M1 Techniques

Question 1

Clinical features of DMD

Answer 1

• Enlargement of calf, gluteal, deltoids
• LL muscles more affected
• Between 6-11 yrs old, strength decreases linearly

Question 2

Causes of DMD

Answer 2

• Lack of dystrophin protein
  
  • Affects DGC/SGC
  • Dystrophin also responsible for signalling

Question 3

What are mdx/dko mice?

Answer 3

• Mdx mice → muscular dystrophy x-linked
• Dko mice → double knock-out mice (Utrophin KO as well)
  
  • More severely affected
  • Complete loss of integrity across ribcage

Question 4

Difference b/w muscles of the mdx and wild-type mice

Answer 4

• Muscles of the mdx mice are larger than wild-type mice
• But they are producing similar force
• ∴ Intrinsically → mdx mice produce less force per cross-sectional area and are more fragile (as they lack dystrophin)

Question 5

Pre-clinical evaluation of efficacy of drugs: 1) Whole body functional tests

Answer 5

• Non/minimally invasive tests
• Overall health or functional capacity
• Assess/monitor treatment → tracks performance
  
  • i.e. running/swimming/climbing
• But generally no definitive (accurate) and provides non-specific measurements of muscle groups

Question 6

a) WBFT: Vertical Hang test

Answer 6

• Latency-to-fall on to a padded mat
• Assess overall muscular strength and endurance
• Pro:
  
  • Natural exercise
  • Simple to perform/evaluate (↑reliability)
  • Assess motivational/volitional aspects too
• Cons:
  
  • Can’t assess specific muscles
  • Crude assessment of strength and endurance

Question 7

b) WBFT: Roper rod

Answer 7

• Spinning wheel → see how long it takes for mice to fall
• Evaluate muscle fatigue and motor performance
• Pro:
  
  • Simple, non-invasive
  • Able to track performance regularly
  • Provides assessment of coordination, motivation, fatigue
• Cons:
  
  • Difficult to identify specific muscle

Question 8

c) WBFT: Grip strength

Answer 8

• Mice holds onto the bar while being pulled on its tail
• Basic assessment of strength
• Able to screen drug for fast or delayed response
• Pro:
  
  • Simple, non-invasive
  • Assess regularly and track
• Con:
  
  • Crude measure
  • Difference between skills of investigators (Someone who knows how to hold the animal compared to someone who doesn’t)
  • Biomechanical advantage (i.e. hunchback) can affect how well the animal holds on, and thus affect reliability

Question 9

2) In vitro/situ/vivo measurements

Answer 9

• Able to look at drug effects on specific muscles
• Able to assess structure and function of the muscles (including fatigue and damage)
• Measure speed of contraction and relaxation

Question 10

a) In vitro

Answer 10

• Muscle out of animal
• Pros:
  
  • Assess functional parameters of muscles directlySp
  • Free from influence of nerve or blood supply
• Cons:
  
  • Less physiological give nerve and blood supply are removed
  • Need to ensure all motor units activated for accuracy of force measurements (technical issues)

Question 11

b) In situ

Answer 11

• Nerve and blood supply intact
• Pros:
  
  • Able to preserve N and BS → able to stim isolated nerve to muscles
  • Able to assess properties of single muscle and its specific adaptations to interventions
• Cons
  
  • More technically challenging
  • More time consuming: limited number of preparations that can be assessed

Question 12

c) In vivo

Answer 12

• Pros
  
  • Can be done in minimally invasive manner
  • Whole muscle group of muscles assessed
  • Use for training/conditioning programs in controlled manner
• Cons
  
  • More technically difficult
  • Equipment can be expensive
  • Not isolated to specific muscle

Question 13

3) Cellular levels

Answer 13

• Cellular levelsMechanically skinned fibres
  
  • Studies E-C coupling/SR release/SR reuptake
• Chemically permeabilised fibres
  
  • Study speed of shortening, damage to fibres

Question 14

Pros and cons of cellular levels

Answer 14

• Pros:
  
  • Study cellular level for mechanistic understanding
• Cons
  
  • Technically difficult
  • Requires expensive equipment

Question 15

Importance of diaphragm

Answer 15

• Function of diaphragm can be a good assessment of drug interventions

Question 16

Other essential analysis

Answer 16

• Not only the structure-function of the muscle is important for assessment
• Assess complementary histological, immunohistochemical, biochemical
• CK levels
• Other pathways: i.e. protein syn vs BD

Question 17

Are dystrophic mice more susceptible to contraction-induced injury?

Answer 17

• Sarcolemmal fragility of mdx mice (i.e. seen from evans blue dye infiltration)
• Greater susceptibility to rupture after shock and stretch
• Compromised costomere → impaired dissipation of force → ↑risk of injury
• Incidence of high muscle fibre branching also contributes to susceptibility

Question 18

What happens when membrane integrity is compromised?

Answer 18

• ↑ Influx on Ca → necrosis/proteolysis of muscle proteins

Question 19

What are contraction clots

Answer 19

• Area of hypercontractility, loss of integrity where fibres are damaged at these points

Question 20

5 theories for muscle fibre necrosis

*My Cat’s Going Very Indie*

Answer 20

1. Mechanical hypothesis
2. Calcium hyothesis
3. Gene regulation hypothesis
4. Vascular hypothesis
5. Inflammatory hypothesis

Question 21

1) Mechanical hypothesis

Answer 21

• Loss of DGC leads to contraction-induced rupture of muscle cell membranes → accumulation of serum proteins
• Exercise in DMD patients and mdx mice → greater muscle damage than unaffected controls

Question 22

2) Calcium hypothesis

Answer 22

• Influx of Ca into cell → overwhelming cell ability to buffer the change
• Overexpress of calpain/caspases

Question 23

3) Gene regulation hypothesis

Answer 23

• Failure of molecules localising to the membrane when DGC is absent → prevent proper signalling molecules recruited

Question 24

4) Vascular hypothesis

Answer 24

• Disruption is membrane proteins → loss of NO signalling → muscles being ischemic and thus cannot repair
• nNOS KO mice do not have muscle disease → nNOS may play a direct role

Question 25

5) Inflammatory hypothesis

Answer 25

* ↑Cytokines and chemokines → overwhelms system → mismatch of muscle repair and extensive fibrosis * Fibrosis in mdx mice: * Limb: little fibrosis → indicating that collagen regulation at post-transcriptional stages mediate extensive fibrosis * Diaphragm: extensive fibrosis

Question 26

Significance of fibrosis

Answer 26

* Fibrosis may create a physical barrier that limits efficacy of drug and cell/genetic treatments * Reversibility to fibrosis → quite irreversible → changes contractile properties of the muscles

Question 27

Therapeutic 'window of opportunity'

Answer 27

* Treat as soon as possible for greatest efficacy * Slow progression of DMD → preserve muscle fibres and prevent fibrosis