Thick Plastic Sections Research Articles

Methylmethacrylate Thin Sections are Superior to Thick Ground MMA Sections for Histologic Evaluation of Complex Submillimeter Metallic Medical Devices

Hard plastic resin embedding is necessary for histology of tissues containing hard and/or metallic medical devices if the device is to be retained within the section for microscopic assessment. Traditional methylmethacrylate (MMA) embedding and sectioning of tissues containing hard and/or metallic devices requires targeted sectioning of the block with a diamond blade saw followed by careful grinding of the MMA‐embedded specimen with a calibrated grinder. This method provides a tissue section for histology that is approximately 50–60 μm in thickness (MMA thick section). However, the process of creating a MMA thick section is somewhat imprecise and results in considerable sample loss due to requisite attrition during MMA block sectioning and grinding procedures. For submillimeter (<1 mm) and/or similarly small, topographically complex medical devices, this level of relative imprecision, specimen attrition, and loss of cellular detail due to inherent thickness of tissue section can result in unacceptable diagnostic slide quality or even complete loss of a valuable specimen during sectioning/grinding procedures. To circumnavigate these challenges, we utilize MMA hard plastic resin embedding of small metallic medical devices followed by acquisition of thin (3–5 μm) MMA sections using a motorized microtome. Thin MMA sections provide superior cellular detail, greater precision of sectioning, and excellent retention of high quality device‐tissue context when compared with traditional MMA thick plastic sections. Although acquisition of MMA thin sections can be limited by larger device sizes and/or composition, MMA thin sections provide superior histologic quality for permissible specimens.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Practicable methods for histological section thickness measurement in quantitative stereological analyses

The accuracy of quantitative stereological analysis tools such as the (physical) disector method substantially depends on the precise determination of the thickness of the analyzed histological sections. One conventional method for measurement of histological section thickness is to re-embed the section of interest vertically to its original section plane. The section thickness is then measured in a subsequently prepared histological section of this orthogonally re-embedded sample. However, the orthogonal re-embedding (ORE) technique is quite work- and time-intensive and may produce inaccurate section thickness measurement values due to unintentional slightly oblique (non-orthogonal) positioning of the re-embedded sample-section. Here, an improved ORE method is presented, allowing for determination of the factual section plane angle of the re-embedded section, and correction of measured section thickness values for oblique (non-orthogonal) sectioning. For this, the analyzed section is mounted flat on a foil of known thickness (calibration foil) and both the section and the calibration foil are then vertically (re-)embedded. The section angle of the re-embedded section is then calculated from the deviation of the measured section thickness of the calibration foil and its factual thickness, using basic geometry. To find a practicable, fast, and accurate alternative to ORE, the suitability of spectral reflectance (SR) measurement for determination of plastic section thicknesses was evaluated. Using a commercially available optical reflectometer (F20, Filmetrics®, USA), the thicknesses of 0.5 μm thick semi-thin Epon (glycid ether)-sections and of 1–3 μm thick plastic sections (glycolmethacrylate/ methylmethacrylate, GMA/MMA), as regularly used in physical disector analyses, could precisely be measured within few seconds. Compared to the measured section thicknesses determined by ORE, SR measures displayed less than 1% deviation. Our results prove the applicability of SR to efficiently provide accurate section thickness measurements as a prerequisite for reliable estimates of dependent quantitative stereological parameters.

Pre and postganglionic innervation of rat adrenal gland by fluorescent tract tracer – Fast blue

IntroductionTo search for pre and postganglionic neurons innervating the adrenal gland by injecting retrograde tract tracer fast blue in the adrenal medulla. MethodsThe motor innervation of rat adrenal gland was studied by a fluorescent tract tracer fast blue. 5 μl of 2% aqueous suspension of fast blue was injected into left adrenal gland. After a survival period of 4–5 days, spinal cord, sympathetic ganglia, suprarenal ganglion, coeliac ganglion and left adrenal gland were dissected out and 15 μm thick plastic sections (JB4 Polysciences) were examined under a fluorescent microscope. ResultsRetrogradely labeled preganglionic neurons were observed in the ipsilateral intermediolateral column of spinal cord from T3 to L2 spinal segments with maximum concentration of labeled neurons from T6 to T11. The labeled neurons were multipolar, spherical or fusiform in shape with transverse diameter 10–20 μm and vertical diameter varying from 12 to 30 μm. Postganglionic labeled neurons were also observed in the left suprarenal ganglion and left sympathetic ganglia (T5 –L2) with maximum concentration from T6 to L1. Labeled neurons varied from 12 to 30 μm in diameter and were randomly distributed throughout the ganglion. DiscussionThe preganglionic neurons from T3 to L2 spinal segments and postganglionic nerve fibers from ipsilateral sympathetic ganglia (T5 –L2) and suprarenal ganglion supplying the adrenal gland might be responsible for the hormone release by regulating blood flow and also by directly innervating the parenchymal cells.

