Gelatin Embedding of Central Nervous System Tissue Improves the Quality of Vibratome Sections

We have developed a procedure for Vibratome (Oxford Laboratories) sections that is particularly valuable for providing uniformly thick, well preserved CNS tissue sections for morphometric applications.     

Embedding of Neural Tissue in Agarose or Glyoxyl Agarose for Vibratome Sectioning

Agarose was used to embed the brain or spinal cord of lampreys or rats before cutting vibratome sections. Agarose embedding was compatible with immunocytochemistry or the use of horseradish peroxidase as a neuroanatomical tracer. Concentrated agarose with high intrinsic gel strength was optimal for embedding glutaraldehyde fixed neural tissue. A quick procedure was to blot tissue and embed in 5% (w/v] Sigma type I-A or Litex type LSL agarose at 45-55 C dissolved in 50 mM neutral-pH TFUS buffer before cutting 50-100 μm vibratome sections. An alternative procedure that improved retention of tissue sections in the agarose was to rinse the tissue in H₂0, blot and embed in 5% (w/v] Sigma type I-A or Litex type LSL agarose at 45-55 C dissolved in H₂0, then equilibrate the block overnight in buffer. Phosphate buffer prevented complete dissolving of agarose. Tissue could be covalently linked to the embedding matrix using a novel aldehyde-derived agarose (NuFix® FMC BioProducts). Slices of spinal cord from neonatal rats could be cut after embedding in 5% FMC Seaprep® agarose in rat Ringer's at 23-26 C.     

A Simple Screening Procedure for Evaluating Central Nervous System Tissue Sections Showing Structural and Cytochemical Alterations of the Blood-Brain Barrier

A simple method for rapidly screening and evaluating many areas of central nervous system tissue before and after flat embedding in Beem capsules is described. This method uses light microscopy to select regions surrounding needle track injuries of brain tissue for subsequent fine structural and enzyme cytochemical analysis of the blood-brain barrier. The mouse cerebral cortex was sectioned with a tissue chopper at 40-50 μm and reacted with diaminobenzidine to demonstrate the presence of exogenous horseradish peroxidase near an injured central nervous system site. Following the enzyme reaction, both osmicated and unosmicated tissue slices were processed for routine electron microscopy, infiltrated with unpolymerized resin, and evaluated on glass slides by light microscopy prior to flat embedding and polymerization. Numerous tissue specimens can be screened in this way for maximum information per tissue slice, and extra tissue samples can be polymerized on the glass slides and conveniently stored for future sectioning.     

Plastic Embedding of thick Celloidin Sections of nerve Tissue

A method is described for mounting Golgi-impregnated and Weigert-stained thick celloidin sections of brain and spinal cord in transparent plastic. Finished mounts have good optical properties and are suitable for macroscopic and microscopic observation. The durability of such preparations makes them superior to similar material prepared by the more conventional methods. Holes of suitable size were cut in matrices of 2.5 × 5 × 3/16 inches Plexiglas. Ward's Bio-plastic was used to form a base for the holes and also as the embedding medium for the sections. Plate glass formed a working substrate and gave a polished surface to the plastic base and later to the top of the preparation. For Golgi material (200μ) the celloidin was removed by dioxane. A dioxane-plastic bath preceded plastic embedding. For Weigert material (30-40μ) celloidin was not removed due to fragility of sections. Prior to plastic embedding, they were subjected first to benzol and then to a benzol-plastic bath.     

Tissue Bath Improvements for the Oxford Vibratome

Modification of the tissue bath on the Oxford Vibratome Sectioning System facilitates simpler and more efficient operation. the basic modification consists of the introduction of a small, approximately 25 ml capacity tissue bath into the larger bath provided by the instrument's design. Temperature in the modified system is controlled by using the instrument's large bath as an ice chamber. This modification allows better temperature control, easier retried of tissue sections, and more efficient use of expensive buffers, reduces the chance of section carry-over from one specimen to another, and requires less cleanup time. When it is advantageous to have the tissue in a large volume of solution, more efficient section retrieval and solution use can still be obtained by substituting a Wire mesh collection basket for the small tissue bath, while using the standard bath in the usual way.     

