Neurology brings together the art of human interaction and expression on one hand with the clinical assessment, advanced neuroimaging, understanding of disease and therapeutics on the other hand. Here are some notes focused on the study of nervous system disease, the natural history and pathological mechanisms; our Neuropathology notes!

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Neuropathology notes:

 

Stains:

• H&E hematoxylin & eosin stain:
  • Basophilic: stains blue/purple, takes up the base (hematoxylin)
  • Eosinophilic: stains pink/red, takes up the acid (eosin)

 

Biopsies:

An approach to Brain biopsies:

PAS-positive macrophages in the brain are found in:

• Whipple’s disease
• Adrenoleukodystrophy, Globoid-cell leukodystrophy, metachromatic leukodystrophy, and other hereditary leukodystrophies
• Acquired leukoencephalopathy due to toxins e.g. organic solvents

Ballooned neurons are found in:

• Argyrophilic grain disease AGD
• Progressive supranuclear palsy PSP
• Corticobasal degeneration CBD
• Parkinson’s disease

Coiled bodies in oligodendrocytes are found in:

• Tauopathies
• Argyrophilic grain disease AGD

Lymphocytic markers:

• Pan B-cell antigens (CD19, CD20, CD22, CD79a),
• CLA leukocyte common antigen or LCA= CD45
• In Diffuse large cell B cell lymphoma:
• CD5+ leads= worse prognosis

 

An approach to Muscle biopsies :

General notes on muscle biopsies:

Suitable biopsies:

• Don’t biopsy a site of previous EMG needle insertion (focal necrosis & inflammation occur)
• Don’t biopsy severely affected end stage muscle
• Don’t biopsy uninvolved muscle
• Don’t biopsy near tendinous insertions (fibre size variation)
• Don’t biopsy gastrocneimus
• i.e. biopsy MRC 3 grade muscle
• Size of biopsy: 1cm X 1cm X 1-2.5cm is sufficient. Don’t manipulate or traumatize the muscle too much.

Staining of frozen sections with 8 stains:

• 5 stains.
• 3 muscle enzyme histochemical reactions

This can be followed by:

• More histochemistry
• Immunohistochemistry IHC
• Electron microscopy EM
• Molecular tests: Western blott, Southern blott, PCR,

 

Hematoxylin & Eosin (H&E) stain :

Muscle fibres size:

• Variation is size (diameter).

Muscle fibre shape:

• Polygonal in adults
• Round:
  • Normal: in infants & very young children
  • Round: in myopathies e.g. muscular dystrophies
• Angulated: small angulated in neuropathy

Muscle fibre structure:

Atrophic fibres:

• “nuclear bags” without much myofibril

Note distribution of atrophy:

• Random
• Fascicular ‘group atrophy’: denervation
• Perifascicular atrophy (perimysium): dermatomyositis

Note type of fibre atrophy (see other stains)
Regenerating fibres:

• Basophilic (blue) fibres
• These occur in myopathies

Necrotic fibres:

• Eosinophilic homogenous cytoplasm, in longitudinal section (loss of striations). Later: vacuolated fibres, infiltration with inflammatory cells (macrophages & lymphocytes).
• Dystrophic, Inflammatory or toxic myopathy.

Split fibres:

• These occur in muscular dystrophies

Rimmed vacuoles:

• Vacuoles rimmed by basophilic material. Inclusion body myositis,

Vacuoles without rims:

• Metabolic myopathies:
  • Glycogen storage diseases,
  • Hypokalemia
• Drugs (lysosomal toxicity): colchicine, chloroquine, amiodarone
• Hyperkalemia periodic paralysis/hypokalemic periodic paralysis

Centromyocytic necrosis:

• Inflammatory cells in the myofibre surrounded by normal sarcoplasm occurs in polymyositis.

Ragged red fibres:

• best seen on Gomori’s trichrome stain. Subsarcolemmal +/-intermyofibrillary aggregates, blue on H&E. Occurs in Mitochondrial disorders & in patients >60 y.o..

Whorled fibres ‘coiled fibres’: central disorganisation of myofilaments.
Location of nuclei:

• Peripheral: normal
• Central: muscular dystrophies, myotinic dystrophy

Connective tissue on H&E:

• Endomysium: increased connective tissue ‘endomysial fibrosis’ occurs in muscular dystrophies.
• Perifascicular (perimysium):
  • Look for inflammation around vessels in inflammmatory myopathies.
  • Look for inflammation involving vessels in vasculitis, destruction of vessel wall, fibrinoid necrosis.
• Epimysium (fascia)

 

Gomori’s trichrome stain:

Stains mitochondria red.