Effect of Storing Temperature on Hepatotoxicity and Nephrotoxicity of Ifosfamide in Female Rat

Problem statement: Ifosfamide (IFO) is a cytotoxic alkylating drug used for the treatment of a variety of cancers, although it has been found to induce certain hematological, hepatotoxic and nephrotoxic side effects. This drug is widely used in developing country, especially Iraq, but without caring too much to the storing temperature due to low drug maintaining facilities. The present work was achieved to investigate the effect of different temperature degrees on the toxicity of IFO on liver and kidney of female rats. Approach: Ifosfamide (60 mg Kg-1 b.wt) stored at different temperature degrees (4, 10 and 25°C) was given to female rats to detect the effect of temperature degrees on hepatotoxicity and nephrotoxicity of this chemotherapeutic drug. A modified technique was used for demonstration the dying kidney cells. Results: All IFO treated rats showed Fanconi syndrome such as increase of serum creatinine, phosphorus, ALT and AST and decrease of glucose as well as an increase in serum Malondialdehyde (MDA) level. Ifosfamide was still maintained its antimitotic action in all the treated groups as revealed by examining the bone marrow smears. The biochemical estimation of ALT, AST and MDA showed significant temperature dependent increase in the toxicity of liver and kidney by this drug. The histopathological alteration in these two organs were come supportive to the above biochemical results. The liver of the rats exposed to IFO stored at 25°C showed higher liver steatosis, hemorrhage and more degeneration of hepatocytes. Similarly, more hemorrhage, degeneration of kidney tubule cells and lymphocytes infiltration in kidney were observed in the 25°C group in comparison to the other groups. A successful modified technique for staining thick plastic sections was employed revealing a specific color for the dying kidney cells. Conclusion: Although it can maintain its antimitotic property, IFO caused more severe hepatotoxic and nephrotoxic side effects if it is stored at temperature degree higher than 4°C.

Use of different morphological techniques to analyze the cellular composition of the adult zebrafish optic tectum

Cellular composition of the adult zebrafish (Danio rerio) optic tectal cortex was examined in this study. Morphological techniques such as 1 μm thick serial plastic sections stained with osmium tetroxide and toluidine blue, modified rapid Golgi silver impregnation, GFAP immunohistochemistry, confocal microscopy, as well as scanning and transmission electron microscopy were used. Neuronal and glial components are described and the layers of the cortex are revisited. Specific neuronal arrangements as well as unique glial/ependymal cells are described. A three dimensional rendering of the astrocytic fiber arrangement in the marginal zone is presented and a composite drawing summarizes the cellular composition of the optic tectum.

Design-based estimation of neuronal number and individual neuronal volume in the rat hippocampus

Tools recently developed in stereology were employed for unbiased estimation of the neuronal number and volume in three major subdivisions of rat hippocampus (dentate granular, CA1 and CA3 pyramidal layers).The optical fractionator is used extensively in quantitative studies of the hippocampus; however, the classical optical fractionator design may be affected by tissue deformation in the z-axis of the section. In this study, we applied an improved optical fractionator design to estimate total number of neurons on 100μm thick vibratome sections that had been deformed, in the z-dimension, during histological processing.For estimating cell number, it is necessary to randomize only the location of section planes. But, in the local stereological methods, like cell volume estimation, the orientation of sections must also be randomized. We present a detailed application of a method for making vertical sections from the hippocampus. The volume of hippocampal neurons was estimated using the rotator principle on 40μm thick plastic vertical uniform random sections and corrected for tissue shrinkage. Application of the proposed new design should result in more accurate estimates of neuron number and neuronal volumes in tissue sections affected by homogenous non-uniform shrinkage

