MITROCHONDRIAL DISEASE Ragged red fibres Modified Gömori trichrome Appropriate methodology Skeletal muscle biopsy - histochemical alterations in heir skeletal muscle = mitrochondiral dysfunction - histochemical MGT - If stained correctly it ragged red fibres will appear which are the hall marks of MD in striated muscle -> mitrochondiral clisters appear as deposits in cryostat sections -> the myofibres with these deposits are called the ragged fibres - due their disrupted structure > usually found in invidiuals older than 50 Mitochondria will appear purple as they are pushed to the periphery GT is a plasma dye (2R chromotropic) and connective tissue dye (fast green FCF, light green or aniline blue), combined with phosphotungstic acid and glacial acetic acid: PA promotes the red staining of muscle and cytoplasm Outline of methodology No fixation - needs active enzymes to work use frozen tissue at pH 3.4 Cut 12 um sections in cryostat from snap frozen biopsy Attach one or more sections to a 22mm cover slip Stain: The modified Procedure
· Immerse sections in harris hematoxylin used as a nuclear stain · Sampel is rinsed with tao water · Immersion in Gomori trichrome - Gomori solution consists of 0.015% fast green FCF, 0.12% chromotrope 2R, 0.65% phosphotungstic acid and 1.0% acetic acid in 100 ml distilled water - muscle cells blue/green, nuclei will be red, mitrochondira purple . Differenctiate in 0.2% acetic acid . Immerse in tt5% alcohol · Clear with xylene · Mount coverslip What you expect to see in infected/normal Background · Effects most organs in the body as it effects the efficacy of mitrochondira and the production of ATP - the metabolically most axtive organs . We need ATP for respiration . Failure of the mitrochondira which is responsible for tt0% of the energy needed b the body to sustain itself and support organ function, therefore a fial in this leads to a reduction in the energy supply which can result in cell injury or cell death - if the process is repeated throughout the body it can lead to rogan failure. . The parts of the bdoy which require the most energy are usually the areas which are most affected by MD: heart, brain, lungs and muscles, eyes, nerves. . Either rinherited or a spontantous mutation in mtDNA or nDNA - alterations in the proteins or RNA molecules within the mitrochondira - 3000 genes ot code one mitrocondrion, mtDNA codes 37, therefore a genetic mutation in the nuclear dna is more common and therefore can easily effwct the development of mitrochondira · Is usually present at birth but can be diagnosed at any stage · One in 5,000 has the genetic mt disease · Symtpms: deoend son which cells a=of the body are effected, symptoms can rnage frome midl to severe, incolve one or multiple organs: poor growth, muscle weakness, muscle pain,
low exercise intolerance, nision or hearing loss, neurologival problems such a sseizures, increased reisk of infection · Diagnosis: review of patent family history, physical exam, neurological scan,