the exploitation of genetically modified animals in which certain neurons express fluorescent protein in all their processes, including the nooks and crannies of their dendritic spines.

Pseudomonas species may appear strikingly filamentous in tissue sections: an important consideration for surgical pathologists and a reminder of the utility of modified silver impregnation methods.

Although tissue culture is the gold standard for diagnosing infection, histologic examination of surgically resected tissue can be a critical component in the diagnosis of tissue infection. The goal of this brief report is to alert surgical pathologists that Pseudomonas species can appear strikingly filamentous histologically and may somewhat mimic the appearance of filamentous bacteria, such Actinomyces or Nocardia, or thin fungal hyphae. A secondary aim is to raise awareness that Pseudomonas can sometimes only be identified histologically through the use of a modified silver impregnation method (Steiner stain). Five cases of filamentous Pseudomonas were encountered in three different surgical pathology subspecialities (ophthalmic pathology, cardiovascular pathology, and dermatopathology) over a 1-year period. All cases were of formalin-fixed, paraffin-embedded tissue, stained using hematoxylin & eosin (H&E) and multiple histochemical stains. Four cases grew Pseudomonas aeruginosa in culture and, in the fifth case, a nonaeruginosa species was detected using polymerase chain reaction-based methods. The markedly filamentous-appearing Pseudomonas organisms were identified in five different tissue sites: vascular graft, enucleation (whole eye) specimen, scleral biopsy, soft-tissue excision, and skin punch biopsy. In one of the five cases the organisms were seen on H&E, and in only two of the five were the organisms seen on Brown-Hopps stain. In all five cases, the organisms were identified on Steiner stain. It is therefore important to recognize that Pseudomonas can appear markedly filamentous, Pseudomonas or other bacterial infection is suspected, the surgical pathologist would be advised to employ the Steiner stain to most consistently detect the organisms.

Modified Silver Impregnation Method for Basal Membranes in Renal Biopsies.

Silver impregnation methods are essential in biopsy interpretation in nephropathology with regard to visualizing the basal lamina and its associated changes. The most widely used methods, mainly Jones methenamine impregnation, are time-consuming in their protocols and require multiple microscopy control points. In this report, we propose an alternative, modified method for silver impregnation with methenamine solution with a significantly shorter protocol time and good staining quality, allowing for proper interpretation of basal lamina changes in the glomeruli and blood vessels. Furthermore, unlike some other modified techniques, our proposed protocol does not include microwaving of the solutions but rather a thermostat is used, thereby reducing fire hazards. Implementing the protocol in our everyday practice has reduced sample processing time while not negatively impacting biopsy interpretation.

A combined use of silver pretreatment and impregnation with consequent Nissl staining for cortex and striatum architectonics study.

Despite a rapid growth in the application of modern techniques for visualization studies in life sciences, the classical methods of histological examination are yet to be outdated. Herein, we introduce a new approach that involves combining silver nitrate pretreatment and impregnation with consequent Nissl (cresyl violet) staining for cortex and striatum architectonics study on the same microscopy slide. The developed approach of hybrid staining provides a high-quality visualization of cellular and subcellular structures, including impregnated neurons (about 10%), Nissl-stained neurons (all the remaining ones), and astrocytes, as well as chromatophilic substances, nucleoli, and neuropil in paraffin sections. We provide a comparative study of the neuronal architectonics in both the motor cortex and striatum based on the differences in their tinctorial properties. In addition to a comparative study of the neuronal architectonics in both the motor cortex and striatum, the traditional methods to stain the cortex (motor and piriform) and the striatum are considered. The proposed staining approach compiles the routine conventional methods for thin sections, expanding avenues for more advanced examination of neurons, blood-brain barrier components, and fibers both under normal and pathological conditions. One of the main hallmarks of our method is the ability to detect changes in the number of glial cells. The results of astrocyte visualization in the motor cortex obtained by the developed technique agree well with the alternative studies by glial fibrillary acidic protein (GFAP) immunohistochemical reaction. The presented approach of combined staining has great potential in current histological practice, in particular for the evaluation of several neurological disorders in clinical, pre-clinical, or neurobiological animal studies.

A revisit to staining reagents for neuronal tissues.