Dual-axis electron tomography: a new approach for investigating the spatial organization of wood cellulose microfibrils

We have employed dual-axis electron tomography to investigate the 3D organization of cellulose microfibrils in plastic resin-embedded, delignified cell walls of radiata pine early wood. The ∼ 1 nm thick tomographic slices produced in this study provided for a resolution of ∼ 2 nm in the cross-section of the slices throughout the 150 nm thick plastic sections. This resolution is sufficient to resolve individual cellulose microfibrils and to map the 3D organization of the cellulose microfibrils within the S2 layer of the secondary cell walls. The individual cellulose microfibrils measure ∼ 3.2 nm in diameter, and appear to consist of a ∼ 2.2 nm unstained core and a ∼ 0.5 nm thick surface layer that is lightly stained. Both individual and clustered cellulose microfibrils are seen surrounded by more heavily stained and irregularly shaped residual lignin and hemicellulose. The tightness of packing of the cellulose microfibrils in the cluster varies along the thickness of the section. These findings demonstrate that dual-axis electron tomography is a technique that can provide new insights into the 3D organization of cellulose microfibrils in plant cell walls.

Dynamics of noise-induced cellular injury and repair in the mouse cochlea.

To assess the dynamics of noise-induced tissue injury and repair, groups of CBA/CaJ mice were exposed to an octave-band noise for 2 hours at levels of 94, 100, 106, 112, or 116 dB SPL and evaluated at survival times of 0, 12, 24 hours or 1, 2, or 8 weeks. Functional change, assessed via auditory brainstem response (ABR), ranged from a reversible threshold shift (at 94 dB) to a profound permanent loss (at 116 dB). Light microscopic histopathology was assessed in serial thick plastic sections and involved quantitative evaluation of most major cell types within the cochlear duct, including hair cells (and their stereocilia), supporting cells, ganglion cells, spiral ligament fibrocytes, spiral limbus fibrocytes, and the stria vascularis. Morphometry allowed patterns of damage to be systematically assessed as functions of (1) cochlear location, (2) exposure level, and (3) postexposure survival. Insights into mechanisms of acute and chronic noise-induced cellular damage are discussed.

Stereological estimates of number and length of capillaries in subdivisions of the human hippocampal region.

The hippocampal formation is a neuroanatomically well-defined region of the brain involved in memory processes. In view of the functional importance of the region and its involvement in a number of brain pathologies, including Alzheimer's disease and temporal lobe epilepsy, a quantitative description of its vascular supply represents an important first step in evaluating the involvement of vascular changes in these phenomena. Unbiased estimates of the length and connectivity of the vascular supply of brain regions have not been described previously. The total number, total length, and distribution of the diameters of capillaries were estimated in the five major subdivisions of the hippocampal formation (fascia dentata, hilus, CA3-2, CA1, and subiculum) in 5 normal males, 52-84 years of age. These estimates were used to derive several other structural parameters. Both the primary and the derived parameters were used to make inter- and intra-individual comparisons. For each of the five major subdivisions from each individual, the volume was estimated using the Cavalieri principle. The total capillary length was estimated on 3-microm-thick plastic isotropic uniform random sections. Using a topological definition of a capillary unit and the optical disector, total capillary number was estimated in 40-microm-thick plastic sections. Length-and number-weighted three-dimensional diameter distributions were obtained from the thin and thick plastic sections, respectively. In each subdivision the total length of capillaries was correlated with previously obtained data on the number of neurons in the same subdivisions of the same individuals. Intersubdivisional differences were observed, in that the hilus of the dentate gyrus had fewer capillaries per unit volume than the other four subdivisions. Interindividual comparisons indicate that the interindividual variances are of a magnitude suitable for sensitive group comparisons. The design-based stereological methods that were used in the analyses can provide a basis for a new unbiased approach to the estimation of vascular parameters in well-defined regions of the brain.