• Rimmed vacuoles: vacuole rimmed by red staining material. Inclusion body myositis,
• Ragged red fibres: Subsarcolemmal +/-intermyofibrillary aggregates, appear red. Occurs in Mitochondrial disorders & in patients >60 y.o..
• Target fibres: central lack of staining, surrounded by dark intermediate zone, surrounded by normal staining peripherally. Denervation.
• Nemaline rods: sarcoplasmic red aggregations

 

PAS stain:

Stains glycogen & mucin. Stains type II fibres well.

• Increase staining: glycogen storage diseases
• No staining: necrotic fibres

 

PAS D stain:

Stains mucin but not glycogen i.e. if something is dark on PAS and on PAS D, it is mucin.

Oil Red O stain:

Stains lipid well.

• Increased staining:
  • Lipid storage diseases
  • Mitochondrial diseases

 

NADH-TR stain (NADH tetrazolium reductase):

Stains type I fibres well. Good for structure:

• Target fibres:
  • Central lack of staining, surrounded by dark intermediate zone, surrounded by normal staining peripherally.
• Central cores:
  • Lack of staining in the centre.
  • Occurs in Central core disease
• Central clearing:
  • Occurs in Denevation
• Ring fibres:
  • Subsarcolemmal peripheral rim of increased staining, myofibrils perpendicular to the axis
  • Occurs in Myotonic dystrophy
• Necrotic fibres:
  • No staining.
  • Occurs in dystrophic, inflammatory or toxic myopathy
• Moth eaten fibres:
  • Ill defined areas of loss of staining.
  • Non-specific: Inflammatory myopathies, malignant hyperpyrexia, denervation
• Lobulated fibres:
  • Irregularities of the intermyofibrillary network

 

ATPase stain:

• Fibre type grouping: denervation & renervation

Type I fibre atrophy:

• Myotonic dystrophy
• Congenital myopathies
• Deneravation

Type II fibre atrophy (usually type IIb):

• Non-specific
• Occurs in Disuse atrophy
• Occurs in steroid induced myopathy
• Lots of other stuff

Type I fibre predominance:

• i.e. more type I than usual:
• Occurs in Congenital myopathies

Type II fibre predominance:

• i.e. more type II than usual:
• Occurs in Amyotrophic lateral sclerosis

Notes:

• Axonal neuropathies cause fibre type grouping. In demyelinating neuropathies fibre type gourping doesn’t occur because the axons and hense the neurotropic factors are intact.

 

COX SDH stain (cytochrome c oxidase, succinate dehydrogenase):

COX stains brown, SDH stains black. COX stains mitochondria.

• COX deficient fibres
• Moth eaten fibres

 

Fibre type	Type I (slow twitch)	Type II (fast twitch)
Mitochondria	High	Low
ATPase at 4.3	Dark	Light, except type IIc fibres
ATPase 10.3	Light	Dark
ATPase 4.6	Dark	Type IIa are light Type IIb are intermediately staining
COX SDH	Dark	Light, unless atrophic
Fast myosin	None	High
Glycogen		Rich

 

Myopathic pattern	Neuropathic pattern
Extensive variation in fibre size	Nests of atrophic fibres
Rounded fibres	Angular fibres
Central nuclei	No central nuclei
Necrotic fibres	No necrotic fibres
Cytoplasmic alteration	Target cells
Interstitial fibrosis	Minimal interstitial fibrosis
Inflammatory cell infiltrates	No inflammatory cells

 

Muscle immunohistochemistry:

 

Fast myosin:

• Present in type II (fast twitch fibres)

Slow myosin:

• Present in type I (slow twitch fibres)

NCAM isoform CD56/Leu19:

• Marker of satellite cells (beneath basal lamina), indicating regeneration of muscle

 

Muscle histochemistry:

Myoadenylate deaminase MAD:
Phosphofructokinase PFK:
Myophosphorylase MPH:

An approach to Nerve biopsies:

Patterns of nerve damage:

Neurapraxia:

• Damage to myelin only, causes conduction block

Axonotmesis:

• Damage to axons with intact myelin & connective tissue. Wallerian degeneration occurs distal to the lesion.

Neurotmesis:

• Damage to axon, myelin & connective tissue i.e. complete nerve damage.