In the early days of deciphering the injured neuronal tissues led to the realization that contrast is necessary to discern the parts of the recovering tissues from the damaged ones. Early attempts relied on available (and often naturally occurring) staining substances. Incidentally, the active ingredients of most of them were small molecules. With the advent of time, the knowledge of chemistry helped identify compounds and conditions for staining. The staining reagents were even found to enhance the visibility of the organelles. Silver impregnation identification of Golgi bodies was discovered in owl optic nerve. Staining reagents since the late 1800s were widely used across all disciplines and for nerve tissue and became a key contributor to advancement in nerve-related research. The use of these reagents provided insight into the organization of the neuronal tissues and helped distinguish nerve degeneration from regeneration. The neuronal staining reagents have played a fundamental role in the clinical research facilitating the identification of biological mechanisms underlying eye and neuropsychiatric diseases. We found a lack of systematic description of all staining reagents, whether they had been used historically or currently used. There is a lack of readily available information for optimal staining of different neuronal tissues for a given purpose. We present here a grouping of the reagents based on their target location: (I) the central nervous system (CNS), (II) the peripheral nervous system (PNS), or (III) both. The biochemical reactions of most of the staining reagents is based on acidic or basic pH and specific reaction partners such as organelle or biomolecules that exists within the given tissue type. We present here a summary of the chemical composition, optimal staining condition, use for given neuronal tissue and, where possible, historic usage. Several biomolecules such as lipids and metabolites lack specific antibodies. Despite being non-specific the reagents enhance contrast and provide corroboration about the microenvironment. In future, these reagents in combination with emerging techniques such as imaging mass spectrometry and kinetic histochemistry will validate or expand our understanding of localization of molecules within tissues or cells that are important for ophthalmology and vision science.

Easy to build cost-effective acute brain slice incubation system for parallel analysis of multiple treatment conditions.

BACKGROUND: Acute brain slices represent a powerful tool for analysis of brain function in physiology and pathology. Commercial systems and custom-build solutions with carbogen (95% O2/5% CO2) aeration, but they are expensive, have a high working volume requiring large amount of substances, and only limited options for treatment in parallel are possible. NEW METHOD: We developed a novel cost-effective incubation system using materials available in every laboratory, allowing parallel incubation of several treatment conditions, thus also reducing the number of experimental animals. Our system incubation parameters were optimized for cortical neuron observation. RESULTS: We tested several different options using 6, 12 or 24 standard culture well plates, combining them with cell strainer baskets inside. The system was placed in a pre-warmed incubator at 37 °C. Carbogen was injected through a 22 gauge needle, positioned between the basket and the wall of the well. Best results were achieved in a 6-well plate. In 12 and 24-well plates bubbles accumulated beneath the basket, displacing it upwards, making it unsuitable for our purposes. The gas oxygenized the medium without mechanically disturbing the slices, protected within the strainer basket, but still allowing optimal diffusion through the 100 µm pores. In a 6-well plate, six simultaneous treatments were possible in parallel. LDH/Cytotoxicity tests showed an acute toxicity of less than 7%. The system lost about 2.5% per hour of the fluid through evaporation, which was replenished every 2 h. Up to 6 h after treatment, however, this evaporation was excellently tolerated by the neurons even without fluid replenishment, most probably due to the anti-swelling effect of the mildly hypertonic medium. We performed two staining procedures, working excellently with this experimental setup, namely - a modified DiI staining and a slice silver impregnation method, both confirming the intact neuronal morphology. Preserved CA3 calcium influx and removal response following KCl depolarization confirmed the normal physiology of the pyramidal neurons 6 h after exposure in the system. COMPARISON TO EXISTING METHODS: The proposed system is much cheaper than the commercial solutions, can be constructed in any lab, allows up to 6 different treatments in parallel, which none of the existing systems allows. Antibiotic presence in the incubation medium and adequate evaporation control is required if longer incubation (> 6 h) is needed. Lower incubation volumes (3-6 ml) allow sparing expensive reagents. Our procedure was optimized for cortical neurons, further fine tuning to meet other specific requirements is possible. CONCLUSIONS: The system we propose allows filling the gap for budget solutions for short to mid-term incubation of acute brain slices.

How dynamic could be the 45S rDNA cistron? An intriguing variability in a grasshopper species revealed by integration of chromosomal and genomic data.