Preventing Curling of Thick Plastic Sections

Preventing Curling of Thick Plastic Sections

Neuronal pathways of three neurosecretory cells from the lateral lobes in Helisoma (Mollusca): innervation of the dorsal body

The neurite distribution of three large neurosecretory cells, namely one canopy cell and two lateral-lobe cells from each lateral lobe of the cerebral ganglia in Helisoma duryi were studied by thick and thin plastic serial sections. These cells from only the right lateral lobe innervate the dorsal body. Neurites from the canopy cell innervate the cell-bodies whereas those from the lateral-lobe cells innervate the cell-processes of the dorsal body. Neurosecretory granules from these neurites are released at their sites of innervation. The neurites of the optic nerve form synapses with lateral-lobe cell 1, and synapse-like contacts with lateral-lobe cell 2 while a neurite of each canopy cell is found within its collateral optic nerve. Based on the anatomy of the lateral-lobe nerve cells and the optic nerve, it is argued that the stimulatory effect of long days on the dorsal body and on egg production is mediated through the lateral-lobe cells and the canopy cell.

A Comparison Between the Effects of Paraffin and Plastic Embedding of the Normal and Obstructed Minipig Detrusor Muscle Using the Optical Dissector

Purpose The purpose of this study was to estimate the relative shrinkage of the normal, obstructed and recovery minipig urinary bladder by comparing tissues from the same bladders embedded in paraffin or plastic. Materials and Methods Optical dissectors were used to make nucleus numerical density estimates in thick paraffin and plastic sections from the same bladder. The ratio of the 2 numerical densities depends only on differences in tissue shrinkage. In 9 minipigs a partial bladder outlet obstruction was created by implanting a 6 to 7 mm. Teflon ring around the proximal urethra. After an obstruction period (median 63 days) the ring was removed and after a recovery period (median 60 days) the animals were sacrificed. Biopsis were taken prior to obstruction, at removal of obstruction, and after recovery and were processed for paraffin and plastic sections to evaluate relative shrinkage. Two control pigs were sham-operated and biopsies taken at the same 3 time points. Results The optical dissector method was found to be an easy way to estimate the relative shrinkage of paraffin-embedded bladder tissue in proportion to plastic-embedded tissue. Both in human and minipig bladders, paraffin embedding caused a relative shrinkage of about 30 percent in proportion to plastic embedding. Both the obstructed and recovery detrusor muscles responded to embedding by either method in a manner indistinguishable from the normal bladder. Conclusion When dealing with stereological evaluation of the detrusor muscle, the relative shrinkage of the embedded normal, obstructed and recovery bladder tissue can be ignored.

Embryonic-maternal cell interactions at implantation in the fat-tailed dunnart, a dasyurid marsupial.

In marsupials implantation occurs about two-thirds the way through the short gestation before which time the embryo is surrounded by the permeable shell membrane which prevents physical contact between the trophoblast and uterine epithelium. Although the trophoblast has been shown to be invasive to varying degrees in several species of marsupials, the ultrastructure of the embryonic-uterine cell interactions at the time of implantation has not been described in this group. Thick plastic sections and transmission electron microscopy were employed to investigate the cellular interactions at implantation in the fat-tailed dunnart (Sminthopsis crassicaudata), a dasyurid Australian marsupial. Our results show that epithelial penetration begins when the embryo is at the late presomite/early somite stage. In the trilaminar region of the yolk sac (TYS), trophoblast cells adjacent to the embryo form desmosomes with uterine epithelial cells and also appear to fuse with them to form hybrid cells, the cytoplasm of which resembles that of trophoblast. Later in the TYS, as the placenta develops, trophoblast microvilli and larger cell processes invaginate, and interdigitate with, the highly folded maternal epithelium but do not invade it. At this time in the bilaminar, or avascular, yolk sac (BYS), multinucleate trophoblast giant cells (TGCs) from an annular region adjacent to the sinus terminalis intrude between, and possibly fuse with, the maternal epithelium. The invading TGCs spread laterally above the residual basal lamina before migrating into the stroma. In this species of marsupial at least, the cell interactions at the time of implantation are similar to those seen in some eutherian species despite the fact that the fetal chorion is of yolk sac rather than allantoic origin.

Estimate of the total number of neurons and glial and endothelial cells in the rat spinal cord by means of the optical disector.

