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Staining Sections of the Central Nervous System
John A. Kiernan, MB, ChB, PhD, DSc Professor
Department of Anatomy and Cell Biology The University of Western Ontario London, Canada N6A 5C1
S
ections well stained with hemalum and eosin (H&E) display the cellular and microanatomical features of most normal and diseased organs. The method is valued especially by pathologists (1), who examine great numbers of slides and appreciate the simple rendition in two colors: a bold blue largely confined to nuclear chromatin, but insipid shades of pink for nearly everything else (2). H&E-stained sections of pieces of normal central nervous system (CNS) provide hardly any information about the cytoplasm of neurons and glial cells, though it is possible to recognize several cell-types on the basis of their stained nuclei. Special staining methods are needed to characterize different types of neurons and to reveal such elements as dendrites, axons, myelin sheaths and the cytoplasmic processes of glial cells(3, 4) (Fig. 1). Some of the traditional techniques, especially for glia, have largely been replaced by immunohistochemistry, but there is still a need for dye-based staining of neuronal cell bodies and myelin sheaths in neuropathology, research in neuroscience and the preparation of teaching materials for human and animal neuroanatomy.
Nissl Staining
H&E shows the shapes and sizes of the cell bodies of neurons but little detail is visible in the perikaryon − the cytoplasm surrounding the nucleus. (Most of the cytoplasm of a neuron is in the dendrites and axon.) In 1884 Franz Nissl (1860-1919), then a medical student in
Nucleus
Internode Node of Ranvier
Myelin Sheath Axon
Synaptic terminal Axon Hillock Preterminal branch Perikaryon Dendrites
Legend : Diagram of a generalized neuron with a myelinated axon. The term “nerve fiber” means an axon together with its myelin sheath. Synaptic terminals may contact the dendrites, penkarya or preterminal axonal branches of other neurons.
Figure 1. Diagram of a generalized neuron with a myelinated axon. The term “nerve fiber” means an axon together with its myelin sheath. Synaptic terminals may contact the dendrites, penkarya or preterminal axonal branchesof other neurons.
Munich, discovered darkly colored granules in neuronal perikarya in sections of brain stained with methylene blue, a cationic (basic) dye. The abundance, size and distribution of the granules varied among different cells, providing data for the description of different types of neurons. Nissl published his technique and results in a series of papers in the mid-1890s (5,6). Within less than 10 years Nissl’s name became an eponym for the cytoplasmic granules in neuronal perikarya and for most of the methods used for staining them with cationic dyes. The terms Nissl body and Nissl substance also evolved. In the early decades of the 20th Century some authors, perhaps disliking eponyms, used the word tigroid, from the striped appearance often seen in the perikarya of large neurons. The science of cytoarchitectonics – parcelation of the gray matter of the CNS into cell groups (nuclei) and layers (laminae) containing distinctive types of neuronal somata – is based on the study of Nissl-stained sections. Advances in electron microscopy and biochemistry in the 1950s showed that the Nissl bodies represented aggregations of rough endoplasmic reticulum, containing numerous ribosomes. Here, mRNA is translated and proteins are synthesized (7). The staining of these structures is due to high concentrations of rRNA.
Cationic dyes currently in favor for Nissl staining are cresyl violet, neutral red, thionine and toluidine blue, typically applied as 0.1% to 0.5% aqueous solutions adjusted to about pH 3. Some differentiation occurs during dehydration, which needs to be carefully carried out. A pale contrasting anionic counterstain is optional and preferably applied before the cationic dye. Blood stains such as Giemsa (8) can also be used to demonstrate Nissl substance.
Figures 2 and 3 show paraffin sections of human brain stem, 10 μm thick, stained with toluidine blue.