To better understand the structure and variability of the 45S rDNA cistron and its evolutionary dynamics in grasshoppers, we performed a detailed analysis combining classical and molecular cytogenetic data with whole-genome sequencing in Abracris flavolienata, which shows extraordinary variability in the chromosomal distribution for this element. We found astonishing variability in the number and size of rDNA clusters at intra- and inter-population levels. Interestingly, FISH using distinct parts of 45S rDNA cistron (18S rDNA, 28S rDNA, and ITS1) as probes revealed a distinct number of clusters, suggesting independent mobility and amplification of the 45S rDNA components. This hypothesis is consistent with the higher genomic coverage of almost the entire cistron of 45S rDNA observed in A. flavolineata compared to other grasshoppers, besides coverage variability along the 45S rDNA cistron in the species. In addition, these differences in coverage for distinct components of the 45S rDNA cistron indicate emergence of pseudogenes evidenced by existence of truncated sequences, demonstrating the rDNA dynamics in the species. Although the chromosomal distribution of 18S rDNA was highly variable, the chromosomes 1, 3, 6, and 9 harbored rDNA clusters in all individuals with the occurrence of NOR activity in pair 9, suggesting ancestry or selective pressures to prevent pseudogenization of rDNA sequences in this chromosome pair. Additionally, small NORs and cryptic rDNA loci were observed. Finally, there was no evidence of enrichment and association of transposable elements, at least, inside or nearby rDNA cistron. These findings broaden our knowledge of rDNA dynamics, revealing an independent movement and amplification of segments of 45S rDNA cistron, which in A. flavolineata could be attributed to ectopic recombination.

Gallyas Silver Impregnation of Myelinated Nerve Fibers.

In the nervous system of vertebrates, nerve impulse propagation is accelerated by the ensheathment of neuronal axons with myelin. Myelin sheaths are molecularly specialized, lipid-rich plasma membrane extensions of Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system (CNS). To visualize myelinated nerve fibers and to allow for the morphological analyses of myelin in the brain and the spinal cord, an efficient method for silver impregnation of myelin has originally been developed by Ferenc Gallyas in 1979, referred to as Gallyas silver impregnation. Gallyas' method is based on the agyrophilic characteristic of myelin to form and bind silver particles, while this process is suppressed in tissues other than myelin. The silver particles are finally enhanced in a developing step ("physical developer"). The main advantage of this method is that it efficiently visualizes both large myelinated fiber tracts and individual myelinated axons. Here we provide our laboratory protocol that is suitable for paraffin embedded sections and the use of light microscopy based on Gallyas' original protocol and subsequent modifications by Pistorio and colleagues.

Spinal Alterations of Reil Insula in Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that involves numerous cellular and biochemical mechanisms resulting in synaptic alterations and extensive neuronal loss. It is primarily characterized by impairment of memory, associated frequently with mood disorders. Continuous studies have shown that insula may be an important target of AD, but neuropathological alterations have not been described extensively. In the present study, we attempted to describe the morphometric and morphological changes of the spines of Reil insula in AD in comparison with normal aging using a silver impregnation technique. We classified spines into 3 types: (1) long neck, (2) short stubby, and (3) other types; and we measured and correlated the length of them in normal controls and in individuals with AD using ImageJ application. Statistical analysis was based on the Student t test on the basis of 360 cells in SPSS v.17.0, and significance was taken as P < .05.

A preclinical study testing "Focused multiple laser beams," a new concept of irradiation with the 1064-nm Nd:YAG laser for skin rejuvenation.

INTRODUCTION: In order to avoid epidermal heat damage, we developed a novel irradiation method termed "Focused multiple laser beams (FMLB)," which allows long-pulse neodymium:yttrium aluminum garnet (Nd:YAG) laser beams to be irradiated from several directions in a concentric fashion followed by focusing into the dermis without epidermal damage. This study aimed to assess whether FMLB achieves the desired dermal improvement without epidermal damage. MATERIALS AND METHODS: The dorsal skin of New Zealand White rabbits was irradiated with FMLB. Macroscopic and histological analyses were performed after 1 hour and 1, 2, 3 and 4 weeks. Real-time PCR analysis of type I and III collagen expression was performed at two and four weeks. RESULTS: Control groups exhibited skin ulcers which were healed with scar formation whereas FMLB groups remained intact macroscopically. Histologically, FMLB group showed increase in dermal thickness at four weeks while the epidermis remained intact. Real-time PCR demonstrated that both type I and III collagen increased at two weeks but decreased at four weeks. CONCLUSIONS: FMLB can deliver the target laser energy to the dermis without significantly affecting the epidermis.

Development of the human oculomotor nuclear complex: somatic nuclei.

