NEUROHISTOCHEMISTRY

BY
B. A. OWUSU
 DEMONSTRATION OF NERVE FIBRES
There are several methods of demonstrating special structures of the neuron.
These structures include:
 axons,
 dendrites,
 terminal boutons (synaptic structures)
 Degenerated axons.
Most of these methods are silver impregnation techniques.

 It is therefore necessary to understand the basic principles underlining the

silver impregnation techniques.
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 SILVER IMPREGNATION TECHNIQUES

Silver methods have been divided into two fundamental groups.

1. The first group uses silver diamine (ammoniacal silver) followed by formalin
reduction.
2. The second group employs a weak solution of silver with a photographic developer as
a reducing agent, followed by gold chloride.

The technical explanation of the two processes has never been well explained but when
viewed from the simple understanding of basic applied sciences one explanation can be
accepted.

During the process of impregnation silver foci are selectively formed on targeted tissue
components. With more silver ions and the presence of reducing agents available, each silver
focus appears to function as a macroscopic aggregate of silver micronuclei which catalyzes the
reduction and further precipitation of silver on and around it.
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 IMPLICATION OF THE PRINCIPLE
The implication is that factors that affect the redox potential of reactants will
affect silver impregnation techniques and these can affect the staining
procedure. The factors are:
 pH,
 temperature,
 concentration of reactants
 presence of interfering chemical components to redox
activities

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Silver impregnation methods therefore have three areas of concern:

1.Reactants that induces the selectivity.

2. Those that cause the reduction

3. Those that intensify the results

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 DEMONSTRATION OF AXONS &
PROCESSES
For axons and neuronal processes in the CNS there are two important stains.
Both of which are applicable on paraffin processed sections.

 Bielschowsky

Marsland, Glees & Erikson methods).

Axons in PERIPHERAL NERVES can be demonstrated by Palm green

method and Linder’s method all on paraffin sections.
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Bielshchowsky's silver stain. 8 µm thick paraffin
embedded sections of mouse cerebellum

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 IMMUNOHISTOCHEMICAL
MARKERS FOR NEURONS
The main markers can be divided into three groups
1. Neuronal cytoskeletal proteins.

2. Neuro-specific cytoplasmic proteins

3. Protein associated with neuro-secretory granules

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 Neuronal cytoskeletal proteins
These are in the form of neurofilament proteins, a form of intermediate filament which is
specific to nerve cells.

Antibodies to neurofilament proteins identify mature nerve cells and there are three types:
 NF70 (L),
 NF150 (M)
 NF200 (H)

Each have different molecular weight and each capable of being modified by
phosphorylation.

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 Neuro-specific cytoplasmic proteins
Protein Gene Product (PGP) 9.5 and neuron specific
enolase (NSE) are examples of proteins which are expressed at
high level in neurons and can be reliably detected by
commercially available antibodies.

They are however not specific for neuronal tissues and should
be used among a panel of antisera for reliable identification.

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Protein associated with neuro-secretory granules

These are used for establishing neuroendocrine differentiation.

Antibodies to these proteins will help stain sites of synaptic
junctions and normal cerebellar cortex.

Chromogranin A is a protein of the dense core matrix of

neurosecretory granule.

Synaptophysin is a membrane glycoprotein seen in pre-

synaptic neuro-secretory granules.
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 DEMONSTRATION OF NORMAL
MYELIN
Myelin is formed by oligodendrocytes in the CNS and Schwann cells in the
PNS.

Each nerve is wrapped up in up to 100 concentric layers of highly

specialized plasma membrane.

Chemically, myelin consists of specific proteins, lipids and cerebrosides.

