Staining Neurological & Muscle

Question 1

Neuropathology Techniques

Answer 1

• Neuropathology: H&E remains the most useful and commonly used stain
• Neuropathology : relies heavily on IHC as well as some special stain.

Question 2

Neurological Tissue Components - CNS and PNS

Answer 2

CNS comprises the brain and spinal cord
PNS comprises motor and sensory neurons.
Neuropathological tissues comprises mainly neurons (cell body, dendrites, and axons) and supporting Glial cells/Neuroglia, as well as meninges and blood vessels.

Question 3

Techniques for Neurons

Answer 3

Neurons - H&E: Yellow (oligodendrocytes), Orange: Astrocytes, Blue: Neurons.

Question 4

Neurons - IHC

Answer 4

• Neurofilament protein (NFP) demonstrates intermediate filaments expressed in mature neurons
• PGP9.5 & NSE – cytoplasmic proteins both strongly expressed in neurons but not specific to nerve cells
• NeuN (Neuronal Nuclear Protein) - a neuron-specific DNA binding protein
• Synaptophysin - a membrane glycoprotein component of pre-synaptic neurosecretory vesicles; useful marker of neuroendocrine differentiation (also stains metastatic neuroendocrine tumours)
• Chromogranin A - a protein of the dense core matrix of neurosecretory vesicles; used predominantly to elucidate neuroendocrine differentiation in tumours

Question 5

Techniques for axons and neuronal processes - Axons - Special stains

Answer 5

• Luxol Fast Blue - stains Myelin; useful for demyelinating diseases such as Multiple Sclerosis and Guillan-Barre Syndrome
• Toluidine blue - stains peripheral nerve

Question 6

Techniques for Glial cells - What are Glial cells?

Answer 6

Supporting cells of the CNS including: Ependymal cells, astrocytes, oligodendrocytes, microglia

Question 7

Axons - IHC

Answer 7

• Myelin Basic Protein (MBP) stains myelin sheaths.
• S100- used in diagnosis of tumours derived from Schwann cells.

Question 8

Glial cells - IHC: GFAP

Answer 8

GFAP (Glial Fibrillary Acid Protein)
Can be used as a marker of brain injury
Can be used to assess whether a tumour is of glial origin

Question 9

Glial cells - special stains

Answer 9

• Cresyl Violet - stains oligodendrocytes

Question 10

Neurodegenerative Diseases - inclusion bodies

Answer 10

Many neurodegenerative diseases characterised by inclusion bodies (accumulations of proteins) that can be demonstrated using IHC.

Question 11

Demonstration of neurodegeneration: Diseases, inclusion bodies and how they are demonstrated using IHC

Answer 11

Disease->Inclusion-> Demonstrated by

Alzheimer’s , Neurofibrillary tangles, Tau protein, ubiquitin/p62

Parkinson’s, Lewy bodies, alpha-synuclein, ubiquitin/p62

Motor Neuron Disease, Motor Neuron disease inclusion, Ubiquitin/p62, TDP-43

Multiple system atrophy, Glial cytoplasmic inclusion, alpha-synuclein, ubiquitin, p62

Picks disease, Pick body, Tau protein, ubiquitin/p62

Question 12

How is Alzheimer’s disease visualised?

Answer 12

via its plaques and tangles using IHC.
Plaques – Beta Amyloid (IHC)
Tangles - Tau protein (IHC)

Question 13

Parkinson’s Disease - Visualisation

Answer 13

Lewy bodies:
- H&E
- Alpha synuclein (IHC)

Question 14

Picks disease - Visualisation

Answer 14

Pick bodies via Tau Protein using IHC

Question 15

Muscle Biopsy samples

Answer 15

Skeletal muscle taken via biopsy needle
Frozen sections needed
Often assessing enzyme activity (disrupted by fixation)

Question 16

Muscle Biopsy samples - staining techniques used

Answer 16

routine H&E is used for general morphology of muscle biopsies, but frequently other Cellular Pathology staining techniques are required:
Special stains
IHC
Enzyme histochemistry

Question 17

Muscle biopsy samples ; Special stains used

Answer 17

• Gomori Trichrome: used to demonstrate abnormal mitochondrial aggregates, the rimmed vacuoles of inclusion body disorders, connective tissues, and ‘ragged red’ fibres suggestive of mitochondrial cytopathies)
• PAS (Periodic Acid Schiff) +/- Diastase: used to demonstrate glycogen in muscle tissues; accumulation of glycogen is indicative of glycogenoses
• Oil Red O/Sudan Black: used to demonstrate accumulation of lipids in muscles which can indicate carnitine deficiency

Question 18

Muscle biopsy samples: IHC. What can IHC detect in myopathy (musclar disease)?

Answer 18

MHC (Major Histocompatibility Complex) class I proteins are overexpressed in some inflammatory - detectable by IHC

Question 19

Muscle biopsy samples: IHC. What IHC stains are used to detect in myopathy (muscular disease)

Answer 19

Dystrophy-related IHC stains include Dystrophin 1, 2 and 3, sarcoglycans, dysferlin, merosin, caveolin, emerin and spectrin).

In normal muscle, Dystrophin is located beneath the cell membrane of muscle fibres.
Dystrophin: rod shape protein that links intracellular cytoskeleton network to transmembrane components of the DGC, including dystroglycan, sarcoglycans and sarcospan.
DCG: dystrophin complex - acts as a membrane stabilizer during muscle contraction to prevent contraction-induced damage

In Becker’s disease this staining is reduced, and in Duchenne muscular dystrophy the staining is absent

Question 20

Enzyme Histochemistry

Answer 20

Branch of histochemistry using specific enzymatic reactions within tissues
Commonly used on muscle biopsies
Frozen sections are essential as the cross-linking nature of additive fixatives (e.g formalin) denatures enzymes of interest within muscle tissues.

Question 21

ATPase staining

Answer 21

Assess fibre type
Alkaline pre-incubation demonstrates type 1 (slow) fibres
Acid pre-incubation demonstrates type 2 (fast fibres

Question 22

NADH staining

Answer 22

Internal fibre architecture
Type 1 dark and Type 2 light

Question 23

What does NADH staining target?

Answer 23

Targets fibres with vacuoles and loss of myofibrillar architecture (neurogenic process)

Question 24

Amyotrophic? Lateral Sclerosis (ALS)

Answer 24

Atrophied fibres stain darkly
Hypertrophied fibres appear larger and paler
using alkaline phophatase..?

Question 25

Alkaline Phosphatase

Answer 25

Identifies regenerating muscle fibres
Aids in diagnosis of acquired inflammatory myopathies
can visualise Immune mediated necrotizing myopathy (IMNM)