Neurohistochemistry I

NEUROHISTOCHEMIST
RY
BY
DR. MAHMOOD ABDULAI SEIDU
TEL. 0208212982
INTRODUCTION
• Neurohistochemistry is the main demonstrative histology
for neuropathological indicators.
• There are lots of Histochemical procedures for investigating

neuropathological complains.
• This is because, nerve cells have a complex morphology and

pathologies associated with them extend beyond
themselves
Components of the nervous system
The nervous system is divided into three sections:
• Central nervous system (CNS) comprising the brain and the spinal
cord.
• The peripheral nervous system consisting of nerves outside the CNS

and communicate with the cns.
• Autonomic nervous system consisting of nerves and ganglia which

are autonomous. (Ganglia are discrete aggregation of neurons cell
bodies outside the CNS)
Two principal parts
CELLULAR COMPONENTS OF THE NERVOUS SYSTEM
The major cellular components of the nervous system are:
• Neurons (nerve cells)
• Supporting cells (glial cells or neuroglia)

• Ependymal cells
• Astrocytes
• Oligodendrocytes
• Microglia
• Schwann cells
The neuronal cells types
The function of neurons is to receive and transmit electrical signals
• Motor neurons convey signals away from the central nervous system to
end organs and relay commands for muscle contractions. They therefore
terminate at motor end plate in skeletal muscles.
• Sensory neurons convey signals to the central nervous system from

specialized receptors.
• Interneurons are connected to several neurons and act as relay centers

STRUCTURE OF NERVE CELLS
Irrespective of their functions the neurons have three elements
common to most of them. They are:
• Cell body which is the main part of the cell containing the nucleus.
• Axon, an elongated process carrying signals away from the cell

body.
• Dendrites, elongated processes that receive inputs via connections

with other nerve cells.
The cell body
The cell body contains:
• Nucleus
• Nissl granules
• Neuro melanin
• Lipofuscin
• Neurofibrils
Nucleus
•The cell body of a

neuron contains a
large nucleus with
dispersed chromatin
and a prominent
nucleolus.
Nissl granules
• Representing RER with ribosomes and rRNA.
• Changes in the pattern of distribution of Nissl granules are

associated with neuronal disease.
• Example: Following neuronal damage, nissl granules move from

around the nucleus to the periphery.
• This is usually associated with swelling of the cell, a process known

as chromatolysis.
Neuromelanin
•A brown granular pigment which is
homogeneous in appearance
•May be found in the neuronal

cytoplasm
Lipofuscin 1
• In old brains, lipofuscin is frequently seen as
yellow/brown granules which are not
homogenous as neuromelanin but stain only
around the edges of the cell body.
• In Immunoperoxidase preparations they may

pick up a varying degree of brown staining .
Lipofuscin 2
•In inherited storage disease such a

Batten’s disease (lipofuscinosis) large
amount of lipofuscin-like pigments
may accumulate in the cytoplasm.
Neurofibrils:
• A complex network of fibrils seen running through the cytoplasm
of neurons in silver stains.
• This is due to the presence of the three main cytoskeletal

components microtubules, neurofibrils (intermediate filaments)
and microfilaments (actin).
• In certain degenerative diseases such as Alzheumer’s Dx

neurofibrillary tangles composed of abnormal filamentous
proteins are found in the cell body.
Axons and Dendrite 1
Axons and dendrites are two main nerve cell processes.
• Axons arise from a conical projection of the neuron
(axon hillock).
• In motor neurons the axon may be as long as one

meter and may be sheathed.
• They also contain neurofibrils but no Nissl granules.

Axons and Dendrite 2
• Myelination is done when large fibres (axons) are wrapped
by a variable number of concentric layers of Schwann cell
plasma membrane.
• Within the CNS myelination is caused by oligodendrocytes
• Dendrites are short multiple processes which make contact

with other neurons at synapses. In contrast to axons they
contain Nissl granules.
Neuroglia
The term neuroglia refers to the supporting cells of the CNS and comprises
four main cell types.
• Ependymal cells
• Astrocytes
• Oligodendrocytes
• Microglia
Ependymal cells
• Ependymal cells form the simple cuboidal epithelial cells
lining the ventricles and spinal cord.
• They are tightly bound together at the luminal surface by

the usual epithelial junctional complexes and have a variable
number of cilia.
• Unlike normal epithelial cells ependymal cells do not rest on

basement membrane but rather the bases of the cells taper,
then break up into fine branches.
Astrocytes
• Astrocytes are identified as star-shaped neuroglia,
which are the most numerous cells in grey matter.
• They have long branched processes which occupy

most of the inter-neuronal spaces.
• It is suggested that in grey matter they probably

mediate some metabolic exchanges between neurons
and blood.
Oligodendrocytes
• Oligodendrogcytes are medium size cells with small
number of short-branched processes.
• They are the cells responsible for myelination of

nerve fibres in the CNS.
• It is known that the short-branched processes are

pedicles that connect the cell body to the myelin
sheaths.
Oligodendrocytes
•They are the most predominant type of glia
cells in white matter.
•In grey matter they aggregate around

neuron cell bodies giving the impression
that they are support cells.
Cross section of myelin sheaths that
surround axons
Microglia
•Microglia are small cells with irregular nuclei
and relatively small cytoplasm which forms
fine highly-branched processes.
•In response to tissue damage, micoglia

transform into large amoeboid phagocytic
cells.
SYNAPSE: Junction between a nerve
cell and another cell
1. Messages travel within the neuron as an
electrical action potential
2. Every axon branches into many fine endings,

each tipped by small swelling called an axon
bulb...bulb lies very close to either the dendrite
or the cell body of another neuron...this region is
called synapse
SYNAPSE: Junction between
nerve cell and other cell
synaptic cleft
3.
a. space between presynaptic

membrane (first neuron) and
postsynaptic membrane (next neuron)
b. passage across requires actions of