Others:

• Uniform demyelination (all nerves, e.g. Charcot-Marie-Tooth)
• Segmental demyelination (some fibres affected others not)
• Focal demyelination (all fibres in one focal area are affected)
• Conduction block (severe form of focal demyelination)

 

General notes on nerve biopsies:

• Sizes of nerves:
  • Myelinated fibres 2-15 microns:
    • Large & small, usually 1:2 ratio
  • Unmyelinated fibres 0.5-2 microns, more abundant than myelinated fibres
• Nerves by myelination:
  • Myelinated:
    • Motor nerves are myelinated

Vibration & proprioception nerves are myelinated

• Unmyelinated:
  • Pain & temperature nerves are unmyelinated

Autonomic nerves are unmyelinated

• Structure:
  • Epinurium

Perinurium
Endonerium

• Stains:
  • Paraffin fixed nerve:
    • H&E (frozen or permanent sections)
      • Permanent sections are good for inflammatory cell infiltrate, granuloma, lymphoma

Vasculitis: transmural inflammation of the vessel wall & fibrinoid necrosis,

• LFB luxol fast blue:
  • for myelin (blue)
• Trichrome stain:
• Bodian stain (a type of Silver stain):
  • for axons
• Congo-red stain:
  • Amyloidosis
• Alcian blue, Cresyl violet, PAS

Immunohistochemistry:

• Neurofilament immunohistochemistry: for axons (large & small)
• S100 protein: Schwann cells & Schwannomas
• CD20: B cells
• CD3: T cells
• EMA: perineural cells & perineuromas

• Osmium: ?what is it embedded in
  • for myelin
• Gluteraldehyde:
  • Semithin (10 micro M) plastic preparations:
    • for myelin & demeylinating neuropathies

Assess population of myelinated fibres i.e. small vs. large myelinated fibres (& myelinated fibre loss)
Demyelination:

• Denuded axons (Normal nonmyelinated axons are 0.5-2 microns in diameter. If a fibre >3 micron is found without myelin then it is a denuded axon)
• Thinly myelin axons i.e. remyelinating i.e. there was previous demyelination
• Giant axons
• Tomacula
• Polyglucosan bodies
• Onion bulb formation
• Axonal sprouting= clusters of Small thinly myelinated axonal sprouts (evidence of chronic axonal degeneration)
• Myelin ovoids may be confused with crush artefact

• G ratio= Axon to entire nerve fibre ration = 0.6
• G Ratio >0.6 implies thinly myelinated axon
• G Ratio <0.6 implies axonal damage

• On frozen nerve:
  • H&E

Modified Gomori Trichrome stain:

• For myelin (pink/red)
• Myelin-digestion chambers
• Tomacula
• Giant axons
• Polyglucosan bodies
• Onion bulb formations (red-tinged myelin surrounded by Schwann cell nuclei)

Central fascicular degeneration, & selective nerve fascicular degeneration: ischemia

• Cresyl-fast violet:
  • Shows metachromatic substance
• PAS:
  • Polyglucosan bodies
• Congo-red stain:
  • Amyloidosis
• EM electron microscopy:
  • Thin (1 micro M)

G ratio= Axon to entire nerve fibre ration = 0.6
G Ratio >0.6 implies thinly myelinated axon
G Ratio <0.6 implies axonal damage
Small thinly myelinated axonal sprouts (chronic axonal disease)

• Teasing technique a.k.a. Teased preparation:
  • =or>100 fibres need to be evaluated

Shows mild demyelination & axonal degeneration
Demyelination:

• Decreased internode distance (remyelination)

• Wallerian degeneration: myelin ovoids are seen

Tomacula (excessive folds of myelin) can be seen (HNPP)

Interpretation:

• Hemosiderin in nerve: old evidence of vasculitis
• Patchy loss of axons: vasculititis

 

Skin biopsy for neuropathy:

• 3mm punch skin biopsy from leg skin
• Anti-protein-gene-product 9.5 protein a.k.a. Anti PGP 9.5 by immunohistochemistry or immunofluorescence, (this stains nerve fibres in the skin)
• Count intraepineural nerve fibres IENF
• Reduced: <8 fibres/mm on dorsum of foot
• If reduced small fibre sensory neuropathy is present (with or without other features of distal symmetric polyneuropathy). If not reduced, distal symmetric neuropathy is still possible.