BACKGROUND: Precise anatomical data on the development of human oculomotor somatic nuclei (OSN) remain rare. DESIGN/SUBJECTS: This study describes the histology of human OSN in 11 preterm and full-term infants aged 20-43 postmenstrual weeks who died of various causes. Celloidin-embedded serial sections were stained with the Klüver-Barrera and other conventional methods including silver impregnation. To evaluate the growth of OSN quantitatively, the author estimated the nuclear volume and the average neuronal area on morphometry. RESULTS: Four subnuclei were identified at 20-21 weeks: the fascicular, principal, dorsal median, and ventral median nucleus. Early tigroid Nissl bodies appeared in presumed motoneurons by 27-28 weeks, then resembled adult Nissl bodies at birth. On silver impregnation, the oculomotor nerve roots, crossed or uncrossed fibers at the midline, and a plexus of efferent or afferent axons in the neuropil were observed at 20-21 weeks. Then, the plexus was elaborated to form a perineuronal net of thin axon terminals by 28-29 weeks. The nuclear volume of OSN exponentially increased with age over 20-43 weeks, while the average of neuronal profile areas linearly increased in each subnucleus; the coefficient of regression was largest in the principal nucleus, and the regression lines nearly overlapped among the other subnuclei. Statistical analysis confirmed that the average neuronal area was largest in the principal nucleus in older cases. CONCLUSION: This study suggests that four subnuclei can be distinguished in human OSN by mid gestation, and that the principal nucleus may be different in neuronal cytoarchitecture from the others.

Adaptación de la técnica de impregnación argéntica de Llombart para la demostración de fibras nerviosas, en cualquier tejido en cortes por parafina / Adaptation of Llombart's silver impregnation technique for demonstration of nerve fibers in any tissue in paraffin sections

En la investigación biológica sigue siendo necesaria la demostración de la inervación periférica en numerosos tejidos y órganos. El objetivo de este trabajo fue rescatar y modernizar uno de los métodos más constantes que hemos probado para demostrar la inervación periférica. La técnica de Llombart para fibras nerviosas se adaptó en cortes por parafina de 7 µm en diferentes tejidos animales. La impregnación argéntica se hizo por goteo en cámara húmeda. Se demostraron en forma constante, precisa y seriada terminaciones nerviosas y corpúsculos sensoriales, neuronas y fibras nerviosas periféricas. A pesar de la alta especificidad para fibras nerviosas, la técnica no compromete el panorama tisular por lo que da bellas imágenes de conjunto. Sin ser una técnica para argentafinidad, demuestra claramente dos tipos de células argentafines en las glándulas adrenales. La adición de los reactivos metálicos en gotas y en cámara húmeda, ofrece una variante sumamente económica.

In Biological research is still necessary for the demonstration of the peripheral innervation in numerous tissues and organs. The aim of this study was to rescue and modernize one of the most consistent methods that we have tried to demonstrate peripheral innervation. Llombart's technique for nerve fibers was adapted by paraffin cuts of 7 µm in different animal tissue. The silver impregnation was done by dripping in a moist chamber. It was demonstrated in a constant, precise and serial form, nerve terminations, and sensorial corpuscles, neurons, and peripheral nerve fibers. Despite being highly specific to nerve fibers, the technique does not sacrifice tissue panorama so it gives beautiful images set. Without being a technique to argentaffin structures, it clearly shows two types of argentaffin cells in the adrenal glands. The addition of the metal reactive in droplets and in a humid chamber provides a very economical variant.

Morphologic and molecular description of Metopus fuscus Kahl from North America and new rDNA sequences from seven metopids (Armophorea, Metopidae).

Most species in the large ciliate genus Metopus Claparède & Lachmann, 1858 lack detailed descriptions based on modern morphologic and molecular methods. This lack of data for the vast majority of species hampers application of a morphospecies approach to the taxonomy of Metopus and other armophorids. In this report we redescribe the large species, Metopus fuscusKahl, 1927 based on in vivo observation, silver impregnation, scanning electron microscopy, and single-cell 18S rDNA sequencing of a freshwater North American (Idaho) population. Metopus fuscus invariably has a perinuclear envelope of endosymbiotic bacteria not found in other species. Unlike the original description of a single row of coarse granules between ciliary rows, the Idaho population has five loose rows of small interkinetal granules. We discuss the possible importance of this character in metopids. We also provide a phylogenetic analysis including seven other new metopid 18S rDNA sequences: Brachonella spiralis, B. galeata, Metopus laminarius, M. setosus, M. striatus, M. violaceus, Palmarella lata. Metopus fuscus and M. setosus form a fully supported clade, challenging previous morphospecies groupings. We discuss some ambiguities of armophorid morphologic terminology in the earlier literature. Our phylogenetic analysis of Idaho metopids indicates that the genera Metopus and Brachonella are both nonmonophyletic.

Dendritic, axonal, and spinal pathology of the Purkinje cells and the neurons of the dentate nucleus after long-term phenytoin administration: a case report.