Tissue Profiling of the Mammalian Central Nervous System Using Human Antibody-based Proteomics

A need exists for mapping the protein profiles in the human brain both during normal and disease conditions. Here we studied 800 antibodies generated toward human proteins as part of a Human Protein Atlas program and investigated their suitability for detailed analysis of various levels of a rat brain using immuno-based methods. In this way, the parallel, rather limited analysis of the human brain, restricted to four brain areas (cerebellum, cerebral cortex, hippocampus, and lateral subventricular zone), could be extended in the rat model to 25 selected areas of the brain. Approximately 100 antibodies (12%) revealed a distinct staining pattern and passed validation of specificity using Western blot analysis. These antibodies were applied to coronal sections of the rat brain at 0.7-mm intervals covering the entire brain. We have now produced detailed protein distribution profiles for these antibodies and acquired over 640 images that form the basis of a publicly available portal of an antibody-based Rodent Brain Protein Atlas database (www.proteinatlas.org/rodentbrain). Because of the systematic selection of target genes, the majority of antibodies included in this database are generated against proteins that have not been studied in the brain before. Furthermore optimized tissue processing and colchicine treatment allow a high quality, more extended annotation and detailed analysis of subcellular distributions and protein dynamics.The brain is the most complex organ in the mammalian body. It processes sensory information from our external environment; produces behavior, emotions, and memories; and regulates the internal body homeostasis. To fulfill these diverse functions the brain harbors a myriad of neuronal networks processing information and connecting input and output systems. Because of the highly specialized functions, each neuron population is neurochemically specified expressing the necessary sets of proteins. Consequently a large number of genes are expressed in the mammalian brain. Based on microarray and in situ hybridization studies it is estimated that ∼55–80% of all mouse genes are expressed in the brain (1, 2) (gene expression during developmental stages and pathological conditions not included). Interestingly 70% of these genes are expressed in different cell populations each covering less than 20% of the brain, indicating the complexity of the brain and the specialization of individual populations of neurons (1).The success of humans as a species relies on our mental abilities, a result of brain development during evolution. The human brain is distinguished from other mammalian brains by its size; especially the neocortex involved in higher cognitive functions is greatly enlarged in humans. Despite this difference, the human brain has many similarities to brains of other mammalian species, and to some extent mammalian brains have a well preserved basic architecture (basic uniformity) (for reviews, see Refs. 3 and 4). Therefore, most human brain nuclei and connections have orthologs in other mammalian species ranging from great apes to rodents.Genetic variation underpins interspecies variation in gene expression and assembly of proteins. The human and rat genomes encode similar numbers of genes of which the majority have persisted throughout evolution without deletion or duplication (5). It is evident that small changes in protein structure and altered expression levels of proteins influence brain development and form the basis of interspecies differences. However, most human genes have orthologs in rodents, and for most cell types in the brain their neurochemical specification has been preserved throughout evolution. Because of genomic homology and similarity in basic layout of the mammalian brain as well as the preservation of neurochemical specification of subsets of neurons throughout evolution, animal models have shown their value in medical neurosciences (6).Advances in science are largely dependent on the processing of available information and the generation of new concepts and are driven by innovation and availability of new technologies. Recently mRNA-based techniques have emerged as an effective tool for genome wide analysis of expression levels in entire organs or disease-affected tissue. Results obtained from these studies are a source for identification of novel key molecules and have a predictive value to estimate changes in protein synthesis. There are several ongoing initiatives focusing on the expression profiles of the mammalian brain. The Allen Brain Atlas has produced detailed in situ hybridization profiles for over 20,000 genes in the mouse brain (1). The Gene Expression Nervous System Atlas (GENSAT) project uses enhanced green fluorescent protein reporter genes incorporated into bacterial artificial chromosome transgenic mice to visualize the expression profiles of the most important genes (7). This strategy can result in the identification of expressing cell types as the detailed morphology of enhanced green fluorescent protein-expressing cells is apparent. The Brain Maps project has a large collection of mammalian and non-mammalian brain maps using “classical” histochemical techniques but also includes a few protein distribution profiles visualized using immunohistochemistry (8).We previously described the possibilities of using antibodies raised against human proteins on rodent brain tissue (9). Here we show the first efforts to produce detailed proteome wide large scale tissue profiling maps of a mammalian brain using an antibody-based proteomics approach. In addition to the available, mentioned information on mRNA levels (Allen Brain Atlas), gene expression profiles (Gene Expression Nervous System Atlas), and detailed neuroanatomy (Brain Maps), antibody-based proteomics provide new information on cellular and subcellular distribution of gene products. This information will increase general knowledge and understanding of the organization and functioning of the brain. The study is based on antibodies generated as part of the Human Protein Atlas program aimed at exploring the protein expression patterns in normal and cancer tissues using tissue microarray-based immunohistochemistry and fluorescence-based confocal microscopy (10).The Human Proteome Resource center aims to produce monospecific antibodies against every human gene. So far, the distribution profiles of 3,000 proteins in 48 human tissues, including four brain areas (cerebellum, cerebral cortex, the hippocampal formation, and lateral subventricular zone), and 20 cancers are available (Human Protein Atlas). The antibodies generated within the framework of this program are based on antigens selected as unique regions for each individual protein, called protein epitope signature tags (PrESTs)¹ (11, 12). Over 5,000 antibodies have been generated and validated using Western blot analysis and protein arrays (13). The smaller size of the rat brain allows analysis of many brain areas and exposure of the antibodies to a very wide variety of proteins. Furthermore tissue can be processed under perfect conditions optimizing tissue antigenicity with flawless tissue morphology.Here we describe the initial large scale mapping of 89 protein distribution profiles in 25 selected rat brain areas. By exposing systematically sampled rat brain tissue to our collection of monospecific antibodies a more detailed protein atlas of the mammalian brain was produced, expanding the four brain areas available in the human protein atlas to 25 brain areas (Fig. 1) involved in higher cognitive functions, sensation, emotion, maintenance of internal homeostasis, sleep, and motor and sexual behaviors. A database portal has been created to show selected images from the various regions of the brain.Open in a separate windowFig. 1.Schematic overview of the 25 selected brain areas. Included are telencephalon (medial septum, lateral septum, horizontal/vertical diagonal band, prefrontal/cingulate/somatosensory/piriform/entorhinal cortex, ventral pallidum, stria terminalis, globus pallidus, caudate putamen, amygdala (basolateral, central, and medial), hippocampus, and dentate gyrus); diencephalon (preoptic area (A), supraoptic nucleus (A), suprachiasmatic nucleus (A), paraventricular nucleus (A and B), arcuate nucleus (B), median eminence (B), and thalamus); mesencephalon (substantia nigra, ventral tegmental area, and raphe nucleus (dorsal and median)); pons (locus caeruleus (C)); and cerebellum.     