The total numbers of neurons and glial and endothelial cells in five rat spinal cords were estimated by stereological techniques. Each spinal cord was divided into 12 slabs of equal length. One transverse and one oblique slice was cut from each slab. The volumes of gray and white matter of each cord were then estimated by point-counting techniques on the transverse slices. By means of optical disectors and systematic sampling, the numerical densities of different cell types were estimated on 35 microns-thick plastic sections from the oblique slices. The total cell number was calculated by multiplying the numerical density by the total volume of gray and white matter. On average there were 15.1 and 21.1 million cells in white and gray matter, respectively. Of the cells in gray matter, 6.4 million were judged to be neurons, 4.3 million to be endothelial, and 10.3 million to be glial. Of the neurons, 1.7 million were located in the cervical region, 2.5 million in the thoracic, 1.6 million in the lumbar, and 0.6 million in the sacro-coccygeal region. The methods used are simple to perform, and the counting necessary to obtain a reliable estimate of cell number from one spinal cord can be carried out during the course of 1 day. The only major problem is reliable criteria for unambiguous cell classification.

The sertoli cell membrane body in the skink

The structure of the Sertoli cell and its physical relationship with the germ cells was studied in laboratory maintained skinks, Eumeces laticeps (Schneider) in January, and September, corresponding to the periods of prenuptial and postnuptial spermatogenesis respectively. Light micrographs obtained using 1 μm thick plastic sections, show the Sertoli cell to have a large polymorphic nucleus located in the basal portion of the cell, and a darkly staining juxtanuclear body. In ultrathin sections, this body consists of a complex array of thin, electron dense membranous structures resembling the endoplasmic reticulum. The lumina of these membranous channels appear empty. Between the channels, there are structures that resemble the expanded cisternae of the endoplasmic reticulum. In some sections, these dilated cisternae are confluent with the channels, indicating that the channels and the cisternae are parts of the same structure. Three organelles, namely, mitochondria, lysosomes and microfilaments are found among the elements of the membrane body. There is no structural modification of the channels where they come in contact with mitochondria, but they are dilated in proximity to lysosomes. In some sections bundles of microfilaments are clearly visible within the diamond shaped region of contact between two channels, suggesting that these organelles are involved in structural or functional organization of the membrane body.

High-voltage electron microscopic studies of the interrelationship between microglial cells and amyloid fibrils in scrapie-infected mouse brains

We have previously demonstrated the usefulness of high-voltage electron microscopy (HVEM) in the study of microvessels and inflammatory cell attachment in the central nervous system (CNS). In the present study, we used HVEM to further explore the interrelationship between microglial cells (MCs) and amyloid deposits in scrapie-infected mice. Scrapie infection in the mouse has been employed as an animal model to study the pathogenesis of amyloid fibril formation. The central question was whether three-dimensional (3-D) stereo-pair reconstruction would offer further insight into amyloid formation by MCs, which is currently the view of our group. Brains or cervical spinal cords from IM mice previously inoculated with 87V scrapie agent were used. One-half-micrometer thick plastic sections were stained with uranyl acetate and lead citrate. Light-microscopy views enabled us to target primary inoculation channels associated with amyloid deposits. Cells located at the periphery of the amyloid were identified as MCs (Fig. 1).

The total number of neurons in the human neocortex unbiasedly estimated using optical disectors

An efficient method is presented for obtaining, in under 4 h, an unbiased estimate of the total number of neurons in the human neocortex, with a coefficient of error on the estimate of approximately 5%. The novel sampling scheme used in this study is unbiased and was designed so that only a small amount of neocortical grey matter had to be removed. Hence, the majority of the cerebral grey matter and all the internal grey matter was left intact for further resampling and analysis. Each cerebral hemisphere was subdivided into the four major neocortical regions, sliced coronally at 7-mm intervals and the volume of the neocortex determined using Cavalieri's principle. Uniform sampling of neocortex was performed in the hemisphere followed by regional subsampling with a varying sampling fraction being taken from each region. Neuronal density estimates were made in thick plastic sections using optical disectors. Shrinkage estimates were made in parallel with the number estimates and found to be negligible. The total number of neocortical neurons in the right hemisphere of five normal 80-year-old men was found to be 13.7 x 10(9) with an inter-individual coefficient of variation of 12%.