SPECIMEN HANDLING IN NEUROPATHOLOGY LABORATORY
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.
INTRODUCTION
• 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, neuropathology laboratory facilitates

the diagnosis of disease processes by preserving
samples in a way which will allow the appropriate
investigative techniques to be performed.
SOURCES OF SAMPLES
The following are the main specimen
sources in the neuropathology laboratory.
• Tumour samples from neurosurgery
• Brain biopsy from neurosurgery
• Whole brain from postmortem
SOURCES OF SAMPLES
The following are the main specimen sources in
the neuropathology laboratory.
• Spinal cord from postmortem
• Peripheral nerve biopsy from
neurosurgery
• Pituitary gland from neurosurgery
BRAIN AND SPINAL CORD
BIOPSIES 1/3
•These will usually be performed for
identification of a tumour or
neurodegenerative disease (including
infections).
•Ideally the samples must be received in the

laboratory as soon as possible.
BIOPSIES 1/3
• 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.
BIOPSIES 2/3
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 gluteraldehyde for electron

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

neutral buffered formalin for paraffin processing.
BIOPSIES 3/3
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.
(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 in a large container capable of

holding up to 4X the volume of the brain of fixative.
• The brain must be suspended from a piece of cord or on cotton

wool.
(AUTOPSY)
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 oedematous brains could take up to 8-12 weeks before they
can be cut.
Notes on fixation of whole brain
• The 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 Bouin’s to speed up the process

PERIPHERAL NERVE BIOPSIES 1-
2
• 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.
2
• 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.
2
• 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)
Handling of peripheral nerve
biopsy
• Biopsies must be received fresh and wrapped
atrumatically in gauze lightly moistened with normal
saline.
• The biopsy is usually about 2-4cm long.
• Using a scalpel blade the traumatized cut ends are

removed and frozen in liquid nitrogen (used for
frozen sections or biochemical analysis).
Handling of peripheral nerve
biopsy
• 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

gluteraldehyde to facilitate processing for electron microscopy and
paraffin
• For both paraffin and resin embedding it is necessary to include

longitudinal and transverse sections
The End

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NEUROHISTOCHEMIST

• There are lots of Histochemical procedures for investigating

• This is because, nerve cells have a complex morphology and

• The peripheral nervous system consisting of nerves outside the CNS

• Autonomic nervous system consisting of nerves and ganglia which

• Neurons (nerve cells)

• Supporting cells (glial cells or neuroglia)

• Sensory neurons convey signals to the central nervous system from

• Interneurons are connected to several neurons and act as relay centers

• Axon, an elongated process carrying signals away from the cell

• Dendrites, elongated processes that receive inputs via connections

•The cell body of a

• Changes in the pattern of distribution of Nissl granules are

• Example: Following neuronal damage, nissl granules move from

• This is usually associated with swelling of the cell, a process known

•May be found in the neuronal

• In Immunoperoxidase preparations they may

•In inherited storage disease such a

• This is due to the presence of the three main cytoskeletal

• In certain degenerative diseases such as Alzheumer’s Dx

• In motor neurons the axon may be as long as one

• They also contain neurofibrils but no Nissl granules.

• Within the CNS myelination is caused by oligodendrocytes

• Dendrites are short multiple processes which make contact

• They are tightly bound together at the luminal surface by

• Unlike normal epithelial cells ependymal cells do not rest on

• They have long branched processes which occupy

• It is suggested that in grey matter they probably

• They are the cells responsible for myelination of

• It is known that the short-branched processes are

•In grey matter they aggregate around

•In response to tissue damage, micoglia

2. Every axon branches into many fine endings,

a. space between presynaptic

b. passage across requires actions of

•Histology is supplemented by other areas

• To this effect, neuropathology laboratory facilitates

•Ideally the samples must be received in the

• In the laboratory a dissecting microscope should

• A small bit should be fixed in gluteraldehyde for electron

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

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

• If the brain is sliced before fixation, considerable distortions

• It should therefore be fixed in a large container capable of

• The brain must be suspended from a piece of cord or on cotton

• The fixative must be changed after 48 hours and after 2 weeks.

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

• 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 Bouin’s to speed up the process

• In modern practice the usual reason for taking

• Moreover, histology of formalin-fixed paraffin

• The biopsy is usually about 2-4cm long.

• Using a scalpel blade the traumatized cut ends are

• Then fix the biopsy on the card in phosphate buffered

• For both paraffin and resin embedding it is necessary to include

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