Estimation of Thy-1 in Cryostat Sections of Nervous Tissue

The conventional assay for measuring cell surface antigens--the quantitative absorption of antibody by tissue homogenates--proved inadequate when used to determine the level of Thy-1 glycoprotein in rat nerves and peripheral ganglia. In this paper we report that the binding of 125I-labelled Fab fragments of a monoclonal anti-Thy-1 antibody to cryostat sections is sufficiently sensitive to give consistent estimates of the Thy-1 level on single samples of even small nerves. Observed levels of Thy-1 were generally higher than had previously been thought, and in particular we found no nerves totally lacking the antigen. The lowest levels (6-10 pmol/mg protein) were in peripheral nerves with a large motor component. Autonomic and sensory nerves had higher levels (15-20 pmol/mg protein). The highest levels were on the optic nerve (34 pmol/mg protein), superior cervical sympathetic ganglion (40 pmol/mg protein), and the cerebellar vermis (46 pmoles/mg protein; the only brain region examined in this study). From a practical point of view, the cryostat assay has the advantage that measurements of Thy-1 can be done on sections from the same series as is used for immunohistochemical localisation.     

Staining Tissue of the Central Nervous System with Luxol Fast Blue and Neutral Red

The tissue is fixed in 10% neutral saline formalin for 1 day to 3 wk depending on the size of the block, dehydrated and embedded in paraffin. The sections are stained at 57° C for 2 hr, then at 22° C for 30 min, in a 0.0125% solution of Luxol fast blue in 95% alcohol acidified by 0.1% acetic acid. They are differentiated in a solution consisting of: Li₂CO₃, 5.0 gm; LiOH-H₂O, 0.01 gm; and distilled water, 1 liter at 0-1° C, followed by 70% alcohol, and then treated with 0.2% NaHSO₃. They are soaked 1 min in an acetic acid-sodium acetate buffer 0.1 N, pH 5.6, then stained with 0.03% buffered aqueous neutral red. Sections are washed in distilled water, 1 sec, then treated with the following solution: CuSO₄·5H₂O, 0.5 gm; CrK(SO₄)₂·12H₂O, 0.5 gm; 10% acetic acid, 3 ml; and distilled water, 250 ml. Dehydration, clearing and covering complete the process. Myelin sheaths are stained bright blue; meninges and the adventitia of blood vessels are blue; red blood cells are green. Nissl material is stained brilliant red; axon hillocks, axis cylinders, ependyma, nuclei and some cytoplasm of neuroglia, media and endothelium of blood vessels are pink.     

Carbowax Embedding for Obtaining Thin Tissue Sections and Study of Intracellular Lipids

Carbowax, a water soluble wax, as an embedding agent is a valuable adjunct to the armamentarium of the tissue technologist. This report is intended to supplement previous publications on the use of Carbowax and to indicate die necessity for preheating and variation of Carbowax mixtures according to the climate.