Phenytoin is a commonly prescribed anticonvulsant drug; however, there is evidence that long-term administration is related to cerebellar ataxia, cerebellar atrophy, loss of Purkinje cells, and hyperplasia of Bergman glia cells. The aim of the present study was to detect and describe any possible alterations of the Purkinje cells, and neurons of the dentate nucleus, as those can be seen with the use of silver impregnation techniques, such as Golgi and Nauta method. The study was performed on a 7-year-old boy who was under phenytoin treatment for more than 3.5 years and had clinical manifestations of cerebellar ataxia. Golgi silver impregnation technique revealed substantial loss of dendritic spines and tertiary dendritic branches, both on the Purkinje cells and the neurons of the dentate nucleus, whereas the Nauta method demonstrated swollen and degenerated axons of Purkinje cells.

Nerve endings of filliform, fungiform and vallate papillae of dorsal tongue mucosa of White-lipped peccary (Tayassu pecari): Neurohistological observations / Terminação nervosa das papilas filiformes, fungiformes e valadas da mucosa dorsal da língua de queixada: observação neurohistológica

The neurohistologic observations were performed using the specimens prepared by Winkelmann and Schmitt silver impregnation method. The tissues were fixed in 10 percent formalin solution and sections of 40µm thickness were obtained by Leica Cryostat at -30ºC. The sections of dorsal mucosa of White-lipped peccary tongue showed numerous filliform and fungiform papillae, and two vallate papillae on the caudal part. The epithelial layer revealed queratinized epithelial cells and the connective tissue papillae of different sizes and shapes. Thick nerve fiber bundles are noted into the subepithelial connective tissue of the papillae. The connective tissue of fungiform and vallate papillae contained numerous sensitive nerves fibers bundles forming a complex nerve plexus.

As observações neuro-histológicas foram realizadas utilizando amostras preparadas segundo o método de impregnação por prata de Winkelmann e Schmitt. Os tecidos foram fixados em solução de formol a 10 por cento e seções de 40µm de espessura foram obtidas em criostato Leica -30ºC. As seções da mucosa dorsal da língua de queixada revelaram numerosas papilas filiformes, fungiformes e duas papilas valadas sobre a parte caudal. A camada epitelial revelou células epiteliais queratinizadas e papilas de tecido conjuntivo de diferentes tamanhos e formas foram observadas. Espessos feixes de fibras nervosas são notados no tecido conjuntivo subepitelial das papilas. O tecido conjuntivo das papilas fungiformes e valadas contêm numerosos feixes de nervos de fibras sensíveis formando um plexo nervoso complexo.

RESEARCH ON THE QUANTITATIVE ANALYSIS OF THE HIPPOCAMPUS—FORNIX—MAMILLOTHALAMIC SYSTEMS AND THE SPATIAL SEQUENTIAL STRUCTURE IN THE HIPPOCAMPUS OF THE RABBIT / 解剖学报

Nine adult rabbits, perfused with 10% formalin, were used in this experiment. Five of them were mounted on the stereotaxic apparatus according to the Sawyer's atlas. Brains were removed and made serial sections including thalamus and hippocampus. The neocortices of the other four rabbits were removed and the hippocampal formations were exposed. Serial sections of the hippocampus were made and stained alternately with Nissl's method and the silver impregnation method of Glees. The results could be summarized as follows.1. The hippocampus measured 28.00-36.00 mm in length, 6.00-8.50 mm in breadth and 2.68-3.39 mm in thickness. The total numbers of the pyramidal cells (CA_1-CA_4), the granular cells in the dentate gyrus and the basket cells were 7.36?10~6, 4.93?10~6, 2.40?10~5 respectively. The ratio of the total numbers among the pyramidal, granular and the basket cells was 30:20:1 approximately. The total number of the CA_1 pyramidal neurones was 4.11?10~6, and that of the CA_3 was 2.48?10~6. The ratio of the numbers of the CA_1 and CA_3 neurones was 1.66:1.2. The arrangements of the granular cells in the dentate gyrus and the apical dentrites of the CA_1 neurones were rather regular and possessed a typical array-like structure.3. The total numbers of the fibers in the column fornix (FN)and the mamillothalamic tract (MT) were 83700 and 58740 respectively. MT:FN=1:1.42. The total number of the neurones in the mamillary nucleus was 113500, and that of the anterior thalamic nucleus was 165600. The ratio of the neurones between these two nuclei was 1:1.46.According to the results mentioned above, the functional significance of the characteristic structure of the hippocampus was also discussed.