Myelin Stains
The myelin sheath consists of compacted spirally wrapped layers of neuroglial surface membranes surrounding the axon of a neuron (Fig. 4). Myelin is rich in phospholipids and basic proteins. Each glial cell (a Schwann cell in the peripheral nervous system, or an oligodendrocyte in the CNS) provides myelin for a length of axon known as an internode. The intervening nodes are the only points at which the axonal surface
Figure 2. Near the ventral surface of the medulla, neurons of the arcuate nucleus are sandwiched between transversely sectioned descending fibers from the cerebral cortex (above) and transversely oriented arcuato-cerebellar fibers (below). Stained with toluidine blue to show nucleic acids and lightly counterstained with eosin Y.
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Figure 4. Transmission electron micrograph of a myelinated axon. Generated at the Electron Microscopy Facility at Trinity College, Hartford, CT. (Courtesy of Wikipedia). Figure 3. Neurons of the external cuneate nucleus in the medulla of the human brain, characterized by rounded cell bodies, often with eccentric nuclei. The axons of these neurons carry proprioceptive signals, derived from the upper limb, to the cerebellum. Stained with toluidine blue to show nucleic acids and lightly counterstained with eosin Y (pink background). 500 nm Schwann cell Myelin sheath Mitochondria Axon
Figure 5. Bundles of myelinated axons leaving the optic tract (lower left) and entering the lateral geniculate body of the thalamus. Stained by Weigert’s chromium-hematoxylin method.
Figure 6. Cerebellothalamic fibers passing around and through the red nucleus of the human midbrain. Stained by Weigert’s chromium-hematoxylin method.
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Figure 8. Myelinated axons in the human inferior olivary nucleus. At the top of the picture is the hilum of the nucleus, composed of fibres that will end in the contralateral cerebellar hemisphere. Stained with luxol fast blue MBS.
Figure 7. Myelinated axons in the decussation of the medial lemnisci in the midline of the human medulla. Stained with luxol fast blue MBS.
Figure 9. Motor neurons in the hypoglossal nucleus of the human brain stem. Stained with Page’s iron-eriochrome cyanine R to show myelinated axons and neutral red to show DNA and cytoplasmic rRNA (Nissl substance). The spaces around the neuronal cell bodies are artifacts due to differential shrinkage that commonly occurs when formaldehyde-fixed central nervous tissue is dehydrated and embedded in paraffin wax.
Figure 10. In the midline of the medulla of the human brain, the nucleus raphes pallidus contains neuronal cell bodies with scanty, peripherally located Nissl substance. The nucleus is named from its appearance as a pale area in a region otherwise occupied by tracts of myelinated axons. Stained with Page’s iron-eriochrome cyanine R to show myelinated axons and neutral red for Nissl substance and nuclei.
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membrane is in contact with extracellular fluid. Action potentials therefore “jump” from node to node, providing much more rapid conduction than is possible in an unmyelinated axon (7, 9). Although it is not part of the neuron, the myelin sheath degenerates if the cell body of its neuron dies, or if the axon is severed.
Sections stained for myelin are valuable aids in teaching the normal anatomy of the CNS. With no or low magnification the tracts of white matter show as dark areas whereas nuclei and other bodies of gray matter are colorless or palely stained. Myelinated fibers are present in gray matter, but at a lower density than in white matter because dendrites and the terminal branches of axons lack myelin sheaths. The principal pathological changes seen in stained white matter are regions of pallor, which may indicate bundles of degenerated axons (as, for example, following destruction of their cell bodies by a stroke) or foci of demyelination containing mostly intact but non-functional axons (as in lesions of multiple sclerosis).