Carbowax embedding provides an easy means for obtaining tissue sections 1 to 3 μ in thickness either with or without previous exposure to fat solvents. These sections are admirably suited for cytological study, particularly of intracellular lipoids.     

TWEAK and the Central Nervous System

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily that acts on responsive cells via binding to a cell surface receptor named fibroblast growth factor-inducible 14 (Fn14). TWEAK can regulate numerous cellular responses in vitro and in vivo. Recent studies have indicated that TWEAK and Fn14 are expressed in the central nervous system (CNS), and that in response to a variety of stimuli, including cerebral ischemia, there is an increase in TWEAK and Fn14 expression in perivascular astrocytes, microglia, endothelial cells, and neurons with subsequent increase in the permeability of the blood–brain barrier (BBB) and cell death. Furthermore, there is a growing body of evidence indicating that TWEAK induces the activation of the NF-κB in the CNS with release of proinflammatory cytokines and matrix metalloproteinases. In addition, inhibition of TWEAK activity by either treatment with a Fn14-Fc fusion protein or neutralizing anti-TWEAK antibodies has shown therapeutic efficacy in animal models of ischemic stroke, cerebral edema, and multiple sclerosis.     

Vimentin in the Central Nervous System

Intermediate filament proteins were identified by two-dimensional gel electrophoresis in urea extracts of rat optic nerves undergoing Wallerian degeneration and in cytoskeletal preparations of rat brain and spinal cord during postnatal development. The glial fibrillary acidic (GFA) protein and vimentin were the major optic nerve proteins following Wallerian degeneration. Vimentin was a major cytoskeletal component of newborn central nervous system (CNS) and then progressively decreased until it became barely identifiable in mature brain and spinal cord. The decrease of vimentin occurred concomitantly with an increase in GFA protein. A protein with the apparent molecular weight of 61,000 and isoelectric point of 5.6 was identified in both cytoskeletal preparations of brain and spinal cord, and in urea extracts of normal optic nerves. The protein disappeared together with the polypeptides forming the neurofilament triplet in degenerated optic nerves.     

Central Nervous System Manifestations of Chickenpox

A study of 57 cases of affection of the central nervous system associated with chickenpox diagnosed and treated at The Hospital for Sick Children in Toronto between 1956 and 1967, inclusive, is presented. The commonest type, the cerebellar variety (50%), had an excellent prognosis. In the next commonest, the cerebral type (40%), the mortality rate was 35% but there was a low incidence of permanent sequelae in the surviving patients. A small group classed as aseptic meningitis was defined and one case of myelitis was reviewed.     

Riboflavin Homeostasis in the Central Nervous System

Abstract: The mechanisms by which riboflavin, which is not synthesized in mammals, enters and leaves brain, CSF, and choroid plexus were investigated by injecting [¹⁴C]riboflavin intravenously or intraventricularly. Tracer amounts of [¹⁴C]riboflavin with or without FMN were infused intravenously at a constant rate into normal, starved, or probenecid-pretreated rabbits. At 3 h, [¹⁴C]riboflavin readily entered choroid plexus and brain, and, to a much lesser extent, CSF. Over 85% of the [¹⁴C]riboflavin in brain and choroid plexus was present as [¹⁴C]FMN and [¹⁴C]FAD. The addition of 0.2 mmol/kg FMN to the infusate markedly depressed the relative entry of [¹⁴C]riboflavin into brain, choroid plexus, and, less so, CSF, whereas starvation increased the relative entry of [¹⁴C]riboflavin into brain and choroid plexus. After intraventricular injection (2 h), most of the [¹⁴C]riboflavin was extremely rapidly cleared from CSF into blood. Some of the [¹⁴C]riboflavin entered brain, where over 85% of the ¹⁴C was present as [¹⁴C]FMN plus [¹⁴C]FAD. The addition of 1.2³μmol FAD (which was rapidly hydrolyzed to riboflavin) to the injectate decreased the clearance of [¹⁴C]riboflavin from CSF and the phosphorylation of [¹⁴C]riboflavin in brain. Probenecid in the injectate also decreased the clearance of [¹⁴C]riboflavin from CSF. These results show that the control of entry and exit of riboflavin is the mechanism, at least in part, by which total riboflavin levels in brain cells and CSF are regulated. Penetration of riboflavin through the blood-brain barrier, saturable efflux of riboflavin from CSF, and saturable entry of riboflavin into brain cells are three distinct parts of the homeostatic system for total riboflavin in the central nervous system.