Its demonstration can be achieved by using immunohistochemical

techniques or conventional methods.
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Immunohistochemical methods of detecting normal myelin
The presence of myelin can be demonstrated by immunohistochemistry with antibodies to
S100 protein and Leu-7 which was originally described as a marker for a subset of
lymphocytes.
Other myelin markers are
 Galacto-cerebroside antibodies
 Myelin basic protein (MBP)
 Myelin associated glycoprotein (MAG)
 Carbonic anhydrase –C
 P0, P1 and P2 proteins
 Alpha/Beta crystalline.
 None of these markers has taking the place of conventional stains in the demonstration of
normal myelin. However, S100 has been very useful in detecting tumours derived from cells
forming myelin in peripheral nerves (Schwann cells).
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Conventional methods of detecting normal myelin
The conventional methods of staining myelin rely on the detection of the
components of the Schwann cell membrane. The main detectable components are
sphingomyelin and cerebrosides.
Sphingomyelin:
Sphingomyelin is a phospholipid found in abundance in Schwann cell plasma
membrane and lipid histochemical stains directed to stain sphingomyelin are used
for this purpose.
Myelin has a strong affinity for haematoxylin after treatment with chromate-
containing fixative, methods in which metallic salts are used to mordant myelin
dyes has been useful.
The dichromate-acid haematin (DAH) method is reasonably specific for choline
containing phospholipids. Interference from proteins, which are also positive for
this stain can be discounted by including a lipid extracted control section
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 Conventional methods of detecting normal
myelin
Cerebrosides
These lipids can be demonstrated by reactions specific for their
carbohydrate molecules.

 Cerebrosides and related lipids are PAS positive.

In this process periodic acid converts 1:2 glycol groups to Schiff’s stainable
aldehydes.

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DEMONSTRATION OF DEGENERATION PRODUCTS
OF MYELIN
In de-myelinating diseases or following neuronal death, the axon and associated myelin
dies.

Myelin loss will occur in such processes as infarction, multiple sclerosis and in several
primary degenerative disease of the central nervous system. In such circumstances the
detection of degeneration products is important in neuropathology.

In its normal state myelin is hydrophilic due to its high content of polar phospholipids but
following degeneration, it becomes hydrophobic with the formation of cholesterol esters.
Both normal myelin and degenerated myelin can be stained by osmium tetra oxide.
However the osmiophilia of the normal myelin can be blocked by pretreatment with strong
oxidizing agents (potassium dichromate) and this phenomenon allows the differentiation of
degenerate myelin from normal ones.(This is the principle of Marchi method for
degenerate
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 LOSS OF NORMAL MYELIN STAINING

Following myelin loss, the degenerate myelin is phagocytized by

marcrophages and over a period of weeks or months they are removed
from the area.

This process results in the lack of normal and degenerated myelin

staining with routine methods

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 COMBINATION TECHNIQUES FOR
DEGENERATE MYELIN
Stains for neutral lipids such as oil red O can effectively demonstrate
myelin degeneration in formalin-fixed and frozen sections during the
period of myelin-lipid phagocytosis.

This is an effective way for looking for tract degeneration when loss of
normal myelin staining is established but no Marchi method is available.

Oil red O can be combined with dichromate acid hematein (DAH) to

stain normal myelin blue to contrast beautifully with the red stained fatty
degenerate myelin lipids within lipophages.

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 DEMONSTRATION OF NISSL GRANULES
Nissl stains are used routinely in neurohistochemistry not only to demonstrate
nissl granules but also to demonstrate the cellular pattern.
The granules are distributed only in the cytoplasm of nerve cells and dendrites.
Nissl granules can be demonstrated by several basic dyes. These include: Neutral
red, methylene blue, pyronin, Azur, thionin, toluidine blue and cresyl fast
violet.
Tissues fixed in alcohol stains particularly well in toluidine blue and cresyl fast
violet.
Cresyl fast violet is however the method of choice for Nissl substances. Nissl
substances are stained purple dark blue, neurons pale purple blue and nuclei
purple blue.
When the emphasis of demonstration is on Nissl granules, the stain should be
acidified with the addition of 0.25% acetic acid
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 DEMONSTRATION OF NEUROGLIAL
CELLS
DEMONSTRATION OF EPENDYMAL CELLS
Ependymal cells are ciliated and are not lined by basement membranes.
These can be demonstrated easily by conventional H and E staining.
Ependymal cells may form tumours of the central nervous system where their
identification is based on the determination of cilial basal body
(blepharoplast). This can be done conveniently by PTAH or iron
haematoxylin.
Immunohistochemically, ependymal cells may express the glial intermediate
filament (GFAP) but do not express the normal epithelial intermediate
filament (cytokeratin)