There are many ways to stain myelin (9); three are illustrated here. In the original (1885) method of Carl Weigert (1845-1904), pieces of nervous tissue were fixed by immersion for several weeks in a potassium dichromate solution, which acts by rendering proteins and many lipids insoluble in water and organic solvents. (Formaldehyde, first used for fixation in 1891, does not have this action on lipids.) The Cr(VI) in dichromate ions is reduced to Cr(III), which is deposited at sites of oxidation of the choline-containing phospholipids of myelin (10). The chromated specimens were dehydrated, embedded in nitrocellulose, sectioned and stained with a solution of hematein (“ripened” hematoxylin), which formed a dark blue complex with the bound Cr(III). Simpler techniques have been used in more recent years, using Fe(III)-haematein (“iron hematoxylin”) formulations (11, 12). These later methods, which are applicable to paraffin sections of formaldehyde-fixed tissue, probably demonstrate the basic proteins of myelin rather than residual phospholipids (9), but they are frequently called Weigert stains. Figures 5 and 6 show anatomical details of two areas in the human brain.
The myelin stain most frequently used in American laboratories is probably luxol fast blue MBS (13). This compound is the salt formed by an anionic dye (disulfonated copper phthalocyanine) and a ditolylguanidinium cation. This hydrophobic ion pair is insoluble in water but soluble in ethanol. Paraffin sections are stained overnight at 55-60°C. The dye anions are attracted to cationic sites in the tissue, coloring all components. The ditolylguanidinium cations remain in solution. Differentiation in aqueous alkali, which extracts loosely bound
dye anions, is continued for 15 to 30 seconds, until gray matter is almost unstained. Basic proteins probably account for retention of the dye in myelin (9). There is a one-hour variant of this technique, in which the dye is dissolved in acidified methanol (12). In pathology laboratories staining with luxol fast blue is usually followed by counterstaining with cresyl violet or neutral red to show Nissl substance. Figures 7 and 8 show staining of the brain stem with luxol fast blue alone.
Myelin sheaths can be stained in 30 minutes with the iron-eriochrome cyanine R method, introduced in Britain by Kathleen Page in 1965 (14). The staining mechanism is probably similar to that postulated for luxol fast blue MBS (9). Unlike many of the dyes used in microtechnique, this one, a hydroxytriarylmethane with metal-binding properties, has many commercial uses, so it is inexpensive and likely to be encountered with any of a wide range of other names, including chromoxane cyanine R, solochrome cyanine R and CI Mordant blue 3. The last name identifies the dye unequivocally, as does its Colour Index number: 43820. Hydrated sections are immersed for 30 minutes at room temperature in a solution containing the dye and a ferric salt. All components of the tissue become strongly colored. Differentiation may be in either alkali, which acts in seconds, or a solution of a ferric salt, which takes 5-10 minutes to remove color from material other than myelin. Figures 9 and 10 show 10 μm sections of brain stem stained by Page’s method for myelin, with neutral red as a Nissl counterstain.
“
H&E-stained sections of pieces of normal
central nervous system (CNS) provide hardly
any information about the cytoplasm of neurons
and glial cells, though it is possible to recognize
several cell-types on the basis of their stained
nuclei. Special staining methods are needed
to characterize different types of neurons and
to reveal such elements as dendrites, axons,
myelin sheaths and the cytoplasmic processes
References
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5. Kiernan JA. Nissl: the man, the stain and the substance.
Microscopy Today 2005;13(6):50.
6. Kleinert R 2001Franz Nissl www.whonamedit.com/ doctor.cfm/2465.html (Accessed 1st November 2009). 7. Bear MF, Connors BW, Paradiso MA. Neuroscience:
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8. Kiernan, JA What is Giemsa’s stain and how does it color blood cells, bacteria and chromosomes? Dako Connection Mag. [Please provide reference]. 9. Kiernan JA. Histochemistry of staining methods for normal
and degenerating myelin in the central and peripheral nervous systems. J Histotechnol 2007;30(2):87-106.
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York: Blakiston, 1954, pp. 403-409.
12. Cook HC. Manual of Histological Demonstration Techniques. London: Butterworths, 1974, 159-163. 13. Kluver H, Barrera E. A method for the combined staining
of cells and fibers in the central nervous system.
J Neuropathol Exp Neurol 1953;12:400-403.
14. Page KM. A stain for myelin using solochrome cyanin.