 

CNS immunohistochemistry:

 

Antibody/antigen	Tumours positive	Tumours negative
GFAP	Astrocytoma, oligodendroglioma, glioblastoma	Meningioma
Synaptophysin
Neurofilament
Chromogranin	Paraganglioma
EMA	Meningioma*	Solitary fibrous tumor
Vimentin	Meningioma, hemangiopericytoma, solitary fibrous tumor
Cytokeratin
S100	Schwannoma	Solitary fibrous tumor
CD99
Smooth muscle specific actin
Desmin
Myoglobin
INI1		Atypical teratoid rhabdoid tumor ATRT*
CD1a	Langerhan cell*,
AE1/AE3
CD34	Solitary fibrous tumor*	Hemangiopericytoma (only mildly),
Ki-67 (MIB index)	Tumors with frequent mitosis

* important association

Lymphocyte markers:

Pan B-cell antigens (CD19, CD20, CD22, CD79a),

CLA leukocyte common antigen or LCA= CD45

In Diffuse large cell B cell lymphoma:
CD5+ leads= worse prognosis

Neuroimmunology:

Multiple sclerosis:

• Plaques within white matter: demyelination, lymphocytes (T & B Cells), activated macrophages/microglia,
• Oligodendrocyte loss
• Axonal loss, gliosis, astrocyte proliferation

 

Paraneoplastic syndromes & antibodies:

 

Syndrome	Antibodies (synonym)
Paraneoplastic encephalomyelitis	Anti-Hu Anti-amphiphysin Anti-CRMP5 (anti-CV2) Anti-PCA-2 ANNA-3 Anti-NMDAR
Paraneoplastic Cerebellar degeneration	Anti-Hu Anti-Yo (PCA-1) Anti-Tr Anti-CRMP5 (anti-CV2) Anti-PCA-2 Anti-Ma1 Anti-mGluR1
Paraneoplastic sensory neuropathy	Anti-Hu Anti-CRMP5 (anti-CV2) ANNA-3
Paraneoplastic autonomic neuropathy	Anti-Hu Anti-nicotinic ACh receptor Purkinje cell antibodies PCA-2
Paraneoplastic intestinal pseudoobstruction	Anti-Hu
Lambert-Eaton Myasthenic syndrome LEMS	Anti-VGCC Anti-PCA-2
Myasthenia Gravis	Anti-ACh receptor Anti-titin

 

Antibodies (synonym)	Paraneoplastic syndrome	Underlying cancer
Anti-Hu* (ANNA-1)	Sensory neuronopathy Autonomic dysfunction Encephalomyelitis Cerebellar degeneration Myelitis	Small cell lung cancer Neuroblastoma Prostate cancer
Anti-Yo (PCA-1)	Cerebellar degeneration
Anti-PCA-2	Encephalomyelitis Cerebellar degeneration LEMS	Small cell lung cancer
Anti-amphiphysin	Stiff-man syndrome Encephalomyelitis	Breast cancer Small cell lung caner
Anti-gangliosides	Sensorimotor neuropathy
Anti-VGKC	Neuromyotonia	Thymoma Small cell lung caner
Anti-VGCC	Lambert Eaton Myesthesenic Syndrome LEMS	Small cell lung cancer
Anti-ACh receptor	Myasthenia gravis
Anti-titin	Myasthenia gravis
Anti-neuronal nicotinic Ach receptor	Autonomic dysfunction and several other disorders
Anti-MAG	Sensorimotor neuropathy	Waldenstroms macroglobulinemia
Anti-Ri	Ataxia Opsoclonus-myoclonus	Breast Gynaecologic Lung cancer Bladder cancer
Anti-Tr	Cerebellar degeneration	Hodgkin’s lymphoma
Anti-retinal a.k.a. Anti-CAR (anti-recoverin protein)	Cancer-associated retinopathy Melanoma-associated retinopathy	Small cell lung caner Melanoma Gynecological
Anti-CRMP5 (anti-CV2)	Encephalomyelitis Cerebellar degeneration Chorea Sensory neuropathy	Small cell lung cancer Thymoma
Anti-Ma1	Brain stem encephalitis Cerebellar degeneration	Lung cancer Other cancer
Anti-Ma2 (a.k.a. anti-Ta)	Limbic brain stem encephalitis	Testicular cancer
ANNA-3	Sensory neuronopathy Encephalomyelitis	Lung cancer
Anti-mGluR1	Cerebellar degeneration	Hodgkin’s lymphoma
Anti-NMDAR	Limbic encephalitis (Associated with orolingual dyskinesias & catatonia like state)	Ovarian teratoma

Other notes:

• VGKC, voltage-gated potassium channels (Kv1) antibodies: These are positive in patients with limbic encephalitis, Morovan’s syndrome. These have differnet targets.
  • Caspr2: contactin-associated protein-antibody-2, a subtype of VGKC found in Morovan’s syndrome

Lgi1: leucine-rich, glioma inactivated 1 protein, a subtype of VGKC found in limbic encephalitis.