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 DEMONSTRATION OF NEUROGLIAL CELLS
DEMONSTRATION OF ASTROCYTES
The identification of astrocytes is important in neuropathology as they react
promptly to local tissue injury.
Example: they increase in size in response to cerebral edema or to metabolic
disturbances such as liver failure. In such circumstances they
characteristically develop prominent pink cytoplasm, the nucleus become
eccentric and the processes prominent.
Such cells are referred to as reactive astrocytes
Astrocytes proliferate when there is damage to the CNS and fill defects left
by loss of specialized nervous tissue with glial fibres.
This is called astrocytic gliosis and its identification is necessary for the
analysis of disease of the CNS
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 ASTROCYTIC GLIOSIS (PROLIFERATION OF ASTROCYTES

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 DEMONSTRATION OF NEUROGLIAL CELLS
Demonstration of Astrocytes
Astrocytes are star-shaped but contrary to common sense the processes are not
easily displayed in H & E sections.
Using Cajal’s gold sublimate method, two types of astrocytes can be identified
(fibrous and protoplasmic types).
Fibrous astrocytes are found mainly in the white matter. They have small cell
bodies with long processes containing metallic impregnated fibrils after staining.
Protoplasmic astrocytes have more processes that are shorter and thicker than
those of the fibrous type.
Immunohistochemical demonstration of astrocytes is mainly by the expression
of glial fibrillary acidic protein (GFAP)
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Purpose of Astrocyte staining
Astrocyte stains are usually performed for one of three reasons

Histological demonstration of astrocytes

Histological demonstration of reactive astrocytes (abnormal) or

astrocytic proliferation (gliosis)

Demonstration of astrocytic differentiation in neoplasm

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 DEMONSTRATION OF NEUROGLIAL CELLS 2
DEMONSTRATION OF OLIGODENDROCYTES
In H & E, oligodendrocytes are identified by their small dense rounded nuclei
and the cytoplasm not distinguishable from the surrounding tissues.
In formalin-fixed paraffin processed sections the cytoplasm may form an
artefactual halo around the nucleus.
Demonstration of oligodendrocytes is merely an anatomical or histological
exercise. It is called for in fresh human tissue, in autopsy material, autolytic
processes frequently lead to poor results.
Immunohistochemical markers for oligodendrocytes are antibodies to
galactocerebroside, myelin basic protein or carbonic anhydrase-C.
Tumours derived from oligodendrocytes do not express these, given rise to
speculations that oligodendroglioma may not actually be a tumour originating
from oligodendrocytes.
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DEMONSTRATION OF NEUROGLIAL CELLS
DEMONSTRATION OF MICROGLIA
Microglia are cells of the mononuclear phagocytic system.
Inactive microglia are static cells in the normal CNS with dendritic morphology called
resting microglia.
Following brain injury these inactive microglia undergo phenotypic changes by expressing
cell surface markers more like peripheral macrophages and become phagocytic.
In conventional stained sections microglia cells appear as rod-shaped nuclei.
In silver stained sections, fixed microglia may be seen to have numerous branching
processes.
Immunohistochemistry is the most reliable method for demonstrating microglia. They stain
positively to antisera which detect macrophages:
 CD 68 – a peripheral macrophage marker
 EMB/11 works in frozen sections only
 KP-1: HLA-DR a class II MHC
 CD 45 is faintly positive
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 Microglia cells appear as rod-shaped nuclei

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 Fixed microglia may be seen to have numerous branching processes.
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SPECIMEN HANDLING IN NEUROPATHOLOGY
LABORATORY

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INTRODUCTION
Due to advances made in neurosciences, histological methods are no longer
the only tools for investigating diseases of the nervous system.
Histology is supplemented by other areas such as microbiology, immunology,
biochemistry and molecular genetics.
However, the histologist is more often than not the one responsible for
receiving and preserving the specimen for distribution to the other areas of
interest.
To this effect, the aim of the neuropathology laboratory is to facilitate the
diagnosis of disease processes by preserving samples in a way which will
allow the appropriate investigative techniques to be performed.
This will usually be under the direction of a clinical pathologist who will
choose techniques with regards to the diseases which are likely to present the
nervous disposition.
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SOURCES OF SAMPLES
The following are the main specimen encountered by the neuroscience
histologist in the neuropathology laboratory.
 Tumour samples from neurosurgery
 Brain biopsy from neurosurgery
 Whole brain from postmortem
 Spinal cord from postmortem
 Peripheral nerve biopsy from neurosurgery
 Pituitary gland from neurosurgery

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BRAIN AND SPINAL CORD BIOPSIES
These will usually be performed for identification of a tumour or
neurodegenerative disease (including infective processes).