Morphometric analysis of experimental spinal cord injury in the cat: The relation of injury intensity to survival of myelinated axons

The pattern of axonal destruction and demyelination that occurs in experimental contusion injury of cat thoracic spinal cord was studied by line sampling of axons in 1 μm thick plastic sections with the light microscope. Injuries were produced by a weight-drop apparatus, with the vertebral body (T9) below the impact stabilized by supports under the transverse processes. The effects of two combinations of weight and height were examined: 10 or 13 g dropped 20cm onto an impact area of 5 mm diameter. Animals were kept for 3–5 months after injury, then fixed by perfusion for histological analysis. The number of surviving myelinated axons was found to vary both with the weight used and with the size of the spinal cord. A measure of impact intensity was derived from the calculated momentum of the weight at impact divided by the cross sectional area of the cord (interpolated from dimensions measured rostral and caudal of the lesion following fixation). At impact intensities > 0.02 kg-m/s/cm 2 there was practically no survival of axons at the center of the injury site, combined with almost complete breakdown of the pial margin. Between 0.08 and 0.2 kg-m/s/cm 2 the number of surviving axons varied between 100,000 and 2000, approximating a negative exponential function ( r = −0.88). The number of axons surviving in the outer 100 μm of the cord varied practically linearly ( r = −0.82) between near normal and < 1% of normal over the same range of injury intensity. The number of surviving axons decreased with depth from the pia, also approximating a negative exponential function, with a 10-fold decrease in density over approximately 500 μm. The average slope of this relation with depth remained similar over the range of injury intensity examined, though the slope appeared inversely related to variation in axonal survival for different individuals at a given intensity. It is argued that the loss of axons is probably determined primarily by mechanical stretch at the time of impact. Its centrifugal pattern may be explained by longitudinal displacement of the central contents of the cord, reflecting the viscoelastic “boundary layer” properties of parenchymal flow within the meningeal tube. This is illustrated with reference to the behavior of a gelatin model under compression. The preferential loss of large caliber axons and the characteristic shift to abnormally thin myelm sheaths (resulting from post-traumatic demyelination) both varied in extent independently of injury intensity and overall axonal survival. These effects are concluded to be the result of independent processes, triggered by traumatic injury, apparently delayed in time, but ultimately superimposed on the initial mechanical disruption.

Ultrastructure of the dorsal motor nucleus of the vagus nerve in monkey with a comparison of synaptology in monkey and cat.

Neurons of the dorsal motor nucleus of the vagus nerve were studied following injections of horseradish peroxidase into the vagus nerve in a monkey (Macaca fascicularis). In frozen sections, the dorsal motor nucleus appeared to be completely filled by labeled medium-sized (20-30 micron in long axis) neurons. Labeled dendrites from these neurons often extended outside the borders of the nucleus into the nucleus of the tractus solitarius. In 1 micron thick plastic sections and ultrathin sections of the dorsal motor nucleus, two distinct types of neurons were observed with the light and electron microscope. Medium-sized neurons with abundant cytoplasm and an oval nucleus were retrogradely labeled with HRP, while small (10-15 micron in long axis) neurons with a paucity of organelles and an invaginated nucleus remained unlabeled. Medium-sized neurons outnumbered the small neurons by approximately five to one. The synaptic organization of the dorsal motor nucleus in monkey was studied and compared with that in cat. The porportions of different types of axosomatic synapses were similar in both species. Terminals containing round vesicles and making symmetrical or asymmetrical contact with the postsynaptic structure were more common than synaptic terminals containing pleomorphic vesicles. In both species, there was a slightly greater synaptic density on the medium-sized neurons than on the small neurons. The synaptic density in the monkey dorsal nucleus was greatest on the smallest dendrites in the neuropil and least on the somata.

Glial proliferation in the irradiated rat spinal cord.

The identity of mitotic cells in the ventral half of the irradiated spinal cord in 13-day-old rats was studied by light and electron microscopy. At this post-irradiation interval, astrocytes as well as oligodendrocytes are markedly reduced in both gray and white matter, and few myelin sheaths are present. Earlier studies showed incorporation of 3H-thymidine into cells identified light-microscopically as neuroglia. In the present study, a number of mitotic cells were identified in thick plastic sections. When adjacent thin sections were examined by electron microscopy, these mitotic cells were identified ultrastructurally as astroglia on the basis of the bundles of filaments in their cytoplasm and the irregular outline of the cell body and its processes. It is apparent from this study that astroglia proliferate prior to the delayed myelination that occurs later in the glial cell deprived ventral irradiated cord.