HistoPath Mito Disorders

Question 1

Describe Mitochondrial Respiratory Chain

Answer 1

• one of the most common 1/5000
• generally progressive & multi-systemic
• typically affect organs with high energy demands –> neuromuscular & neurological presentation most common

Question 2

What are some example symptoms of MRC Disorders ?

Answer 2

• seizures
• liver failure
• deafness
• respiratory failure
• thryoid disease
• diabetes

Question 3

What might patients with mitochondrial myopathy present with?

Answer 3

• histochemical alterations in their skeletal muscle which indicate mito dysfunction
• in some patients, muscle biopsies can appear normal

Question 4

Describe the procedure for muscle biopsy

Answer 4

• sample should be places in lightly moist gauze, closed in an airtight container & then placed on wet ice for transport
• optimal cutting temperature compoumnd is used to embed tissue sample prior to frozen sectioning
• Isopentane is used as freezing agent
• muscle is cut transversely, perpendicualr to horizontal plane

Question 5

What can happen if a muscle sample is stored in damp conditions?

Answer 5

• if the specimen is soaked in saline, or wrapped in gauze that is too damp, liquid will be absorbed into the tissue & ice crystals will form during freezing
• this causes artefact which makes interpretation of sample difficult if not impossible

Question 6

What is Modified Gomori trichrome stain ? (MGTS)

Answer 6

• Histological stain used to test for certain forms of mitochondrial myopathy
• MGTS will stain
  mitochondria = red,
  nuclei = black
  cytoplasm = blue/green
  myofibrils = green

Question 7

Describe Modifed Gomori Trichrome Stain

Answer 7

• MGTS specifically allows for the detection of ragged-red fibres (RRF)
• RRF due to accumulation of abnormal mitochondrial below the plasma membrane of the muscle fibre
• leading to appearance of red rim & speckled sarcoplasm
• indicates mitochondrial dysfunction

Question 8

Describe Citrate Synthase

Answer 8

• mitochondrial marker enzyme
• increased CS activity in muscle tissue from mitochondrial patients reflects increased mitochondrial number
• compensatory response to mitochondrial OXPHOS defect

Question 9

What are the 3 fibre types that make up human skeletal muslce ?

Answer 9

• slow oxidative/Type I
• fast oxidative/ Type IIa
• Fast-glycolytic/Type IIB

Question 10

Describe COX Stain

Answer 10

• cryostat-cut muscle section is 1st treated with COX stain
• stain contains DIA & cytochrome c (Cyt C)
• DIA acts as the electron donor to reduce Cyt C which is oxidised by COX
• DIA -> Indamine
• normal COX activity produces a brown stain on the muscle fibre

Question 11

What is Indamine?

Answer 11

Brown insolubl epolymer

Question 12

Describe SDH Staining

Answer 12

• muscle section is then stained for SDH activity
• Succinate is oxidised creating Succinate dehydrognease
• electrons are donated (reduction)
• creates Nitro Blue Tetrazolium

Question 13

Describe Nitro Blue Tetrazolium

Answer 13

• pale yellow colour & water soluble

Question 14

Describe Formazan

Answer 14

• purple blue colour
• water insoluble

Question 15

What does positive staining for COX activity present as ?

Answer 15

• it prevents the precipitation of formazan
• meaning the muslce fibres appear as brown
• fibres that are COX deficient will appear blue as formazan can precipitate

Question 16

Describe Microscopic anatomy

Answer 16

allows for examination of ultrastructural changes in the mitochondrion using electron microscopy studies

Question 17

Describe the steps of muscle prep for electron microscopy

Answer 17

• primary fixation
• secondary fixation
• dehydration
• infiltration & embedding

Question 18

Describe Primary Fixation

Answer 18

• Glutaraldehyde is used to preserve the ultrastructure of the skeletal muscle tissue

Question 19

Describe Secondary Fixation

Answer 19

• Osmium tetroxide is two-fold, it cross-links lipids preserving their structure
• effectively stains the tissue by adding electron-dense material, enhancing contrast

Question 20

Describe Deydration

Answer 20

• water must be removed from the tissue through graded alcohol dehydration to allow for the infiltration of embedding media
• ETOH washing

Question 21

Describe Infiltration & Embedding

Answer 21

• the solvent within the sample needs to be replaced with a miscible resin (embedding media)
• this will polymersie to a hard, but easy to cut material
• this step is infiltration
• Eppxy resin is widely used for morphological studies
• embedding gives support to the tissue

Question 22

Describe the normal structure of mitochondria

Answer 22

• double membraned organelles with inner & outer mitochondrial membranes
• IMM bends inwards & can be divide into 3 regions - inner boundary membrane, the crista junction & the cristae membrane

Question 23

Describe Ultrastructural Mitochondrial Defects

Answer 23

• particularly the highly conserved inner mitochondrial membrane cristae where a number of MRC enzymes reside - linked to imparied mitochondrial function
• caused by mutatuon in nuclear/mitocondrial DNA (1/5000)

Question 24

Describe Paracrystalline Inclusions (Pis)

Answer 24

• ‘parking lot inclusions’
• caused by an accumulation of the enzyme mitochondrial creatine kinase (CK)
• CK is involved in energy buffering using phosphocreatine to convert ADP -> ATP
• CK increase & reaches critial conc. where it crystallises along the inner membrane

Question 25

Describe Concentric Cristae

Answer 25

- causes --> impairment of MICOS which is a mulit-subunit protein complex present in the inner mitochondrial membrane involved in mito architecture & constructure of IMM

Question 26

What can hyper branched mitochondria indicate?

Answer 26

- can reflect cellular stress

Question 27

What can cause mitochondrial swelling?

Answer 27

- impared MRC function - decreased ATP level - both can cause mitochondrial swelling & eventual cell death