Ideally the samples must be received in the laboratory as soon as possible.

The samples are usually very small and sticky and the tendency for the
specimen to dry in transit should be acknowledged and prevented.

In the laboratory a dissecting microscope should be used to facilitate the

identification of grey matter, white matter and abnormal tissue in brain.

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BRAIN AND SPINAL CORD BIOPSIES
ACTION ON RECEPTION OF SPECIMEN
If rapid diagnosis is required, a small part of the tissue is taken for
frozen section by pre-freezing in chilled isopentane, then frozen in
liquid nitrogen in a supporting medium and then to the cryostat for the
frozen section.

A small bit should be fixed in glutaraldehyde for electron microscopy

A bulk of the sample should be fixed in an adequate volume of 10%

neutral buffered formalin for paraffin processing.

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BRAIN AND SPINAL CORD BIOPSIES
Preparation of a smear for cytological examination
It may be necessary to prepare a smear for cytological examination.
This technique allows for rapid observation of several areas of the
biopsy. By this, all cell types in the CNS can be identified.

Preparation: A small piece of the biopsy is placed at one end of a

clean plain glass slide and a second glass slide is used to crush the
specimen and then drawn across the slide to produce a uniform smear.

The only limitation to this method is where the tissue is hard and can
not be crushed.
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PERIPHERAL NERVE BIOPSIES
Nerve biopsies are highly prone to histological artifacts due to handling and this
should be kept at minimum. In modern practice the usual reason for taking
peripheral nerve biopsies is a neuropathy of unknown origin.
Under these requests most of the changes seen in nerves are subtle to the effect
that paraffin sections and light microscopy lack the resolution required for
diagnosis.
Moreover, histology of formalin-fixed paraffin processed peripheral nerves often
shows marked distortions and shrinkage.
When it becomes very necessary to fix and process by paraffin method, then
Heidenhain-Susa or Bouin’s are appropriate and should not be fixed for more
than 24 hours.
This will prevent the sample from being brittle. This may probably allow the
examination of nerve fibres to determine myelin pattern. The most useful
investigation can be done by high resolution histology (electron microscopy)
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 Handling of peripheral nerve biopsy
 Peripheral nerve biopsies must ideally be received fresh in the laboratory and wrapped
atraumatically in gauze lightly moistened with normal saline to prevent drying.
The biopsy is usually about 2-4cm long. Using a new scalpel blade the traumatized cut
ends are removed and frozen in liquid nitrogen after being cooled in chilled isopentane
(used for frozen sections or biochemical analysis).
The main portion of the sample is allowed to adhere to a piece of card by pressing it
down on it with slight pressure. The biopsy will adhere to the card within 30 seconds
Then fix the biopsy on the card in phosphate buffered glutaraldehyde to facilitate
processing for electron microscopy.
Following glutaraldehyde fixation, myelin will be hard enough to be cut into smaller
blocks.
 For both paraffin and resin embedding it is necessary to include longitudinal and
transverse sections

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 BRAIN AND SPINAL CORD (AUTOPSY)
In autopsy, whole brain can be removed for analysis. If the brain is sliced
before fixation, considerable distortions can occur. It should therefore be fixed
(uncut) in a large container capable of holding up to 4X the volume of the brain
of fixative. In fixation, the brain must be suspended from a piece of cord.
Fixation
Fixation of whole brain is usually adequate in 10% formol-saline although
some laboratories prefer 15-20% formalin which they claim, penetrates faster.
The fixative must be changed after 48 hours and after 2 weeks.
Most brains will be hard to cut after 3-4 weeks although necrotic and
edematous brains could take up to 8-12 weeks before they can be cut.

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 Notes on fixation of whole brain
• Formol saline can be buffered to neutral pH with phosphate buffer

• An excess of magnesium carbonate can be added to 10% formol saline to achieve an almost
neutral pH

• Other fixatives can be used depending on what is required to be demonstrated.

• Formol ammonium bromide for neuroglial cells

• Formol calcium for the study of phospholipids

• Formol alcohol and Bouins to speed up the process

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 THANK YOU, ANY QUESTIONS???

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